Qatar Foundation Annual Research Conference Proceedings Volume 2016 Issue 1

Background: Chronic kidney disease (CKD) is characterized by progressive decline in renal function; if left untreated, it ultimately results in end-stage renal disease (ESRD), a condition that demands either dialysis or kidney transplant for survival. CKD and ESRD are associated with a multitude of complications ranging from increased hospitalization to accelerated cardiovascular events and mortality. Currently, type-2 diabetes and hypertension are the two major risk factors for CKD. With the increasing incidence and prevalence of these conditions globally, the patient population with CKD is expanding worldwide. According to local sources, CKD affects about 13% of Qatar's population, and the prevalence of ESRD, the advanced phase of CKD, in Qatar was found to be 212 per million patients. The increased risk of complications associated with CKD in conjunction with its high prevalence in Qatar and in the rest of the world, necessitates its prevention and management as a high national and international priority. Elevated urinary albumin excretion (commonly referred to as proteinuria) is not only a hallmark of renal disease, but also strongly associated with the development and progression of CKD. Albuminuria is thought to induce endoplasmic reticulum (ER) stress, consequently triggering AKT pathway and resulting in inhibition of AMP-activated kinase (AMPK). AMPK, a fuel sensor present in cells, is primarily involved in the regulation of fatty acid oxidation and ATP synthesis. Inactivation of AMPK was found to trigger mTOR (mammalian target of rapamycin) pathway, and subsequently inhibit autophagy (a defense mechanism) and induce epithelial-to-mesenchymal transition (EMT). These signaling changes eventually accelerate renal cell apoptosis, and manifest into CKD. Thus, the objectives of this study are: 1) to standardize and characterize an in vitro model of albumin-induced renal cell injury using normal rat kidney proximal tubular (NRK-5E) cells, and 2) to explore the effect of AMPK activation on ER stress, AKT, mTOR, EMT, autophagy and apoptosis that are thought to mediate renal cell injury during proteinuria using the developed in vitro model of albumin-induced renal cell injury. Methods: NRK-52E cells were grown to 60% confluency and then serum-starved for 24 hours to arrest cell proliferation. Cells were then exposed to albumin, at concentrations ranging from 1 to 30 mg/ml, for 24 to 72 hours. At specific endpoints, cells were assessed for induction of ER stress and alterations in the status of AKT, AMPK, mTOR and autophagy and changes in cellular senescence via x-galactosidase (an enzyme that is expressed in senescent cells) staining. Following standardization of albumin-induced renal cell injury model, studies were performed in the presence and absence of AMPK activator metformin (1 mM) for 24 to 72 hours. Cells were then assessed for alterations in the status of AMPK, AKT and mTOR, and the markers of ER stress, EMT, autophagy and apoptosis. Results: Exposure to albumin for 72 hours caused a dose-dependent increase in cellular senescence in NRK-52E cells. In contrast, cells exposed to albumin for 24 and 48 hours did not reveal any marked changes in cellular senescence. A 4-fold induction in ER stress marker CHOP and the EMT marker a-SMA was noted. Moreover, higher concentrations of albumin, particularly 30 mg/ml, caused severe induction of ER stress and EMT, marked by 20-fold increase in CHOP and 6-fold increase in a-SMA respectively. Similarly, the phosphorylation of AKT and P70S6K (a downstream target of mTOR) was increased by more than 1.5-fold in cells subjected to albumin treatment. In addition, albumin treatment caused a dose-dependent reduction in AMPK phosphorylation and about 66% decrease in the expression of autophagy marker LC3-II. The above changes were observed in conjunction with prominent dose-dependent induction of apoptotic markers - caspase-3 and caspase-12 ranging between 1.5 to 3.5-fold and 3 to 5-fold respectively in cells exposed to albumin. In contrast, metformin co-treatment restored the levels of phosphorylated AMPK, and suppressed activation of AKT and P70S6K in NRK-52E cells exposed to albumin. Notably, metformin also prevented albumin-induced EMT; this was marked by a 50% decrease in a-SMA and a 60% increase in E-cadherin expression. In addition, 2.5-fold increase in LC3-II expression was noted. Intriguingly, the pro-apoptotic protein CHOP was induced following treatment with metformin; nonetheless, the expression of apoptotic markers caspase-12 and caspase-3 was reduced by 80% and 70% respectively, indicating that metformin protected the cells against albumin-induced apoptosis. Conclusion: Albumin treatment induces ER stress, and activates AKT, EMT and apoptosis, with concomitant decreases in autophagy and inactivation of AMPK in renal tubular cells. Activation of AMPK via metformin treatment suppresses AKT and mTOR activation, and prevents EMT and apoptosis, but increases autophagy and ER stress in renal tubular cells. Further studies are required to understand the mechanisms by which metformin differentially modulates ER stress and apoptosis in renal cells under proteinuria. Together, our findings suggest that AMPK activation via metformin could serve as a potential therapeutic strategy to prevent and/or treat the development of CKD in patients with established proteinuria.

Introduction: Polypharmacy (or the use of multiple medications at the same time by the same patient) could expose the patient to health risks and add an extra burden on the life of these patients in addition to the burden of illness. The Living with Medicines Questionnaire (LMQ) was developed to assess the burden of polypharmacy from the patient's perspective. This tool includes items relating to the use of medication and expressed as statements for which the respondents indicates his/her agreement using five-points Likert-type. There is a need to make available such a measure to contribute information generated from the Arabic-speaking world and to share research findings through an Arabic version of the LMQ, which is culturally equivalent to the original English tool. Objectives: We aimed at translating and culturally adapting the Living with Medicines Questionnaire into the Arabic context through a structured process utilizing best practices in translation and cultural adaptation. Methods: As means for adhering to best practice, permission to use the LMQ was sought from the developers, and a protocol for its translation and cultural adaptation was developed using guidelines developed by the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) for the translation and cultural adaptation process for patient-reported outcomes measures. Two forward translations were produced, compared, and reconciled into the first reconciled version. This version was then back translated into English and compared with the original tool leading to the second reconciled version. The emerging Arabic version of the LMQ was cognitively tested among purposively selected individuals to test the linguistic and cultural equivalence and produce the final Arabic translation. The results were documented and shared with the developers of the LMQ. Results: A comprehensive protocol, with the potential to inform future similar studies elsewhere, was developed and used as a guide to produce an Arabic version of the LMQ that is representative of the original tool, and suitable for the Arabic culture. No major issues were found in the demographics section of the questionnaire or the instructions to answer the questions. Issues identified and related to cultural and linguistic equivalence of some terms were resolved by re-wording some items in the tool. A total of seven people were purposively selected to be interviewed in order to assess the LMQ Arabic version in areas related to comprehension, time burden, and acceptability. Individuals were selected with consideration to balanced gender distribution, age, ethnicity/nationality, education, and all with Arabic language as their mother tongue. The cognitive debriefing exercise generated comments regarding the original tool's construct and its Arabic equivalent, which were communicated to the developers of the LMQ for their consideration while conducting further comparative studies. Conclusion: Through following methods based on best practice, we have joined the international efforts into the development of the first questionnaire aiming to measure medication burden in the Arabic-speaking region. We now make available a culturally equivalent Arabic translation of the Living with Médicines Questionnaire for use in Arabic speaking countries in research and/or clinical practice. However, further validation tests are needed to be conducted among Arabic-speaking population.

Antibiotic Susceptibility and Plasmid Profile of Vibrio vulnificus isolated from mussels in Qatar Vibrio vulnificus infections are the worldwide public health problems associated with illnesses resulting from consumption of raw or partially cooked seafood and exposure with the contaminated sea water. Infections of V. vulnificus are reported in many different countries in America, Europe and Asia notably in USA, South Korea, Taiwan, Malaysia and Saudi Arabia. The aim of this study is to isolate and identify V. vulnificus from Mussels in Qatar and detect antibiotic susceptibility and plasmid profiles. A total of 87 Mussels, 50 from Doha and 37 from Akhor were examined for the presence of V. vulnificus using Thiosulfate-citrate-bile salts-sucrose agar (TCBS) and Chromagar Vibrio (CV), 18% of Doha samples were positive and 13.5% of Alkhor samples were positive, 9 of 14 isolates from Doha and 5 of 14 isolates from Alkhor. The antibiotic susceptibility tests were performed for 12 antibiotics by the disc diffusion method. For molecular identification of the isolates, the 16S ribosomal RNA gene fragment was amplified by polymerase chain reaction (PCR) and the nucleotide data were subjected to Basic Local Alignment Search Tool (BLAST) for analysis, Sequence comparison with public databases resulted in 96-100% similarity with V. vulnificus in 15 (60%), 99-100% similarity with V. paheamolyticus in 5 (20%); and 5 (20%), were identified as the non-vibrio species, The analysis of evolutionary relationships among isolates in this study was represented in seven cluster groups. The cluster A referred to non-vibrio isolates whereas the clusters B, C, E, F, and G included V. vulnificus.

V. paraheamolyticus was only represented in the cluster D. 16S rDNA-based identification confirmed the conventional identification for isolates. The morphological and biochemical test detected V. vulnificus in 32% of the Mussels samples all of which showed resistance for two to eight antibiotics. Plasmids were found in 88% of the isolates with 10 different profiles having two to four plasmids. Based on 16S rRNA gene sequence homology 15 isolates were identified as V. vulnificus, five as other Vibrios and five as nonvibrios. We have observed a marked difference between morphological methods and molecular methods in identification of V. vulnificus indicating the in adequacy of the morphological technique in discriminating the Vibrio species. The occurrence of V. vulnificus in the Mussels samples is quite high so consumption of uncooked and semicooked Mussels should be avoided in order to prevent food-borne infection by this pathogenic bacterium. There are observed a marked difference between morphological methods and molecular methods in identification of V. vulnificus indicating the in adequacy of the morphological technique in discriminating the Vibrio species. The occurrence of V. vulnificus in the cockle samples is quite high so consumption of uncooked and semicooked cockles should be avoided in order to prevent food-borne infection by this pathogenic bacterium. Antibiotic susceptibility. Bacterial isolates showing resistance to more than six antibiotics contained more than three plasmids. Plasmids are known to carry antibiotic resistance genes. Otherwise, V. vulnificus isolates in which no plasmids could detected also has resistance to some of antibiotics, that mean antibiotic resistance genes may also be carried on the chromosome of bacteria.

Introduction: Angiosarcomas are rare and malignant neoplasms that involve abnormal proliferation and migration of cancerous endothelial cells. Angiosarcomas can arise in any region of the body, but tend to be found in the skin, soft tissue, and liver. They are associated with high mortality rate due to their aggressiveness and high rate of metastasis. Therapeutic strategies for treating angiosarcomas involve use of cytotoxic drugs and radiotherapy. However, patient-resistance to these approaches is commonly reported. Hence, there is a need to further understand molecular mechanisms underlying angiosarcomas and explore novel therapeutic targets. Furthermore, because abnormal angiogenesis underlies nearly all types of cancer, a better understanding of cellular pathways regulating cancer endothelial cell function may also lead to development of novel anti-angiogenic therapies. The PI3K/AKT/mTOR signaling pathway plays an important role in regulating cell proliferation and is also regarded as one of the most commonly dis-regulated pathway in cancer. Inhibitors have been developed targeting these signaling proteins and their therapeutic potential has been evaluated in different studies. However, there is little known about the relevance of this signaling pathway to angiosarcomas. Therefore, in this study, we explored the anticancer therapeutic potential of targeting the PI3K/AKT/mTOR signaling pathway using inhibitors of PI3K, AKT or mTOR in a murine VEGF-dependent angiosarcoma cell line. Methods: Cell culture: MS1-VEGF cells (mouse endothelial cells capable of inducing angiosarcomas) were used. Cells were maintained in Dulbecco's Modified Eagle Medium, which was supplemented by 5% fetal bovine serum, 1% penicillin/streptomycin, and 11 mM glucose. Concentration-response experiments: The inhibitors used were: PI3K - LY294002 (10 μM); Akt – AKTi 1/2 inhibitor (AKTi, 10 μM); and mTOR - Temsirolimus (4 μM). LY294002 is a selective inhibitor of PI3K, but is not yet in clinical use. AKTi 1/2 is a non-ATP competitive inhibitor of Akt isoforms 1 and 2. Temsirolimus is a specific inhibitor of mTOR, and has been clinically used for the treatment of advanced renal cell carcinoma. The treatments were conducted over a 48 h period, with DMSO serving as the solvent control. Experiments was repeated at least three times using cells cultured in 12 or 6-well sterile tissue culture plates (Falcon). Cells were incubated in a 5% CO2 incubator at 37 °C. At the end of the treatment period, cell counts were performed and trypan blue dye exclusion was used to assay cell viability. This was done by pipetting a small volume of cell suspension mixed with trypan blue onto a dual chamber counting slide (Bio-Rad TC20 Automated Cell Counter). Proliferation Assay: In order to further assess the effectiveness of the inhibitors, proliferation assays were performed. Cells plated in 96-well plates with the inhibitors for 48 h were treated with the CellTiter 96 Aqueous One Solution reagent (Promega) and incubated in the CO2 incubator at 37 °C for 3 h. A PerkinElmer 2104 plate reader was used (EnVision software) to measure absorbance (at 492 nm). Western blots: Western blots were used to detect the expression of several proteins linked to the PI3K/Akt/mTOR pathway and also to study changes resulting from treatment with inhibitors. Primary antibodies recognizing mTOR, AKT, PI3K, eNOS, LC3B, Beclin-1, ERK, Survivin, and Bcl-2 were used. All proteins probed have known roles in cell proliferation/apoptosis/autophagy. Images were captured using GeneSnap software on a PerkinElmer Geliance 600 imaging system. Quantitiy One (Biorad) was used to analyze Western blot data. Flow cytometry: Cell cycle analysis was performed on the LSR Fortessa analyzer (BD Biosciences) by staining fixed and permeabilized cells with propidium iodide. Data was processed with FACS Diva 8.0 software (BD Biosciences). Data analysis: All the data was analyzed using the statistical software GraphPad Prism 5.0 (GraphPad Software, Inc. CA, USA). Data is presented as mean ±  SEM. Statistical analysis was performed with using ‘t’ test or one-way analysis of variance (ANOVA). Post-hoc comparisons between groups after ANOVA were performed by Tukey's multiple comparison tests. ‘p’ values less than 0.05 were considered to be statistically significant. Results: Incubation of MS1 VEGF cells with LY294002, Akt-i ½ or Temsirolimus caused a reduction in cell number, indicating reduced cell proliferation. The Akt-i ½ was the most effective among the three and also caused a strong reduction ( < 25% viability) in cell viability (unlike LY294002 and Temsirolimus). Proliferation assays performed in 96-well plates indicate a reduction in MS1 VEGF cell proliferation with all three inhibitors, and Akt-i ½ was again the most effective in these assays. Cell cycle analysis revealed a robust increase in ‘sub G0/G1 population’ after treatment with Akt-i ½ (Control - 8% vs Akt-i ½ - 43%), suggesting an increase in cell death. To investigate the mechanisms underlying the actions of the inhibitors, western blot experiments were performed. The data (n = 4) demonstrated down-regulation of the anti-apoptotic Bcl family protein Bcl-2 and the inhibitor of apoptosis protein survivin after treatment with Akt-i ½ (but not LY294002 or Temsirolimus). Treatment with Akt-i ½ also reduced the phosphorylation of Akt and ERK proteins. Furthermore, there was a strong increase in the expression of the autophagy marker LC3B-II after treatment with Akt-i ½. Immuno-staining experiments confirmed aggregation of LC3B-II after Akt-I ½ treatment, suggesting an induction of autophagy. Inhibition of autophagy by 3-methyladenosine (3-MA) reversed Akt-i ½-induced LC3B-II puncta formation and also significantly enhanced Akt-i ½-induced cell toxicity. This suggests that autophagy induction acts as a cell survival mechanism after Akt inhibition. To investigate if Bcl-2 and survivin could be downstream effectors of Akt-i ½, experiments were performed using specific inhibitors: YM155 for survivin and TW37 for Bcl-2. A combination of YM155 and TW37 induced robust changes in cell cycle, increased the ‘sub G0/G1 population’ (Control - 9% vs. TW37 + YM155 - 38%) and reduced the proportion of cells in G0/G1, S as well as G2/M phase. Discussion: The data revealed promising anti-proliferative actions for LY294002, Temsirolimus and Akt-i ½ in MS1 VEGF cells. The drug, Akt-i ½, drug was particularly effective and also substantially reduced cell viability. The data also suggest that Bcl-2 and survivin may be critical components of the anti-proliferative action of Akt-i ½ thus making it a highly effective agent. The data also revealed that the cells induced autophagy as a survival mechanism when Akt was inhibited. In conclusion, multiple signaling pathways and proteins regulate MS1 VEGF cell proliferation and survival, which are targeted by Akt-i ½. Future studies: Future investigations will focus primarily on direct evaluation of apoptosis by flow cytometry and if possible experiments in nude mice to evaluate in vivo drug efficacy.

Pulmonary arterial hypertension (PAH) is a progressive, debilitating and fatal condition [1]. Current PAH therapy relies on vasodilator drugs, which are seriously limited by their systemic side effects. We suggest that advances made in the field of nanomedicine could be used to improve the utility of drugs to treat PAH. Whilst not currently used clinically, metal organic frameworks (MOFs) such as the Material from Institute Lavoisier (MIL) class, are good candidates since (i) they can be tailored to accommodate different types of drugs including those with the molecular weights of PAH medications (MW; 300-500) [2], (ii) are biocompatible and biodegradable [3]; (iii) have a large internal surface area and low density with commensurate high drug loading capacity; (iv) are thermal and mechanically stable; and (v)have a long drug release period with the ability to incorporate different functional groups [2, 4-6]. However, the idea that nanomedicines can be used to treat PAH is very new and the use of MOFs in this regard is untested. Thus, we must first: (i) validate their chemical structure/stability, (ii) establish MOF cytotoxicity and effect on inflammatory responses in relevant cell types and (iii) investigate their behaviour in vivo model. In PAH, endothelial cells are critical cells to target. This is because the role of endothelial cells in releasing a delicate balance of vasoactive hormones is disrupted in PAH where cardioproective mediators such as nitric oxide and prostacyclin are reduced whilst the release of the constrictor peptide endothelin (ET)-1 is increased. Indeed, current PAH drugs work to boost nitric oxide and prostacyclin pathways and to block ET-1 receptor signalling. Aim: The aims of this work are to (i) synthesis and characterise MOFs designed to accommodate PAH drugs; (ii) to investigate the cytotoxic effect of MOFs in a comprehensive range of cell models relevant to PAH in vitro and the toxicity and distribution of MOFs in vivo. Methods: The nanoporous iron MOF (MIL-89), and a polyethylene glycol formulation (MIL-89 PEG) were prepared as previously described [2], then characterized using infrared spectroscopy (IR), powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Endothelial cells grown from human blood progenitors of control subjects and PAH patients were cultured as we have done previously [7] and the effect of MOFs on viability determined using the AlamarBlue® Assay. In addition the effect of MOFs on endothelial cell inflammatory function was determined by measuring the release of the cytokine CXCL8 and on markers of PAH disease by measuring ET-1 using specific ELISAs. To investigate the effects of MOFs in vivo rats were injected with the MOF MIL-89 (50 mg/kg) in glucose solution twice a week for various times up to two weeks, while the control group was injected with glucose solution only. Animals were killed and tissues including; blood, heart, lung, brain, thymus, liver and kidney as well as urine and faeces were collected at days 1,3,7,10 and 14. Results: MIL-89 and MIL-89 PEG retained functional groups, and were crystalline, spherical and stable in air up to 200 °C. Neither preparations caused toxicity in cells grown from control donors or patients with PAH at concentrations up to 10 μg/ml (Fig. 1). Interestingly, both preparations of MOFs displayed anti-inflammatory effects; inhibiting CXCL8 and ET-1 release from endothelial cells from healthy donors as well as from PAH patients (Fig. 1). These MIL-89 had no affect on body weight of the rats and did not cause any gross changes in their lungs (Fig. 2). Conclusion: Both MIL-89 and MIL-89 PEG represent non-toxic potential drug-carriers with predicted molecular capacity for the current PAH medications, which include treprostinil sodium, bosentan and sildenafil. Furthermore, they both display some evidence of anti-inflammatory properties in vitro that may be of therapeutic benefit in the treatment of PAH. MIL-89 had no overt toxic effects in vivo, although these will need to be explored in more detail in future studies.

References

1. Archer, S.L., E.K. Weir, and M.R. Wilkins, Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. Circulation, 2010. 121(18): p. 2045-2066.

2. Horcajada, P., et al., Porous metal-organic-framework nanoscale carriers as a potential platform for drug delivery and imaging. Nat Mater, 2010. 9(2): p. 172-178.

3. Huxford, R.C., J. Della Rocca, and W. Lin, Metal-organic frameworks as potential drug carriers. Curr Opin Chem Biol, 2010. 14(2): p. 262-268.

4. Kızılel, S.K.a.S., Biomedical Applications of Metal Organic Framework, 2010, Department of Chemical and Biological Engineering: Koc- University, Rumelifeneri Yolu, 34450, Sarıyer, Istanbul, Turkey.

5. Ferey, G., et al., A chromium terephthalate-based solid with unusually large pore volumes and surface area. Science, 2005. 309(5743): p. 2040-2042.

6. Horcajada, P., et al., Metal-organic frameworks as efficient materials for drug delivery. Angew Chem Int Ed Engl, 2006. 45(36): p. 5974-5978.

7. Reed, D.M., et al., Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources. Biochem Biophys Res Commun, 2014. 455(3-4): p. 172-177.

A bacteriophage is a virus that infects a bacteria, it uses the bacteria as a host to further replicate by controlling the replication and protein synthesis machinery of the cell. They are composed of a protein capsule and DNA or RNA genome. Bacteriophages are used in the treatment of bacterial infections, known as the phage therapy. This results from phages invading the bacteria, as they undergo a lytic cycle, where the replication and protein synthesis machinery is used to produce virions, that later cause the cell to lyse, thus killing the bacteria. This technique has higher efficiency compared to the use of antibiotics as bacteria can develop a resistance against antibiotics, while remaining susceptible to the infection of the phage. In addition, phages can evolve and adapt to new mutations that might arise in the bacteria. Lastly, phages might be engineered to contain a survival gene needed by the bacteria to ensure that the bacteria will replicate and synthesize the new virons, leading to lysis and death.

This research explores the potential of the use of bacteriophages in the treatment of water, to disinfect the water from microbial bacterial and other bacteria that were used to detoxify the water. Phages were previously extracted from Qatar's sand and were used as the infecting agents. The model host chosen is Arthobacter bacteria, which is a denitrification bacteria, as it is similar to the bacteria used in water treatment. Denitrifying bacteria reduce nitrates to nitrogen-containing gases, allowing for the recycling of nitrogen back to the atmosphere.

The goal of my research is to analyze the genome of a phage extracted from Qatar's Sand. A culture of Arthobacter grown in Smeg media was used as a host. Unlike the normal optimal growth temperature of Arthobacter at 32 °C, it was shown that higher phage titer was produced when the culture was grown to saturation in smeg media at 37 °C. In addition, the culture was initially grown in luria Broth which is rich in nutrients including tryptone, yeast extract and NaCl. However, low phage titer was produced compared to switching the media to smeg media, which contains Middlebrook 7H9 broth base with supplements of albumin, dextrose and salts.

After manipulating the growth conditions of the host culture to obtain the highest titer, the optimum conditions were found to be a saturated culture of Arthobacter grown in Smeg media at 37 °C. Later, lysis conditions of the phage were optimized by varying several factors. First, the type of the top agar and plates used for pour plating were varied. Several top agars were used this includes LB top agar, LB top agar in 1 mM CaCl2 and PYCA top agar. It was concluded that the top agar that resulted in the highest titer was LB top agar in 1 mM CaCl2. Several plates have been used for pour plating this include LB plates and PYCA plates. Plaques formed using LB plates produced unclear plaques, this unclearness is an indication of persistence of the lysogenic state and not the lytic state. In the lysogenic state the phage genome gets incorportated into the host genome and replicates along with the cell cycle, thus remaining in a dormant state. On the other hand, in the lystic cycle, the phage uses the replication and protein synthesis machinery to produce more phages, that will later lead to lysis. In contrast, PYCA plates containing CaCl2 formed clearer plaques, indicating dominance of the lytic state. Therefore, addition of calcium in the top agar and the plates aided in the phages’ shift from the lysogenic cycle to the lytic cycle. This correlates with previous work, where calcium was shown to be essential for the penetration of the phage's genome into the host.

Finally, the incubation temperature of the plates containing the infected host Arthobacter by Qatar's sand's phage was varied. Initially the plates were incubated at 32 °C, however no plaques were formed. When the temperature was later increased to 37 °C, lysis was observed and a high phage titer was obtained.

After obtaining a high titer lysate, the DNA was isolated and a sample of the phage was sent to obtain an electron micrograph image of the phage. A restriction enzyme digest was preformed on the isolated DNA and the profile created was compared to the profile of lambda phage. Both the phages are lytic and lyse at similar temperatures, which allows for comparisons.

Lastly, motifs were identified between the Qatar's sand bacteriophage and T7 bacteriophage using blast tools in a previously extracted and sequenced Qatar's sand phage, primers will then be designed to verify if they exist in the actual phage.

Future work will focus on the analyzing the sequence of the phage to identify potential lysis genes, In addition, strengthening the lytic ability of the phage by cloning the gene of lysozyme into a T7-based vector system and test its adaptability to temperature using Arthrobacter as the host system.

Diabetes is a disease that occurs when there is an abundance of glucose in the blood stream and the body cannot produce enough insulin in the pancreas to transfer the sugar from the blood to other areas in the body for energy. Type 1 and Type 2 diabetes are both prevalent in Qatar however, Type 2 diabetes is more common and it is mainly what is causing the epidemic Qatar is facing. Type 1 diabetes is inherited and is not related to eating habits or lifestyle, and it is diagnosed in juvenile years. However, Type 2 diabetes can be caused by obesity, unhealthy eating habits, and lack of exercise and overall fitness. It is treatable but not curable and can be treated by maintaining a healthy diet, regular exercise, insulin intakes, and oral medication.

Diabetes is the 3rd most common cause of death in Qatar after road accidents and heart disease (http://www.worldlifeexpectancy.com/country-health-profile/qatar). In fact, in 2011, Qatar ranked 6th for the highest percentage of diabetes in the world. The amount of overweight adolescents in Qatar is very large resulting in higher diabetes rates amongst children and young adults. The percentage of overweight children doubled from 17% in 1997 to 35% in 2007 according to The Peninsula newspaper. Awareness is crucial at this age because most of the human body fat is put on during teenage years when the body is constantly growing and it is very important to maintain a healthy weight at this time.

Students at the secondary school level should understand whether or not they are at risk of developing this disease compared to ordinary people. Each student should know their chances of risk due to their family relations, history, and habits. In Qatar we find that the parents of many students our age face diabetes because of the habits of their parents and so on, therefore all of us are at a higher risk. The fact that fast food is so available and practical keeps people, especially those at a young age constantly consuming such unhealthy products. With the statistics of obesity so alarmingly high, education and awareness is the only key to solving the problem. We plan on increasing the diabetic awareness at our school by conducting surveys, presenting posters, interviewing students, as well as working hand-in-hand with the Qatar Diabetes Association (QDA) to be as successful as possible.

Although many efforts are being made by the government, many students do not know enough about the danger that they may be putting themselves in, and the consequences that result because of their actions. Many students at this age are allowed to choose what they eat and their diets are not regulated by parents anymore, therefore they need to learn to make proper choices. The high temperatures in Qatar also make it inconvenient to exercise outside, however there are numerous alternatives. The goal of this research is to enlighten our peers with this given information and compare their knowledge of diabetes before and after. Awareness is the first step to prevention, and that needs to begin at a young age. Research Methods: The first step to our project on spreading awareness about diabetes is to know the level at which the students are already aware. Therefore, using Google Forums, we will create a survey with questions about how much the students already know about diabetes, if they know of family history, if they have been tested, if they know the symptoms, etc. Once we have fully created the survey, we will email the link to all of the high school students at our high school asking them to each fill out the survey with the appropriate responses. In order to insure that we have accurate gatherings, we will each interview an equal amount of students randomly and record their answers to work from there.

After receiving 189 responses from the high school students we began to analyze the results. Only 22% of the students had been tested for or against diabetes. Out of those who had been checked 48% had checked more than a year ago. 58% of the students knew of family members with diabetes. Most had checked the correct symptoms for diabetes along with a lot of unrelated ones like coughing or vomiting. Also for the things you could do to prevent diabetes, 8% responded with “washing your hands”, 30% responded with “not smoking”, and surprisingly even 3% responded with “dressing modestly”. After witnessing the high percentage of wrong responses, it was made clear that we definitely had to increase our effort in educating our peers.

Once discussing the open ended question of “What do you know about diabetes?” we ranked each student's response out of 10 (Based on the below scale). Then, we performed a chi-squared statistical test to compare our expected value of 7/10 to 3.323/10 which was the average student rank. This proved a significant difference between the levels of awareness we expected from the students, to what we actually received.

The data from the survey proved the indeed lack of awareness among the students of the high school level. We created fun awareness posters and posted them on glass doors to the cafeteria, science wing and specific lockers where they were most visible to the student population. Results and Discussion: Upon the completion of our presentation to the 10th grade health class, we conducted a second survey regarding what the students had learned and the quality of our presentation. The table below shows the responses received from the students after the presentation. Conclusions: Unfortunately at the beginning of this research, we were shocked at noticing the extreme lack of awareness amongst our student population. Considering the high rates of diabetes in the State of Qatar, we expected a basic understanding about the disease especially since many people here are prone to it. It is very alarming that which current trends it is expected that 73% of men and 69% of women would have reached obesity in the country by 2030. Indeed it can be stated that a lot of work needs to be done.

One of the major purposes of this research was to gain insight about this issue and it is quite obvious that these high rates are leading to a huge problem in the nation. Fortunately Qatar has been working hard to increase awareness with events like National Sports Day and hosting events like the 2022 world cup to promote fitness in the country. As mentioned previously, since diabetes type 2 is the major concern because it is developed later in life and is preventable, young students are the target for spreading information too. Habits develop during the adolescent years so spreading the word among students at this age is critical.

This research was very successful in promoting awareness in our own school, but from what we have learned, there is a long way to go in order to decrease the high statistical quantities of diabetes rates. Benefit to students: The Qatar National Research Strategy (QNRS) principle were designed to increase the quality of research occurring in Qatar. The third most important area of research in Qatar was health and under the Addressing National Health Priorities column, Type II diabetes was first in importance. We feel that by following the research plan addressed by H.H. Sheikha Moza that our research could have a strong impact on the research culture in Qatar indeed.

Some of the specific benefits to the students participating in this project include the following;

- How to conduct literature survey

- An increased knowledge and understanding about Diabetes, its causes, preventions and symptoms

- The seriousness of the issue worldwide and in Qatar

- The realization that the awareness of the students they spoke to increased so much that they can now give a lecture about the topic to their peers

- Presentation skills, video, poster, lecture

- Survey design

- How to conduct statistical analysis using statistical software

- Incorporating creative ways in educating their peers

- Communicating with professional societies such as Qatar Diabetes Association

- Took part in the joint program of Qatar Diabetes Association Sports Day event for the Action on Diabetes Campaign

- Interviewing skills as the asked questions of students and health care professionals working with diabetes

- Time management with creating a Letter of Intent and Proposal Report on time

- How to work as a team and how to divide the tasks to cover all requirements efficiently and effectively

- It is noteworthy to mention that the entire research methodology and the final report were fully prepared and conducted by the two students involved. The teacher's role was solely advisory

Qatar National Research Strategy (QNRS) mentions that diabetes is the 3rd most important area of research for QNRS.

Presenting the information about Diabetes to the high school student body, will not only benefit the students and teachers themselves in insuring their health and being aware of what may happen if they do not control some unnecessary desires, but it will inform them about the SSREP program in Qatar. As students participating in a research project during our secondary school years, presenting to our peers will allow them to realize the benefits that come from conducting research projects and inspire them to contribute in the following years.

Acknowledgement

Qatar National Research Fund – SSREP Program

Streptomyces are Gram positive aerobic bacteria from the phylum Actinobacteria with close to 570 known species. They are popular for providing a variety of compounds having medicinal properties including - antibiotics, antifungals, antitumor amongst others. Various researches in the past have tested these properties of Streptomyces sp. and species including Streptomyces avermitilis, Streptomyces venezuelae, Streptomyces aureofaciens, Streptomyces clavuligerns and Streptomyces erythrens have been found effective in producing these varied compounds. For instance, S. avermitilis produces avermectins which are used to treat river blindness while S. venezuelae secretes chloramphenicol. Additionally S. venezuelae has been suggested as ideal test organism for studies based on physiology and also for analysis of differentiation on biochemical basis (Chater, 2013). Although a high number of metabolites of Streptomyces are now available in the health care industry as effective drugs for a variety of diseases, increasing number of cases of antibiotic resistance is threatening global public health. The emergence of resistance has resulted in drug ineffectiveness and there is a wide search for suppressing these strains. Such resistance has been identified to have occurred due to both phenotypic and genotypic modifications (Suzuki, Horinouchi, & Furusawa, 2015). Properties of Streptomyces and increasing cases of antibiotic resistance have fuelled research to identify more and more species of Streptomyces and to look out for novel metabolites released from them. Other reasons for the need of identifying newer compounds include the breakout of new diseases in the second half of the last century, incompetence in fighting naturally resistant bacteria such as P.aerogenosa that causes fatal infections and the toxic effects resulting from consumption of currently available antibiotic drugs (Sanchez & Demain, 2011).

Hence this research attempted to study antimicrobial properties of three Streptomyces species isolated from the desert soil of Qatar. The antimicrobial properties were assessed firstly through direct testing against five test organisms - Escherichia coli and Pseudomonas sp. as Gram-negative bacteria, Candida albicans as fungi, Staphylococcus aureus and Streptococcus faecolis as Gram-positive bacteria. The three strains designated as sp. A, sp. B and sp. D exhibited good inhibition of the test organisms. Acetone, ethanol, ethyl acetate and methanol were used to prepare extracts of the three species and were used to re-assess antibacterial properties and also determine anticancer and antifungal properties. Antimicrobial properties were re-tested using disc-diffusion and puncture method while anticancer properties were studied by subjecting HCT-116 cancer cells to two different concentrations of extracts – 0.05%(v/v) and 0.5%(v/v). Acetone extracts showed some kind of inhibitory pattern hence a third concentration of 5%(v/v) was tested. Antifungal properties were examined by testing all extracts at 10%(v/v) against Aspergillus niger and Penicillium sp. Acetone extracts of all three species A,B and D displayed high inhibition of Aspergillus niger with 99.07% ±  0.12, 99.2% ±  0.01 and 99.19% ±  0.00 inhibition percentages respectively, and also inhibited growth of Penicillium sp. with 82.62% ±  1.62, 79.63% ±  0.11 and 87.44% ±  0.2 inhibition percentage respectively (% as compared to acetone control). These extracts were then re-tested at two other concentrations of 2.5%(v/v) and 5%(v/v). While the extracts at these concentrations were effective against Aspergillus niger, they could not inhibit growth of Penicillium sp.

References

Chater, K. F. (2013). Streptomyces. In S. M. Hughes (Ed.), Brenner's Encyclopedia of Genetics (Second Edition) (pp. 565–567). San Diego: Academic Press.

Sanchez, S., & Demain, A. L. (2011). 1.12 – Secondary Metabolites. In M. Moo-Young (Ed.), Comprehensive Biotechnology (Second Edition) (pp. 155–167). Burlington: Academic Press.

Suzuki, S., Horinouchi, T., & Furusawa, C. (2015). Suppression of antibiotic resistance acquisition by combined use of antibiotics. Journal of Bioscience and Bioengineering(0). doi: http://dx.doi.org/10.1016/j.jbiosc.2015.02.003

Background and Purpose: Great advances have occurred in the field of robotics in the past few years. The integration of robotics in our daily life became not only limited to manufacturing or industrial usage, but also in health care delivery, aerospace, humanitarian aids and others. Most of the existing robots systems rely on the programmer to set the rule it plays within the working environment or rely on a trainer to teach the system what should and need to be done and where they are ought to move. Other robotic systems might involve more intelligent systems to explore and handle tasks within their environment. Most of these systems are usually situated to work within well organized and planned environment. Having modifications on any of the parameters of the environment might produce unpredictable consequences. Depending on the complexity of the system and how intelligent it is, the consequences might be unfavorable in achieving the goals intended and reducing oneself-damages.

Species in nature represents rich source of innovative ideas and creative concepts that can be investigated by researchers. Nature has been inspiring scientists into developing new ways of looking at things, by observing the various living organisms' behaviors in their own habitats. Behavior-based roboticists are concerned with the development of robots based on observing and the studying of neuroscience, psychology and ethology of animals in nature. Humans, animals and plants physiology is yet another rich source of researching potential (Fig. 1). For example, reflexes in living organisms represent a means of survival in the outer environment and means of regulating internal body operations. If we could observe and try to mimic some of the reflex behaviors, we could end up with a machine (E.g. Robots) that has the ability to avoid dangerous situations and keep the outer structure intact.

Figure 1: The potential of reflex systems in intelligent robotics. Objective: Adopting an intelligent reflex system in the robot system similar to that found in humans, animals, and plants can have significant advantages on the overall behavior of the system. A reflex system can improve the risk avoiding capabilities in the unfavorable scenarios. Design: The approach toward reflex based robotic system involves the intensive investigation and review of the fundamental concepts found in the reflex systems of human, animals, and plants. Attention to details, such as the behavior of the organism when subjected to a certain stimulus and the latency it takes for the reflex arc to execute the right response, are among the most important things to consider when trying mimicking the behavior of a living organism. A deduced conceptual model should be based on the distinguishing components found in the reflex arc. An actual design based on this proposed model, will include the basic components that can be achieved by using electronic/mechanical components that are at the same time analogous in function to the ones found in the reflex arc. For example, to mimic the temperature sensing capabilities of a human hand, a simple one-point temperature sensor will not be sufficient to give a desirable realistic result. Instead, a sophisticated flexible array that is capable to sense the temperature at any point must be used. Another design consideration is the controlling method to be used. Will it be centralized or decentralized or a mix of both? Regardless of the answer the controlling mechanism involved should be independent of a central controller (i.e. the brain) and it must be localized to achieve the desirable fast response as that founded in the reflex arc. Conclusion: The reflex based robotic system will be unique and innovative for the applications intended. The system can be incorporated with pre-existing systems to add value especially in the field of medical robotics and more specifically in prosthetics. Artificial reflex systems will add great value, protective feature and life-like sensation for a smarter prosthetic artefacts. With the implementation of the reflex arc at the right latencies and order, the gap between artificial and the actual hand should get narrower.

Acknowledgment

This publication was made possible by the support of an NPRP grant from the Qatar National Research Fund (NPRP 7-673-2-251). The statements made herein are solely the responsibility of the authors.

Keywords

Intelligent Robotics, Reflex System, Prosthetics

Pain is an emotion and neuropathic pain symptoms are modulated by supraspinal structures such as the amygdala. If the central nucleus of the amygdala is often named as the “nociceptive amygdala”, little is known on the role of the basolateral amygdala. Here, we monitored the mechanical nociceptive thresholds of in a mouse model of neuropathic pain and infused modulators of the glutamate/GABAergic transmission in the BLA via chronically-implanted can nulas. First, we found that NMDA-type glutamate receptor antagonist (MK-801) exerted a potent anti-allodynic effect whereas a transient allodynia was induced after perfusion of bicuculline, a GABAA receptor antagonist. Potentiating GABAA receptor function using diazepam (DZP) or etifoxine (EFX, a nonbenzodiazepine anxiolytic) fully but transiently alleviated mechanical allodynia. Interestingly, the anti-allodynic effect of EFX disappeared in animals incapable of producing 3alpha-steroids. DZP had a similar effect but of shorter duration. As indicated by patch-clamp recordings of BLA neurons, these effects were mediated by a potentiation of GABAA R-mediated synaptic transmission. Together with a presynaptic elevation of miniature IPSC frequency, the duration and amplitude of GABAA mIPSCs was also increased (postsynaptic effect). The analgesic contribution of endogenous neurosteroid seemed to be exclusively postsynaptic. This study highlights the importance of BLA and of the local inhibitory/excitatory neuronal network activity while setting the mechanical nociceptive threshold. Furthermore, it appears that promoting inhibition in this specific nucleus could fully alleviate pain symptoms. Therefore BLA could be a novel interesting target to develop pharmacological or non pharmacological therapies.