Qatar Foundation Annual Research Conference Proceedings Volume 2016 Issue 1

Photosensitive molecules such as quinones in drugs may become activated by exposure to UV-A light (320–400 nm) of the solar spectrum and cause damages to biological materials such as amino acids, nucleic acids, lipids, etc.. 9,10-anthraquinone (AQ) and 1,4-naphthoquinone (NQ), based drugs are commonly used as antibacterial, antifungal, antiviral, antitumor and antimalarial. Synephrine (SY) is used in traditional Chinese medicine, mainly for anti-hypotensive, nasal and ophthalmic decongestant. Currently it has been used in treatment of digestive disorders, in emergency treatment of asthma, and more importantly used as anti-depressant. The study of photo induced interactions of the excited quinones and drug molecules having electron donating ability is expected to have some relevance in physical pharmacy. In this in-vitro study, our aim is to understand the photo induced reaction when the quinones AQ and NQ coexisted with SY in an organic medium and exposed to UVA light. The photo induced interaction between SY and above quinones in their triplet states has been studied using nanosecond laser flash photolysis in organic medium, ethanol. The triplet excited states of the above quinones, AQT and NQT were produced by excitation with 355 nm, 5 ns laser pulse, in the presence of SY and under both deoxygenated and oxygenated conditions. In this wavelength only the quinones can be excited to triplets but not the SY, leaving it always in its ground state. Transient absorbance of the products formed was monitored in the 300–700 nm wavelength range. Both quinones, in their triplet excited state have revealed similar reactivity towards the ground state SY. Kinetics probed at the characteristic wavelengths of the triplets and ion radicals show that the formation of the quinone radical anion and the decay of the corresponding triplet are synchronous. This confirms that the photo-induced excited triplets, AQT and NQT have been quenched by SY through electron transfer process forming AQ•–, SY•+ ion pair. The lifetime of the decay of the triplets of the quinones and the growth lifetime of the radical anion of the same quinones are about 3 μs under deoxygenated condition. The presence of oxygen in the medium influences the decay of the triplet and anion radicals of the AQ, but does not affect the decay of the triplet or anion of NQ. In the presence of oxygen, AQ•– radical anion interacts with oxygen and forms superoxide anion radicals, O2•–. The overall result from the current investigation is an evidence for the controversial role of SY in oxidative stress. The ability of SY to quench the photo excited quinones could give some insight to mitigate the drug induced photosensitivity and also could broaden the clinical usage of SY in other therapeutic areas.

Calcific aortic valve disease (CAVD) is the most common valvular disorder, affecting approximately 25% of the population aged over 65 years. The formation of calcific nodules on the aortic surface of the leaflets contributes to a progressive obstruction of the left ventricular outflow and leads ultimately to heart failure (Stewart et al., 1997). While CAVD has been described historically as a passive degenerative process, it has now emerged as a highly regulated pathology presumably triggered by a combination of conventional cardiovascular risk factors, mechanical and hemodynamic cues. As an important hemodynamic force, fluid shear stress (FSS) is the frictional force acting in the direction of blood flow on the leaflet endothelium. FSS is experienced by the ventricularis when blood flows past the leaflets during systole and on the fibrosa when blood pools into the sinuses during diastole. Previous studies showed that FSS affect the molecular mechanisms that eventually lead to formation of calcific nodules for CAVD. (Sun, Rajamannan, & Sucosky, 2013). A severely calcified aortic valve needs to be replaced with an artificial valve. Mechanical or prosthetic valves are durable but come with the lifelong anticoagulant treatment. An alternative approach is tissue engineered valves within which aortic valve cells can grow, enhancing the biocompatibility of the valve (Bezuidenhout, Williams, & Zilla, 2015). No matter the artificial valve is mechanical, prosthetic or tissue engineered, when placed into the patient, it should interact with blood flow with minimal disturbance in order for not causing additional complications. Therefore, these valves need to be tested experimentally for the hemodynamic performance. In this study, we have developed an experimental system to investigate hemodynamics for artificial aortic heart valves. The system is composed of Aptus pulsed duplicator system and GE Vivid-q ultrasonic medical imaging system. Aptus pulsed duplicator generates the flow environment for left ventricular outflow tract. Artificial aortic valves can be placed inside the system and these valves are exposed to natural blood flow environment (i.e. natural pressure, heart rate, ejection fraction). GE Vivid-q system enables us to visualize how valve leaflets open and close in each pulse via b-mode ultrasound imaging. M-mode enables us to measure valve orifice size at peak ejection. Doppler mode on the other hand enables us to measure flow velocities through valves, which then are used to estimate FSS levels. Pressure measurement probes inside the pulse duplicator gives simultaneous pressure readings which are then used to calculate pressure difference across the valve which is another parameter to define valve function. Aptus bioreactor platform is equipped with a holder system that enables testing virtually any material (including woven and knitted fabrics) as a scaffold for heart valve leaflet. The holder is build out of transparent PDMS to enhance optical visibility. The valve geometries that will be tested can be chosen at different wall thicknesses and other dimensions. For example, geometry of the sinuses and leaflets can be freely chosen/designed. This enables comparison of different prototypes for the optimized tissue engineered scaffolds. We will compare the performance of the scaffolds that are designed in our lab with commercially available prosthetic valves. In conclusion, we were able to develop an ultrasound based experimental flow system that will enable us to evaluate the tissue engineered heart valve scaffolds in natural heart environment.

References

Bezuidenhout, D., Williams, D. F., & Zilla, P. (2015). Polymeric heart valves for surgical implantation, catheter-based technologies and heart assist devices. Biomaterials, 36, 6–25. doi: http://dx.doi.org/10.1016/j.biomaterials.2014.09.013

Stewart, B. F., Siscovick, D., Lind, B. K., Gardin, J. M., Gottdiener, J. S., Smith, V. E., … Otto, C. M. (1997). Clinical Factors Associated With Calcific Aortic Valve Disease fn1. Journal of the American College of Cardiology, 29(3), 630–634. doi: http://dx.doi.org/10.1016/S0735-1097(96)00563-3

Sun, L., Rajamannan, N. M., & Sucosky, P. (2013). Defining the Role of Fluid Shear Stress in the Expression of Early Signaling Markers for Calcific Aortic Valve Disease. PloS one, 8(12), e84433. doi: 10.1371/journal.pone.0084433

Background

The X-linked inhibitor of apoptosis (XIAP) is a promising molecular target for the design of novel anticancer drugs aiming at overcoming apoptosis-resistance of cancer cells. Recent studies demonstrated that the BIR3 domain of XIAP where caspase-9 and Smac proteins bind is an attractive site for designing small-molecule inhibitors of XIAP. Embelin, identified primarily from the Embelica ribes plant, is one such compound shown to exhibit chemopreventive, anti-inflammatory, and apoptotic activities via inhibiting XIAP activity.

Material and Methods

Reagents

Embelin was purchased from Tocris (Cambridge, MA). TRAIL was purchased from Alexis Corporation (Lausen, Switzerland). Antibodies against Caspase 3, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, Propidium Iodide Staining Solution, Annexin V Binding Buffer, Mitochondrial Membrane Potential Detection (JC-1) Kit, Stain Buffer (FBS), Annexin V-FITC antibody, H2AX (pS139)-Alexa Fluor 647 antibody, Rabbit Anti- Active Caspase-3- BV605 antibody and PARP Cleaved Form-AF700 antibody were obtained from BD Biosciences (NJ, USA). CellROX Deep Red Reagent was obtained from Molecular Probes, Life Technologies (CA, USA). (3-(4,5-dimethyl thiazol-2yl)-2,5-diphenyl tetrazolium bromide) solution (MTT) powder, DAPI, and N-Acetyl-L-Cysteine were obtained from Sigma-Aldrich (MO, USA). Apoptotic DNA ladder Kit was procured from Thermo fisher Scientific (USA).

Methodology

We used following assays and methods for this study.

Cell culture

Leukemic cell lines K562 and U937 leukemic cells were cultured in RPMI 1640 medium supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin at 370C in humidified atmosphere containing 5% CO2. All experiments were conducted in 5% serum.

Cell viability

Experiments were performed following treatment with various doses of embelin with and without pre-treatment with NAC for 24 hours using MTT assay.

ROS Production

CellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The cell-permeable reagent is non-fluorescent or very weakly fluorescent while in a reduced state and upon oxidation exhibit strong fluorogenic signal. The signals from CellROX Deep Red Reagent are localized in the cytoplasm and measured by flow cytometry (Excitation 640 nm/Emission 665 nm). K562 cells were treated with embelin for indicated time periods and finally analyzed by flow cytometry.

Apoptosis

Apoptosis was measured using annexinV-FITC/PI staining and analyzed by flow cytometry. Cells were treated with embelin in the presence and absence of NAC for 24 hours. Following treatment, cells were harvested, washed with PBS and stained with annexin V-FITC/PI for 20 minutes at room temperature and apoptosis was measured by flow cytometry.

Western blot

Following treatment with embelin and NAC for 24 hours, cells were lysed and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies.

Results

The results from our study showed that Embelin causes a dose dependent inhibition of cell proliferation in K562 and U937 leukemic cells. Anti-proliferative activity of Embelin correlated with induction of apoptosis. In addition, Embelin treatment of K562 cells decreased the constitutive phosphorylation/activation of AKT followed by the upregulation of proapototic protein Bax. Embelin also induced loss of mitochondrial membrane potential, as determined by JC1 staining, with subsequent activation of caspase-3 and polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. Pretreatment of K562 cells with N-acetyl-L-cystein, a scavenger of reactive oxygen species (ROS) prevented Embelin mediated apoptotic effects. Embelin also suppressed K562 derived progenitor colony formation, suggesting its antileukemic effect. Finally our data also showed that co-treatment of subtoxic doses of Embelin and TRAIL potentiated anticancer activity in leukemic cells.

Conclusion

Altogether, these findings suggest that Embelin causes inhibition of cell proliferation and induction of apoptosis via generation of ROS in leukemic cells, which raises the possibility that Embelin alone or in combination of chemotherapeutic agents may have a future therapeutic role in leukemia and possibly other malignancies with up-regulated XIAP pathway.

Keywords

Apoptosis, CML, ROS

The overlap stage is one of the most time- and space-consuming steps in de novo genome assembly. The huge size of output of Next Generation Sequencing (NGS), represented by small segments of multiple copies of the original genome, creates a serious computational challenge. The target is to find overlaps between each pair of sequences (reads) in order to build an overlap-based graph which constitutes the input for the assembly stage in which longer sequences (contigs) are sent to output. Finding overlaps between each pair of the input reads can be performed by solving the all-pairs suffix prefix problem (APSP) for these sequences. Such a problem, if not addressed effectively by designing new algorithms, data structures, and parallel processing techniques, can create an obstacle to the target of making genome assembly possible even with limited resources. This work presents a survey for previously presented solutions for APSP. We demonstrate and classify these techniques. Showing recent results regarding the time and space consumption for these solutions, we draw a conclusion regarding the best direction to tackle this critical problem. All-pairs suffix prefix is a well-known computer science problem. For a group of sequences G =  {S1, S2, S3, …. Sk), finding all pairs suffix prefix is to find the longest suffix prefix match for each ordered pair in G. For instance, let G =  {AACCGT, TAAAC, ACCCTA}. The solution to all-pairs suffix prefix for G is: (1,2) = {T}, {1,3} = {}, (2,1) = {AAC}, (2,3) = {AC}, {3,1} =  {A} and (3,2} = {TA}.

The first optimal solution for APSP was introduced by Gusfield et al. (D. Gusfield, 1992). The algorithm was based on the generalized suffix tree and takes O(n+k2) time, where n is the total length of all k strings. The suffix tree data structure is one of the most important tools for string matching and its applications in bioinformatics. A suffix tree of a sequence S is an index structure in which each suffix of S is stored as a path from the root to a leaf. Obviously many suffixes will share partial path before they end in different leaves.

Despite the fact that suffix tree's performance in solving many genome analysis problems is considered optimal, suffix tree is expensive in term of space. A pointer-type suffix tree typically requires O(n log n) space, whereas the original text over an alphabet of size requires only O(n log) bits. In addition, suffix tree has a bad locality of memory reference, which causes a remarkable slowdown in cached processor architectures.

A practically faster and less space-consuming solution for APSP was presented by Ohlebusch and Gog in 2010 (E. Ohlebusch and S. Gog, 2010), using the enhanced suffix array (M. Abouelhoda, 2004). Suffix array has emerged as a substitute to suffix tree in order to avoid its high space consumption, and to achieve better locality. The suffix array SA of a string S is an array of integers including the positions of the lexicographically sorted suffixes of S; i.e., for any two integers 0 i’ < i’’ <  n, S[SA[i’]] is lexicographically less than S[SA(i’’)]. Several indices were introduced in the last decade as compressed versions of suffix trees and suffix arrays. Those indices were considered a miracle since they don't just offer indexed searching, but also extract any text substring from the original text. With these self-index data structures, the original text is unnecessary and can be discarded. The term self-index highlights the fact that text is not stored explicitly but it can be derived from the index. FM index and RLCSA are examples for compressed suffix array, while Sadakane tree is a good example for compressed suffix tree.

Simpson and Durbin (J.T. Simpson and R.Durbin, 2012) used the FM index (P. Ferragina, 2004) to solve APSP in an indirect way as follows. The index is constructed for all strings after concatenating them in one string. The index is then queried by the reads, one by one, to find prefix-suffix matches. The time complexity of this algorithm is not as optimal as the one of (D. Gusfield, 1992), because one examines more suffixes than the output size. (This limitation stems also from the fact that the FM index lacks structural information to run the algorithms of (D. Gusfield, 1992) or (E. Ohlebusch and S. Gog, 2010) on it.). However, its space consumption is much less than that of the previous algorithms. The resulting assembler was called SGA.

Rachid et al. (M. H. Rachid Q. M., 2014) presented two solutions to solve APSP using a Sadakane suffix tree. The Sadakane suffix tree is a self-index and fully functional in a similar way to the uncompressed version of suffix tree. It offers the typical suffix tree operations such as checking if a node is a leaf, moving to the next sibling, using a suffix link, or even performing lowest common ancestor queries which can be expensive in term of time with other compressed data structure such as FM. Rachid et al. (M. H. Rachid Q. M., 2014) took advantage of the different components of this tree to solve APSP. The authors also utilized these components in building two different parallel implementations of these solutions. Rachid et al. (M. Haj. Rachid, 2014) also presented a solution to APSP using RLCSA. RLCSA is a compressed suffix array, which is introduced in (J. Sirén, 2008), (J.Sirén, 2010), and (V. Mäkinen, 2009). Both solution achieved considerable space reduction, but with a big slowdown.

Readjoiner (G. Gonnella & S. kurtz, 2012) was recently proposed as an efficient genome assembler that, in the overlap stage, finds suffix-prefix matches with a minimal length ℓ by grouping all relevant suffixes in buckets. Each bucket is identified by a common prefix for all suffixes inside it. Then, after sorting suffixes inside each bucket, it finds suffix-prefix matches using the lcp-intervals concept which is introduced in (M.I. Abouelhoda, 1994). The overlap stage in Readjoiner achieved best time and space consumption over all earlier solutions. Rachid and Malluhi (M. H. Rachid Q. M., 2015) presented a solution, called SOF, for APSP using a compact prefix tree. A prefix tree is a tree in which every read is represented by one path from the root to a leaf. The internal nodes represent common prefixes between reads. SOF could not achieve better results than Readjoiner using a single thread; however it shows better scalability and better time and space consumption in a multithreading environment. Figures 1 and 2 show the time and space consumptions for 7 different solutions to APSP when running on randomly generated data (M. H. Rachid Q. M., 2015).

Figure 1. Time consumptions for 7 solutions running on randomly generated data

Figure 2. Space consumptions for 7 solutions running on randomly generated data

We conclude that using suffix tree/array-based solutions, with standard or compressed forms, is not the best direction to solve APSP, despite the fact that they may possess an optimal time complexity. It turns out that other techniques which are based on LCP interval can achieve practically superior results in term of time without the large space which a suffix tree/array requires.

Bibliography

D. Gusfield, G. L. (1992). An efficient algorithm for the all pairs suffix-prefix problem. Inf. Process. Lett, 41(4):181–185.

E. Ohlebusch and S. Gog. (2010). Efficient algorithms for the all-pairs suffix-prefix problem and the all-pairs substring-prefix problem. Inf. Process. Lett, 110(3):123–128.

G. Gonnella, & S. kurtz. (2012). Readjoiner: a fast and memory efficient string graph-based sequence assembler. BMC Bioinformatics, 13:82.

J. Sirén, N. V. (2008). Run-Length Compressed Indexes Are Superior for Highly Repetitive Sequence Collections. in Amihood Amir; Andrew Turpin & Alistair Moffat, pp. 164–175.

J. Sirén. (2010). Sampled Longest Common Prefix Array. CoRR abs.

J. T. Simpson and R. Durbin. (2012). Efficient de novo assembly of large genomes using compressed data structures. Genome research, 22(3):549–556.

M. Abouelhoda, S. K. (2004). Replacing suffix trees with enhanced suffix arrays. Journal of Discrete Algorithms, 53–86.

M. H. Rachid, Q. M. (2014). Using the sadakane compressed suffix tree to solve the all-pairs

suffix-prefix problem. BioMed research international.

M. H. Rachid, Q. M. (2015). A practical and scalable tool to find overlaps between sequences. BioMed Research International, 12.

M. Haj. Rachid, Q. M. (2014). A space-efficient solution to find the maximum overlap using a compressed suffix array. Biomedical Engineering (MECBME), 329–333.

M. I. Abouelhoda, S. K. (1994). Replacing Suffix Trees with Enhanced Suffix Arrays. Journal of Discrete Algorithms, 2, 53–86.

P. Ferragina, G. M. (2004). An alphabet-friendly fm-index. In SPIRE, 150–160.

V. Mäkinen, G. N. (2009). Storage and Retrieval of Individual Genomes. in Serafim Batzoglou, ed., ‘RECOMB’, Springer., pp. 121–137.

Background

Duncan-Hewitt and Austin (2005) argue that pharmacist education has shifted from apprenticeship into higher education. This shift created “a gap between education theory and education practice”. Therefore, the role education theory plays in educational practices should be examined.

Objectives

Analyze the PharmD programme at QU through the lens of a theory-informed CoPF comprising of six elements: enablers, challenges, curriculum, teaching strategies, assessment and outputs.

Methodology

Using a a case study methodology, the CoPF was used to examine the PharmD programme at QU. Data from Interviews and focus groups with programme stakeholders and programme documents were thematically analyzed using NVIVO software.

Results

The CoPF has proved useful in identifying, systematically, the elements of design needed for a fully integrated PharmD programme. Key CoPF elements existed in the PharmD at QU, whilst others were missing. For example, practitioners have not been involved in designing the programme, as anticipated in the Enablers, and they lack understanding of education theory, identified in the Challenges. This has led to the lack of collaboration in curriculum, teaching and assessment between faculty and preceptors. This has subsequently limited one of the outputs of CoP, which is the integration between theory, practice and research.

Discussion

Some elements were identified as significant to have in place for an effective PharmD programme which aligns with CoP. Specifically the collaboration between practitioners and faculty in the design phase, in education process comprising curriculum, teaching and assesment in order to reach the ultimate output of integration between theory, practice and research.

References

Braun, V. & Clarke, V., 2006. Using Thematic Analysis in Psychology Qualitative Research in Psychology, 3, 77–101. Bristol: University of the West of England.

Duncan-Hewitt, W & Austin, Z. 2005. Pharmacy Schools as Expert Communities of Practice? A Proposal to Radically Restructure Pharmacy Education to Optimize Learning. Am J Pharm Educ American Journal of Pharmaceutical Education, 69(3):54.

Lave, J. & Wenger, E. 2001. Legitimate peripheral participation in communities of practice. Supporting lifelong learning: Perspective on learning, 1:111–126.

Yin, R.K. 1994. Case study research: design and methods. Thousand Oaks: Sage Publications.

Background

Cystic fibrosis (CF) is a genetic disease affecting 70,000 individuals worldwide and results from mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR). In the lungs, the mucociliary clearance mechanism is impaired and the airways of CF patients are often colonized by bacteria, yeasts and filamentous fungi. Among clinically significant fungi, Candida spp. are the most common yeasts but their prevalence rates vary greatly according to the different studies. It is still controversial as to whether Candida spp. are transient or persistent colonizers of the respiratory tract of CF patients. Candida dubliniensis is pathogenic yeast of the genus Candida which is phenotypically closely related to C. albicans. It is emerging yeast in the respiratory tract of patients with CF.

Aims and objectives

To determine the frequency of C. dubliniensis recovered from lower respiratory samples of CF patients and compare between pediatric ( ≤  18 year) and adult (> 18 year) CF patients. The secondary objective was to evaluate whether CF patients have persistent C. dubliniensis isolated in their lower respiratory secretions.

Methods

A prospective study of 52 CF patients (38 pediatric and 14 adult CF patients) over a period of 14 months. Each CF patient had at least two lower respiratory secretions either an outpatient or in-patient setting with interval 3–5 months between specimens during the study period. Sputum samples, deep pharyngeal swabs (taken from patients who did not produce sputum), and bronchoalveolar lavage (BAL) samples were collected and immediately delivered to the Mycology Laboratory at Hamad Medical Corporation, Doha, Qatar. Patients were excluded from the study if they had only one respiratory sample. Respiratory secretions were cultured for Candida species and identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Clinical data including body mass index (BMI) and the FEV1% (the volume of air forcefully exhaled in one second and then converted to a percentage of normal) were collected. Descriptive statistics and unpaired t test were used to analyze the data using SPSS 21software.

Results

Candida isolates were obtained from 40 CF patients (76.9%). There were 56.2% (77/137) of respiratory specimens positive for Candida species. C dubliniensis was the most prevalent Candida sp. 65%(50/77) isolated from 29 CF patients more often from adults than children (91.3% vs 53.7%; respectively). Other Candida spp. isolated was C. albicans 27.2%(21/77), C. tropicalis 6.5%(5/77) and C. glabrata 1.3%(1/77). In CF patients, two or more Candida spp. were never isolated from the same respiratory specimen. During the study period, C. dubliniensis was isolated repeatedly in 11 (27.5%) CF patients and transient isolates were found in 13 (32.5%) patients. C. dubliniensis was recovered from the first respiratory specimen followed by other Candida spp. in subsequent samples in 5 (12.5%) patients. C. dubliniensis has no significant effect on BMI and the FEV1% during the study period in each persistent and intermittent C. dubliniensis group respectively (P>0.05). None of the CF patients received antifungal therapy. The prevalence of C. dubliniensis was higher in adults harboring Pseudomonas aeruginosa, while in pediatric patients C. dubliniensis coexisted with Staphylococcus aureus.

Conclusion

The present study reports the high and frequent occurrence of C. dubliniensis from the lower respiratory secretions of CF patients and has no effect on both BMI and FEV1%.

Introduction

This study was held on the in vitro tests for the bacteriophages and their efficiency comparing with the antibiotics susceptibility in destroying bacteria. Because the development of the resistance to chemotherapeutic agents is becoming an increasing problem. Antimicrobial phage therapy trials have demonstratedphage infection of the increasing incidence resistantbacteria against most or all known antibiotics especially (hospital-acquired) infections.

Rational

This had the potential to facilitate more rational thinking about the phages as antimicrobial therapy for the increasing incidence of bacteria resistant against most antibiotics.

Objectives

Isolating and Identifying the bacteriophage, examining the bacteriophage efficiency against bacteria and comparing them with the antibiotics susceptibility.

Methods

Bacteria isolation and identification: Escherichiacoli and Staphylococcus aureus were isolated from Soba Stabilization Station and subjected to test against bacteriophages isolated from the same location. Susceptibility of isolated bacteria: Toward antibiotics and bacteriophages was determining. One of the virus stocks was Chosen and dispended in the serial dilution the virus sample was mixed with a dense bacterial culture and melted with soft agar and then spread over the surface of a base agar plate and used to infectbacteria. The plaques produced were then counted according to the number adjusted for the dilution to investigate bacteriophage specificity toward thespecific bacteria. Protein profiles of the bacteria and their correspondings phages were done by Sodium dodocyl sulphate polyacrylamid gelelectrophoresis (SDS-PAGE). The Microsoft Excelprogram was used for the statistical analysis, and thebioinformatics programmes UN – SCAN – IT version5 and ImageJ 136b were used for the proteinmolecular mass weight analysis.

Results

Escherichia coli: E. coli showed sensitivity towards: Ciprofloxacin, Pefloxacin, Ofloxacine, Tetracycline, Amikacin, Gentamicin, Piperacillin and Ceftizoxime, the largest inhibition zone was shown with Ciprofloxacin as 29 mm diameter. E. coli wasresistant to Chloramphenicol, Cefotaxime, Co-Trimoxazole and Ampicillin. Staphylococcus aureus: he S. aureus was sensitiveto Lincomycin, Cloxacillin, Ciprofloxacin, Tetracycline, Ofloxacine, Ampicillin/ Sulbactam and Cephalexin and the largest inhibition zone was shown with Lincomycin as 42 mm diameter. S. aureus showed resistant towards: Roxythromycin, Gentamicin, Pefloxacin, Cefotaxime, and Co – Trimoxazole. In broth media the affection of the bacteriophage interactions with bacteria showed increasing of the bacteriophages and decreasing of bacteria due to culture clearance, where the readings of the turbidity for the first and second infection showed statistical significant of E. coli phages samples’ transmission due to place of samples collections; from the anaerobic and facultative ponds P>0.05, facultative and maturation P < 0.05 and anaerobic and maturation P>0.05. Whilst, the S. aureus phages samples’ transmission from the anaerobic and facultative P < 0.05, facultative and maturation P < 0.05 and anaerobic and maturation P>0.05. On solid media the affection of the bacteriophage was recognised by the phage plaque formation on bacterial cultures. The antibiotics susceptibility against the bacteria showed statistical significant P < 0.05 for E. coliand P < 0.05 for S. aureus. The phage proteins were separated by Sodium Dodecyl Poly Acrylamide Gel Electrophoresis technique, the protein profiles of E. coli bacteriophage showed three major bands with molecular weight mass of 47, 34 and 16 kilo Dalton and 34 and 20 kDa for S. aureus phage, the band of 35 kDa was the common shared peak between the phage and the bacterial host due to the bacteriophage lytic cycle. The efficiency of isolated phage against E. coli and S. aureus showed remarkable inhibition of growth of the bacteria at both solid and liquid media this might bedue to physio-chemical changes and difference inmotility of these two bacteria. The mechanical action of bacteriophage on selected bacterial species dependon their receptors that adsorb them to their hosts, there lation between the bacteria and their corresponding phages.

Conclusion

The study showed approximately similar results for the mechanical action of the bacteriophage on the selected bacteria species and the mode of action of antibiotics. It is preferably to manipulate bacterial infections by the Bacteriophage therapy in case of the antibiotic resistant bacteria.

The key challenge with Epidemics includes its ability to spread rapidly and impact several large communities with high fatality rates. These characteristics make it difficult and very expensive to manage epidemics once it has broken out. History is replete with several cases of epidemic out breaks spreading from the point of origin and being transmitted to other areas resulting in the deaths of several thousands and in some cases millions of the inhabitants. The distance of the spread in the early 19th century was limited due to the limitations in travel speed and distance but this did not reduce the death rates of these diseases. The small pox disease was recorded to have killed more than 20% of the population of Athens in Greece. The great plague of London was recorded to have started in China in 1334 but spread along the trade routes wiping out entire towns. Florence in Italy lost a third of its entire 90,000 residents in the first six months with Europe losing an estimated 25 Million people. In 1633, Massachusetts which hitherto had been free from small pox became infected as settlers from France, Great Britain and the Netherlands brought the communicable disease with them resulting in the death of several millions. Other infectious diseases which have resulted in epidemics resulting in the deaths of several thousands of people in other regions of the world include the HIV/AIDS, H1N1, flu pandemic, the Severe Acute Respiratory Syndrome among other epidemics, these epidemics are usually spread by virtue of the cross border migration of the disease hosts. The UAE is gradually becoming the global holiday resort of choice while the UAE and Qatar are becoming the major transit hubs to India and China both of which account for over one billion of the world's population. Both regions are also bounded by Saudi Arabia which plays host to the world when they come to perform the holy pilgrimages to Mecca. This makes Qatar a location with a high potential for epidemic out breaks and necessitates the development of a robust real-time health monitoring systems capable of tracking possible epidemic causing diseases before they spread to the general population. The most effective means of containing the spread of epidemics is by monitoring the populace and quarantining any suspected victims or patient with a view to treating the case in isolation and controlling/preventing the spread of the epidemic to the general population. The transmission model for epidemics is represented by the branching process which shows the patient zero is the primary source of the diseases and the rate of spread of the disease is determined among other factors by the contagion probability of the disease.The current health management system does not provide a means of automatically identifying a likely epidemic and informing the relevant agencies to ensure the disease is contained by quarantining the patient zero. This work presents the development of an integrated health management system deployed as an application rining on laptops and tablets to be used by Doctors during consultation with patients for monitoring diseases occurrence in real time. It monitors patients and tracks in real time, the different diagnosis, patient location and possible epidemics by tracking the symptoms reported by the patients as they are examined in the consulting rooms and the results of the differett tests and examinations ordered by the doctors. It provides a means of alerting relevant healthcare authorities and all the other hospitals as soon as a case is identified in any consulting room. It harvests key diagnosis and monitors the number of occurrence of the reported ailments, the dispersion mechanisms and the possibility of the disease resulting in an epidemic. This data is transmitted and collated at a central health management unit. Different thresholds are set for the different ailments and dispersion mechanisms and when the thresholds are exceeded, the appropriate response mechanisms are deployed to the affected areas. When any of the known epidemics are detected in any consulting room, a high alert is sent to all the doctors in that hospital and containment systems deployed immediately to the affected hospitals for quarantining the patient and taking the necessary steps to ensure that the disease doesn't spread to the community. In the event of an epidemic, the application will generate the required emails to the relevant government agencies and send an SMS to the responsible parties to ensure appropriate action is taken. Relevant agencies in different countries can deploy this system and share information between their airports, immigration agencies, health management agencies etc, whenever any of such epidemic causing diseases break out or is reported in any of their hospitals. This will enable the different governments set up and deploy the relevant response teams and tools to ensure that the disease does not cross into their country and in the event that it arrives at the airport, the patient is immediately quarantined and investigated and the necessary medical treatment administered.

Background & objective

In order to survive the effects of the stress conditions, such as hypoxia and glucose starvation (GS) that exist in a tumor microenvironment, the regulatory mechanisms that control metabolism in cancer cells undergo change so that sufficient energy sources are available for proliferation, migration and invasion - thus facilitating metastasis. Via what is referred to as The Warburg Effect cancer cells primarily derive energy by metabolizing glucose through enhanced glycolysis even in the presence of an ample amount of oxygen. Thus, in principle, the non-specific cytotoxicity of traditional cancer chemotherapeutic agents will be reduced via a therapeutic strategy that targets the altered metabolism that is unique to cancer cells. Therefore, we predict that by using drugs, targeting cancer cell metabolism in combination with therapeutic strategies, which cause energy stress in cancer cells will result in a synergistic effect and enhance the likelihood of selectively killing cancer cells. Metformin, the most frequently prescribed, anti-diabetic drug has been shown to exhibit anti-cancer activity in different types of cancer cells thus supporting epidemiologic data that is suggestive that type 2 diabetic patients treated with metformin seem to be less likely to develop various types of cancers. Since angiogenesis is a key function of endothelial cells and aberrant angiogenesis is key to survival and growth of a tumor, we studied the effect of metformin on glucose-starved (GS) cancer microvascular endothelial cells with special reference to the Akt/mTOR pathway that is known to be dysregulated in many forms of cancer.

Materials & methods

In the present study mouse mile sven 1 vascular endothelial growth factor endothelial cells (MS1-VEGF; CRL-2460,

from ATCC, USA, of micro-vascular endothelial origin) cells were subjected to GS for 48 h in the presence & absence

of metformin (2 mM). Metformin treated and non-treated normal glucose (11 mM) exposed cells were used as suitable controls. MS1-VEGF cells were produced by overexpressing the primate VEGF-121 in the MS1 endothelial cell line that was derived from mice pancreatic microvasculature and immortalized with temperature sensitive SV40 large T antigen (1). These cells generate well-differentiated angiosarcomas in nude mice model (1). Following the experimental protocol cell lysates were prepared, total protein estimation was carried out and thereafter western blot analysis was performed to assess the levels of Sirt1, pAkt (S473), acetylated-p53 (K379), pmTOR (S2448), pRaptor (S792), p4E-BP1 (T36/47), pS6 (S235/236), pS6 (S240/244) and cleaved caspase-3. The band densities of the western blot images obtained were then quantified using the basic Quantity One software (Biorad, Inc. CA, USA). Trypan blue exclusion assay was carried out for cell viability analysis while MTS assay was carried out to analyze the cell proliferation status. Propidium iodide staining followed by FACS analysis on a BD LSRFortessa system (BD Biosciences, CA, USA) was performed for cell cycle 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 using one-way analysis of variance (ANOVA) and post-hoc comparisons between groups were performed by Tukey's multiple comparison tests. ‘p’ values less than 0.05 were considered to be statistically significant.

Results

Glucose starvation for 48 h in MS1-VEGF cells reduced cell proliferation and showed a significant increase in the levels of pAkt (S473) and a significant decrease in the levels of acetylated-p53 when compared to normal glucose exposed cells. mTOR is known to be phosphorylated at S2448 by Akt. The GS induced increase in the levels of pAkt (S473) can be related to the observed significant increase in the levels of pmTOR (S2448). The increase in the levels of pmTOR (S2448) also caused increase in the levels of downstream pS6 (S235/236) and pS6 (S240/244) in the glucose starved cells when compared to normal glucose exposed cells. Treatment with metformin (2 mM) for 48 h in MS1-VEGF cells subjected to GS significantly reduced cell viability. This can be related to the significant decrease in the levels of Sirt1 and pAkt (S473), which in turn would contribute to the increase in the levels of acetylated-p53 and decrease in pmTOR (S2448) levels in the metformin treated glucose starved cells when compared to non-treated glucose starved cells. Inhibition of mTOR pathway was confirmed by the significant decrease in the levels of downstream p4E-BP1 (T36/47), pS6 (S235/236) and pS6 (240/244) in metformin (2 mM) treated glucose–starved MS1-VEGF cells when compared to non-treated cells subjected to GS. In addition, the levels of pRap (S792), a negative regulator of mTOR activation, significantly increased in metformin treated glucose starved cells when compared to non-treated glucose starved cells. Inhibition of the Akt/mTOR pathway provides an explanation for the decrease in cell viability as evident by the accumulation of cells in the G2/M phase of the cell cycle. In addition to G2/M arrest, a significant decrease in the G0/G1 phase was observed while the cells in the sub-G0/G1 phase increased indicating cell death in the metformin treated glucose starved cells when compared to the non-treated glucose starved cells. The significant increases in the levels of acetylated-p53 and cleaved caspase-3 indicate that the cells have entered the apoptosis pathway. Treatment with 2 mM metformin also reversed the glucose starvation induced pro-survival autophagic response as evidenced by a decrease in the levels of LC3A-II and LC3B-II and marked reduction in the formation of LC3B stained punctae, when compared to non-treated glucose starved cells. Knockdown of AMPK revealed that this effect of metformin on GS induced autophagy is independent of AMPK.

Conclusion

Our findings indicate that using metformin in combination with agents that modify cancer cell metabolism (such as glycolytic inhibitors) is a therapeutic strategy that will selectively promote cancer cell death.

This work was supported by the Qatar National Research Funds (QNRF): National Priorities Research Program (NPRP: 4-910-3-244, awarded to Dr. Chris R. Triggle), a Junior Scientist Research Experience Program (JSREP 03-016-3-009, awarded to Dr. Samson Mathews Samuel). We thank Ms. Aleksandra M. Liberska (Flow cytometry supervisor) and the Flow Cytometry Facility within the Microscopy Core at Weill Cornell Medicine-Qatar for contributing to these studies. The Core is supported by the “Biomedical Research Program at Weill Cornell Medicine-Qatar”, a program funded by Qatar Foundation.

Reference

[1] Arbiser, J. L., Larsson, H., Claesson-Welsh, L., Bai, X., LaMontagne, K., Weiss, S. W., Soker, S., Flynn, E., and Brown, L. F. (2000) Overexpression of VEGF 121 in immortalized endothelial cells causes conversion to slowly growing angiosarcoma and high level expression of the VEGF receptors VEGFR-1 and VEGFR-2 in vivo. Am J Pathol 156, 1469–1476.