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.

Introduction

Warfarin has been the cornerstone oral anticoagulant for more than 60 years. Direct oral anticoagulants (DOACs) have been introduced to the market since 2008. In Qatar, dabigatran was introduced in 2011 followed by rivaroxaban in 2014 and both DOACs are currently used along with warfarin for the treatment and prophylaxis of different thromboembolic diseases. Despite the perceived advantages of the DOACs and their proven efficacy and safety in randomized controlled studies, their use is not as well-established as it has been expected likely due to the lack of antidote and their high cost. Little is known about the prescription pattern of oral anticoagulants and the extent to which DOACs replaced warfarin in Qatar.

Aim

In this study, we aim to explore the trends in oral anticoagulant use in Qatar over the past 5 years and to what extent did DOACs replace warfarin. We also aimed to determine the appropriateness of DOACs use based on the dose and the indication.

Methods

From electronic medical records, we collected all anticoagulant prescriptions dispensed as in- or out-patient from 2011 to 2015 in all Hamad Medical Corporation (HMC) hospitals. Prescriptions were stratified by the year, the medication prescribed and the dose. For every calendar year, we calculated the number and percentage of patients using each one of the anticoagulants prescribed. We also compared the clinical and demographics characteristics of patients prescribed warfarin versus those prescribed DOACs. Descriptive and inferential statistics were performed using SPSS.

Results

The attached table shows the change over the years in the number of patients prescribed warfarin versus those receiving DOACs. Overall, the percentage of patients receiving DOACs increased gradually from approximately 0.5% in 2011 to 20% in 2015. About 40% of patients receiving DOACs were previous warfarin users. DOACs were appropriately used in more 80% of the patients. Year/ Drug Warfarin Number (%) Dabigatran Number (%) Rivaroxaban Number (%) 2011 2078 (99.47%) 11 (0.53%) 2012 2364 (97.52%) 60 (2.48%) 2013 2567 (90.04%) 285 (9.06%) 2014 2823 (83.3%) 320 (9.44%) 246 (7.26%) 2015 2065 (80.54%) 196 (7.64%) 303 (11.82%).

Conclusion

DOACs have been gradually replacing warfarin in Qatar in a trend that is similar to other countries as well. However, warfarin use remains essential in more than 75% of patients requiring oral anticoagulant. It is important to continue educating healthcare providers to ensure appropriate use of these novel agents.

Several lines of evidence implicated the pathophysiological role of the endoplasmic reticulum (ER) stress in obesity-induced insulin resistance and diabetes. Using a targeted transcriptomic profiling approach consisting of the Heat Shock Response RT2 Profiler PCR Array, we previously reported impaired expression of DNAJB3/Hsp-40 cochaperone in obese and diabetic human subjects that was restored by physical exercise. In addition to DNAJB3/Hsp-40, three other genes showing differential expression between lean and obese human subjects were identified and GRP78 is the subject of our current investigation. Using histochemistry, immunofluorescence and western blots, our data show that GRP78 protein is increased in the adipose tissue obese subjects and thus, confirming the initial transcriptomic approach. More interestingly, higher levels of circulating GRP78 protein were found in obese compared to lean subjects and they correlated negatively with VO2, Max but positively with CRP and the obesity indicators such as BMI, body fat, and waist circumference. As GRP78 is the master regulator of the Unfolded Protein Response (UPR), we investigated the status of three arms of the UPR namely ATF6, IRE1a and PERK. Consistent with the finding on GRP78, our data indicated a marked increase in the expression and activity of these three arms in the adipose tissue from obese subjects. We finally tested the hypothesis that physical exercise could modulate the expression and release of GRP78 and its downstream targets. Here, we provide for the first evidence that physical attenuated significantly the endogenous expression and release of GRP78 with a concomitant reduction in the activity of IRE1a and eIF2a. Taken together, these results strongly suggest that exercise alleviated ER stress in obese subjects through attenuation of GRP78 signaling network.

Background

Foreign Body Aspiration (FBA) is a serious common problem in children, which needs prompt diagnosis and management; delays can result in devastating consequences. Physicians often struggle with the all too important, yet elusive, decision of “To bronch, or not to bronch” patients who present with a suspected FBA. In most cases, the history is often vague, with only subtle, if any, physical and chest radiograph abnormalities. With this study, we aim to use our local experience in Qatar to retrospectively analyze broncho-scopically proven cases of FBA in an attempt to determine the key statistically significant clinical predictors of foreign body aspiration in children.

Objective

To develop a clinical algorithm with a scoring system for aiding physicians to accurately predict patients with FBA, needing bronchoscopy, based on the patient's historical, physical and radiological findings at presentation.

Methods

This is a retrospective observational study, including all patients, aged 0 to 14 years, who were admitted to the pediatric department of Hamad medical corporation, Qatar between January 2001 to January 2011 with a diagnosis of suspected FBA. All patients underwent bronchoscopy, either flexible or rigid or both. Detailed data regarding the history, presenting clinical signs and symptoms, physician documented physical exam assessment as well as radiological findings were collected and analyzed. The bronchoscopy records were reviewed for bronchoscopy details like type (Flexible vs rigid), size and instrumentation used, outcomes including presence, type and location of foreign body (FB) and pre and post procedure complications. The focus of the data analysis was to determine the predictive accuracy of the various variables in diagnosing FBA. For this, the sensitivity, specificity, positive and negative predictive values of these parameters were calculated, using bronchoscopy diagnosis of FBA as the point of reference. Univariate and multivariate logistic regression methods were used to determine their statistical predictive value.

Results

Based on the inclusion criteria, a total of 300 children were included in this study. Male, female ratio of 1.2:1. The mean age was 2.1 ± 1.7 years. 46.3% of the patients were Qatari nationals. These 300 patients, cumulatively underwent a total of 410 bronchoscopies, which included both flexible and rigid bronchoscopies (88 patients underwent 2 bronchoscopies, 17 had 3, 4 patients had 4 and one patient underwent 5 bronchoscopies). A FB was found in the airway of 91 of these 300 children (30.3%). In all of these cases, the FB was successfully removed, in 67 cases (∼75%) with a rigid bronchoscopy and in 23 patients (∼25%), using a flexible bronchoscopy. 1 patient required a thoracotomy. Post bronchoscopy complications were reported in only 2.6% cases, with no procedure related mortalities. The most common site of FB lodging was the right main stem bronchus (47.3%), followed by left main stem bronchus, bilateral main bronchi, carina and trachea. The rest were recovered from segmental smaller airways. Organic FB accounted for 62.6% of the FB removed, with peanuts being the most common type. A complete list of the clinical signs and symptoms based on parent's history, physical exam findings and radiological findings in both groups of children i.e. those with a recovery of FB during bronchoscopy (FBA positive) and those without a FBA (FBA negative), can be viewed in Table 1. Using multivariable logistic regression analysis controlling for all other potential predictors, we found that the risk factors with the strongest association with FBA are witnessed choking crisis (adjusted OR 2.1, 95% CI 1.03–4.3.8; P = 0.041), noisy breathing/stridor/dysphonia (adjusted OR =  2.7, 95% CI 1.22–6.17; P = 0.015), new onset, recurrent or persistent wheeze (adjusted OR 4.6, 95% CI 1.77–11.76; P = 0.002) and unilateral reduced air entry (adjusted OR 2.9, 95% CI 1.53–5.52; P = 0.001). No significant interactions were found between the various otherhistorical signs and symptoms, radiological and physical examination findings. Figure 1 shows the cumulative proportion of children with proven FBA according to the above 4 risk factors. Only 8% of the children without any of these risk factors had a proven FBA, the likelihood increased significantly with an increasing number of risk factors. When all 4 risk factors were present, the likelihood of FBA was a 100%. 242 of these 300 patients were also analyzed and grouped based on whether they had normal or abnormal physical or radiological findings on presentation. (The remainder of the patients did not have a documented exam or a chest radiograph on record review). The results were as follows: in children with both abnormal physical and radiological findings, 47.2% had a proven FBA. If only one was abnormal (i.e. either physical exam or chest x-ray), the likelihood of FBAreduced to 32–33.3%. In children who presented with a completely normal physical exam and chest radiograph, only 7.4% had a FB removed by bronchoscopy (Fig. 2). Based on the above results, we designed a clinical algorithm, with a scoring system (Fig. 3), to aid in determining those children who require bronchoscopy to rule out FBA, and those who can be discharged on close follow up, without any intervention.

Conclusion

Accurately diagnosing FBA in children who need quick intervention will always be challenging. A high index of suspicion is required, along with a comprehensive historical account, detailed physical exam and a chest radiograph. The ultimate decision for bronchoscopy is based on the physician's clinical judgment as there is no 100% diagnostic predictor of FBA, perhaps with the exception of a radio-opaque foreign body. Our proposed clinical algorithm hopes to empower physicians dealing with such cases to accurately predict patients with a high likelihood of FBA and initiate prompt management, as well as avoid unnecessary intervention in those who have a very low probability of FBA.

Chemical composition of fingerprints includes the presence of salts, ions, fatty acids, lipids, amino acids, nucleobases, nucleotides, and nucleic acids. Many methods used to detect latent fingerprints on porous surfaces such as paper exploit the reactivity of amino acids with a number of different reagents, including ninhydrin and lawsone (2-hydroxy-1,4-naphthoquinone; HNQ). HNQ, also known as hennotannic acid, is a natural yellow-orange dye present in the leaves of henna plant (Lawsonia inermis) as well as in the flower of water hyacinth (Eichhornia crassipes). It is used as natural dye to color hair and skin. HNQ has been shown to react with amino acids, accordingly used to detect fingerprints on paper. HNQ has also been shown to be a sensor for the detection of anions (changes its color from yellow to orange-red in the presence of anions such as OH-, CN-, etc.). The presence of nucleobases, nucleotides and/or nucleic acids that are also part of the chemical composition of fingerprints, has not been exploited to detect latent fingerprints. In this study we propose utilize the reactivity of HNQ, nucleobases and nucleotides to detect and enhance the detection of latent fingerprints. HNQ reacts with amines and form Schiff-bases that are expected to be strongly colored, whose colors can be further enhanced upon exposure to a base (NH3) or anions (OH- and/or CN). In this regard, we have synthesized a series of HNQ-amine derivatives using microwave green synthetic methods (reactants dissolved in methanol and the reaction carried out at 150°C for 5 min). The compounds formed in the above reactions are strongly colored as judged visually and by spectrophotometry. Under similar conditions, adenine and guanine bases or adenosine and guanosine nucleosides (with amine substituents) also react with aromatic aldehydes to form Schiff-bases. The aromatic aldehyde derivatives of these purine nucleobases or nucleosides are expected to be highly fluorescent. Again, we have synthesized a series of aromatic imine derivatives of adenine, guanine, adenosine and guanosine as above. As judged visually and by spectrophotometry, these compounds exhibit strong fluorescence. The spectroscopic characterization of the compounds described above is on-going. Further, we have used these compounds to detect and enhance detection of latent fingerprints. The latter involved (a) application of powdered HNQ, adenine, guanine, adenosine or guanosine on latent fingerprints or cyanoacrylate coated latent fingerprints, (b) powder deposition aromatic amines or aldehydes, (c) followed by microwave synthesis of Schiff-base derivatives directly on the surface of the fingerprints using a commercially available domestic microwave oven (medium power for 3–10 min). The resulting fingerprints developed as above with HNQ and aromatic amines are strongly colored. Further exposure of thus formed colored fingerprints to NH3 strongly enhanced their color. The fingerprints developed as above using purine nucleobases or nucleosides with aromatic aldehydes were strongly fluorescent. Through this study, we have successfully synthesized Schiff-base derivatives of HNQ and aromatic amines as well as purine nucleobases or nucleosides and aromatic aldehydes. Further, we also suggest a novel method for the detection of latent fingerprints and its successful application on nonporous surfaces using the newly synthesized compounds.

Introduction

Dissatisfaction with health care performance is an important source of information about health care reforms as perceived by the public as it is associated with negative beliefs about health system. Previous studies have shown that dissatisfaction with health care has a long-term negative impact on the health care users' relationship with healthcare providers, health related behaviors, and health outcomes. In addition, a recent study conducted in Qatar, showed that approximately 24% of the studied population who used health care in the past 12 months prior to the study were dissatisfied with health care services provided in the country. Given that dissatisfaction with care can negatively impact on help-seeking behaviors, this finding could have grave public health implications. This has been witnessed in the context of high prevalence of chronic health conditions in Qatar where long-term relations with healthcare professionals are necessary for better chronic disease management, reduced disease-related complications, and mortality. This study aims to identify the sources of dissatisfaction with medical care among adults, Qataris and white collar migrants aged eighteen years or older.

Methods

This study is based on secondary data from a larger national survey, which was conducted during the fall of 2012 for the purpose of collecting household-based information on health services utilization and health-related expenditures. Disproportionate stratified probability sampling was employed to select a representative sample of households. A final sample of 3,080 completed face-to-face interviews (1,528 Qataris and 1,552 White Collar Migrants) using computer assisted personal interviewing (CAPI) method for a raw response rate of 78.1%. The sample included individuals who may or may not have used Qatar's health care system during the 12 months prior to survey administration. Respondents were asked to discuss the reasons for their discontent with healthcare services in Qatar by selecting pre-coded categories of dissatisfaction including: Waiting time to see the provider, language used to communicate, clarity of how things are explained to the patient, poor services provided (such as cleanliness, reception, respect, and parking), inability to choose provider or doctor, high costs and other reasons to be specified by respondents. A total of 711 open-ended responses to the “Other” category were translated, coded and analyzed qualitatively. “Crowdedness”, “staff and physicians' incompetence”, “medical errors”, “discrimination”, “disrespect”, and “lack of staff and services” are all themes that emerged as reasons for dissatisfaction. Analysis Arabic responses were translated into English and researchers discussed any dissimilar results until an agreement was reached on all translated responses. Upon reviewing the responses, themes, which were different from the pre-specified answer choices of the questionnaire, emerged. The researchers then coded the responses by assigning codes to each response, then compared against each other. Coding discrepancy was discussed until an agreement was reached. The codes of the open-ended responses were later merged with those of the pre-specified categories and the corresponding frequency for each coding category was calculated using STATA. The Alberta Quality Matrix for Health was used to guide the analysis of the themes based on the six dimensions of health system quality.

Results

The analysis of the open-ended responses that probed into reasons for respondents' dissatisfaction revealed thirteen categories of dissatisfaction that were related to four different dimensions of quality of healthcare, based on the Alberta Quality Matrix for Health. The most common dimension of dissatisfaction with health care in Qatar was accessibility, which refers to the provision of health service in the most optimum setting and within “reasonable time and distance”. Safety was the second most common dimension reported by the respondents. This construct relates to minimizing any threats that could cause harm. Acceptability, such as the provision of respectful and patient-centered health services was the third dimension identified, followed by efficiency, which is mainly related to the optimal use of resources, to achieve the best desired health outcomes.

Conclusion

Identifying the roots of dissatisfaction with health care services among distinct social groups can be achieved by analyzing responses to simple open-ended questions in routinely administered population health surveys. This is important for monitoring the quality of care in heterogeneous population contexts as well as engaging the public in the process of developing a world-class health care system as per Qatar's national vision of 2030. This research highlights priority needs to be addressed by the Qatari government in order to increase health care satisfaction as part of the quest for better health care in the country.

The Developmental Origins of Health and Disease (DOHaD) concept describes how the environment impinges on intra-uterine development and early childhood and how it induces changes in the development that have long term impact on later health and disease risk. Environmental exposures including parental lifestyle and diet, obesity and chemical exposure have been shown to modulate disease risk. The effects do not simply disrupt development or induce disease themselves, but can affect how rapidly disease develops. Epigenetic changes are plausible molecular mechanisms that can explain disease development trajectories. We hypothesize that early life exposures can alter DNA methylation patterns, thereby predisposing the child to develop respiratory allergy (RA) later in life. We have used two longitudinal birth cohorts and biobanked samples in Belgium to discover and confirm DNA methylation signatures that can differentiate between RA cases and controls. Blood and saliva samples of 11-year old children from the discovery cohort were analyzed using Illumina Methylation 450K BeadChips (20 RA cases and 20 controls). Saliva is proposed as a DNA source that can be easily collected in a decentralized manner in children. In addition, we analyzed biobanked cord blood samples of the same children in order to detect if altered DNA methylation marks could be detected at birth, as a proxy for intra-uterine changes. The discovered methylation signatures were studied in blood and saliva samples in a second cohort of 5-year old children (N = 78). We installed an analytical pipeline for sample processing and data analysis. R-based software was used for data normalization and Comb-p tool was used to identify differentially methylated regions (DMR). The Illumina Methylation 450K BeadChips showed that the methylation status was comparable between blood and saliva, with ± 11% of the probes having a differential methylation pattern (Padj < 0.05 and |Δβ| >0.1). The results suggest that saliva is a suitable biofluid for genome-wide DNA methylation studies and that there is a substantial overlap with blood profiles. A comparison between RA cases and controls revealed 83 DMR in blood of the 11-year old children; CB 26 DMR in cord blood and 5 DMR in saliva. Two DMR were identified as being in common between blood, saliva and cord blood samples using a Venn diagram analysis. The DMR were located in genes involved in IL4 signalling, Th2-response and phagocytosis; pathways that are implicated in RA disease. These 2 DMR were confirmed by iPLEX MassArray analysis in the same birth cohort (N = 40) as well as in a second cohort (N = 78). This project provided novel insights in the molecular mechanisms that may predispose children to chronic disease development, such as RA. We have identified DNA methylation marks in saliva and blood of children that are relevant for RA. We have also observed these methylation marks in the children's biobanked cord blood taken many years before RA development. We are among the first to show the utility of saliva to study DNA methylation changes in children. Or results contribute to the DOHaD concept that has very important implication for many societies and for global health policy.

North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081, United States Abstract Chronic hyperglycemia is an important risk factor involved in the onset and progression of secondary complications of diabetes. Aldose reductase (AR) has been implicated in the etiology of diabetic eye diseases, diabetic cardiomyopathy and/or nephropathy. High glucose levels activate AR, which is one of the key rate limiting enzymes to use NADPH to reduce glucose to sorbitol in the polyol pathway. The depletion of NADPH is correlated to the production of GSH which increases intercellular oxidative stress. Since inhibition of AR plays an important therapeutic value in preventing and or alleviating diabetic complications. At present there are many AR inhibitors such as Sorbinil, Dilantin, Epalrestat, Tolrestat, Zopolvestat and Minalrestat. However, most of these compounds have serious side effects such as Steven-Johnson syndrome and hypersensitivity reaction. In recent years nutraceuticals have garnered interest for their potential as dietary and natural health promoting properties. Hence, the purpose of this study was to investigate the inhibitory activity of phloretin from apple, ( − )-epigallocatechin 3-gallate (EGCG) from green tea and [6]-gingerol from ginger against aldose reductase as indicator or their potential in the alleviation of diabetic complications. These three compounds were selected out of 9 bioactive compounds based on their activity in cell culture assays. Human retinal pigment epithelial (HRPE) cells were subjected to different concentrations of glucose (10–100 mM) for 1 and 4 days during which cell viability and aldose reductase activity were evaluated. Results show that cell viability decreased to 93 ±  3%, 83 ±  6%, 65 ±  4% and 39 ±  3% at 10, 25, 50 and 100 mM of glucose on day 1 with further drastic decrease in cell viability to 73 ±  5%, 61 ±  3%, 35 ±  2%, and 11 ±  6%, respectively, on day 4 compared to the untreated cell. The activity of aldose reductase was found to increase 5 folds at 10 mM from day 1 to day 4, whereas at 25, 50 and 100 mM concentrations of glucose the AR activity was increased to almost 2 folds. The specific activity of aldose reductase was found to be 8.92 ±  1.6 U/mg protein at 100 mM glucose on day 4. The apparent Michaelis constant (Km) of the substrate glyceraldehyde and NADPH was estimated to be 4.5 mM and 68.96 μM, respectively. Pre-treatment of cells with phloretin, EGCG and [6]-gingerol at 25 μM improved cell viability to 72 ±  4%, 77 ±  5% and 78 ±  4%, at day 1 respectively, whereas EGCG further improved cell viability on day 4 to 89 ±  5%, but phloretin and [6]-gingerol could marginally increase to 76 ±  3%, 81 ±  4% when compared to the untreated cells. The three compounds could inhibit AR activity up to 90 % at 12.5 μM of phloretin, EGCG and [6]-gingerol with IC50 values of 4.1 μM, 3.7 μM, and 2.4 μM, respectively. The enzyme inhibition kinetics showed non-competitive mode of inhibition for phloretin and EGCG, since it did not alter the Km but the maximum velocity (Vmax) decreased in the presence of the compounds, whereas [6]-gingerol indicated uncompetitive type of inhibition against AR, where it decreased both Km and Vmax upon binding. The above cell culture findings were further validated in mouse model. Male C57BL/6J mice 5 weeks old, were divided into eight groups of 11 mice each. The animals had free access to food and water and were fed with either a standard laboratory diet (8604 Teklad Rodent diet, Harlan, TM) or a high fat diet (HFD) (TD 110716, Teklad Research Rodent Diet, Harlan, TM). Animals were randomly assigned to the different treatment groups (N =  11 / group): (i) Normal diet, (ii) HFD, (iii) HFD + EGCG 25 mg/kg, (iv) HFD + EGCG at 75 mg/kg, (v) HFD + phloretin 25 mg/kg, (vi) HFD + phloretin 75 mg/kg, (vii) HFD + [6]-gingerol 25 mg/kg, (viii) HFD + [6]-gingerol 75 mg/kg. All test solutions were prepared freshly every day prior to use and were administered intraperitoneal injection (i. p.), once daily and three times in a week over a period of sixteen weeks. Body weight was recorded every day, while blood glucose levels were determined weekly with a commercially available micro-draw blood monitoring system. After sixteen weeks, the animals were sacrificed; heart, eyes and kidney were examined for AR activity. The results shows that the HFD group had developed diabetes, with blood glucose levels 260 ±  27 mg/dL, whereas the control with normal diet showed about 130 ±  20 mg/dL. The groups treated with EGCG, phloretin and [6]-gingerol at 75 mg/kg significantly decreased blood sugar levels to 128 ±  8, 125 ±  15 and 132 ±  9 mg/dL, respectively. The groups treated with EGCG, phloretin and [6]-gingerol at 25 mg/kg also decreased the blood sugar levels to 198 ±  15, 180 ±  16 and 170 ±  19 mg/dL, respectively. The eye lens of the mice from HFD group had developed cataract and the AR activity increased to 4 fold compared to the group fed with normal diet. All the compounds at 75 mg/kg, prevented/delayed the formation of cataract by 80%, whereas dosing at 25 mg/kg showed signs of cataract but AR activity was decreased to two folds when compared to the HFD group. The HFD enhanced the aldose reductase activity to two and three folds in the heart and kidney. Data from this study suggest the promising potential of these dietary compounds in providing cytoprotection and inhibiting a key enzyme associated with the onset of diabetes-related complications. Upon validation of this benefit in vivo, such dietary compounds could be of interest to dietary supplements and pharmaceutical industry as complementary treatment of secondary complications in diabetic patients.

Fine needle aspiration (FNA) biopsy is an established procedure by which to sample thyroid nodules to ascertain etiology and produce a diagnosis conveying risk of malignancy with recommended patient follow-up. This procedure is well-tolerated and endorsed given the accessibility and vascularity of the thyroid gland. FNA cytopathology has proven efficacious for the primary assessment of thyroid nodules. Well-differentiated papillary thyroid carcinoma (PTC) and benign lymphocytic (Hashimoto) thyroiditis (HT) are distinct thyroid lesions that may be reported with diagnostic confidence based on their characteristic cytomorphologic features. However depending on the adequacy of FNA sampling and the morphology of aspirated cellular material, thyroid nodules with coexisting PTC and HT may pose diagnostic pitfalls. This may be dependent upon: (a) the architectural nature of the coexisting lesions in-vivo; (b) whether both lesions are adequately sampled through FNA; and (c) which of the cell types and cytomorphologic features appear in predominance to support interpretation. Such cases may prove diagnostically challenging in adult patient assessments particularly in the setting of hypothyroidism which may also include multi-focal hyperplastic nodularity. Likewise, diagnostic problems may arise in the pediatric setting as thyroid nodules are relatively rare; marked lymphocytic pleomorphism and anisonucleosis may raise concern of lymphoproliferative malignancy; and as intra-thyroid lymphadenopathy can occur. This two-lesion entity is therefore impactful in both the adult and pediatric thyroid FNA practice. This report aims to raise awareness of this entity through a case of thyroid FNA in a 27 year old female, with ultrasonographic, cytopathologic and histopathologic findings. We conducted an English language literature review to explore the experience pertaining to the coexistence of PTC and HT as identified through FNA of the thyroid, and reviewed clinical, prognostic and ancillary testing data that may facilitate diagnosis. Incidentally, as the Sidra Medical and Research Center in Doha, Qatar, is a comprehensive woman's and pediatric hospital, we also investigated the published pediatric experience in correlation with this case. Thyroid FNA Case/

Materials and Methods

The cytopathology service (at King Abdulaziz Medical City Hospital, Riyadh, Saudi Arabia) assisted in an ultrasound-guided thyroid FNA procedure on a 27-year old female presenting with isthmus and bilateral lobe nodules to rule out malignancy. She had no remarkable medical history or previous thyroid disease. Image-guided FNA was performed using 22-gauge stylet needles without syringes attached. Under ultrasound guidance, the needle was advanced as close as possible to the nodule (with the stylet engaged) then the stylet was removed and the needle advanced then into nodule. The passes were performed in different directions inside the nodule. Two needle passes were performed on the right thyroid and isthmus nodules, but three passes were performed on the left thyroid nodule. Aspirated material from each pass was promptly and carefully smeared between two glass slides whereby, with mirror-image smearing, one slide was fixed in 95% ethanol for Papanicolaou staining, and the second slide remained air-dried for Diff-Quik staining. All needles were rinsed with saline, but no cell blocks were prepared due to insufficient needle rinse material. Ultrasonographic Findings: No nuclear medicine studies were performed prior to the thyroid FNA procedure for this patient. Thyroid, isthmus (Fig. 1): Transverse gray-scale ultrasound showed a predominantly solid, well-defined, non-calcified nodule with heterogeneous echo-texture, measuring 1.2 ×  1.8 cm in diameter. Thyroid, right lobe (Fig. 2): Transverse gray-scale ultrasound showed a predominantly solid nodule with ill-defined margins located in the lateral aspect of the mid right thyroid lobe, with heterogeneous echo-texture, measuring 1.0 ×  0.45 cm in diameter. Thyroid, left lobe (Fig. 3): Transverse color Doppler ultrasound showed an ill-defined, non-calcified hypo-echoic avascular nodule measuring 0.6 ×  0.6 cm in diameter. Fine Needle Aspiration Microscopic and Diagnostic Findings: All smears revealed an adequate cellularity of follicular cells. Upon low power microscopic analysis, the smears from the left and right nodules revealed scattered small clusters of benign follicular cells and isolated Hurthle cells in backgrounds of predominating polymorphous lymphocytes. However, the smears from the isthmus nodule included scattered variably-sized clusters/groups of disorganized abnormal follicular cells with pseudo-papillary architecture, marked cellular crowding and depth of focus, against a background of predominating lymphoid cells of various levels of maturity including immature and mature forms, scattered plasma cells, scattered robust Hurthle cells with abundant fragmented eosinophilic cytoplasm, erythrocytes, and proteinaceous/cellular debris with clotting artifact (Figures 4, 5 and 6). With high power microscopy, these follicular cells showed abnormal nuclear features with moderate anisonucleosis: round/oval shapes, irregular envelope contours, evenly-dispersed coarse chromatin clumping, and moderate hyperchromasia, but without marked nuclear envelope wrinkling. Intranuclear invaginations and grooves were rare (Fig. 7). Follicular cell cytoplasm was moderately-abundant and micro-vacuolated, with a blue-grey staining reaction through Diff-Quik staining and basophilic with Papanicolaou staining (Figures 6, 7 and 8). Fragments of connective tissue were also noted intermixed amongst the abnormal follicular cells, as were rare, intact, micro-follicles (Fig. 8 and 9). Deposits of thick colloid were scattered with thin colloid being scarce (Fig. 10). Based on these microscopic findings, cytopathology interpreted the left and right thyroid nodules as being consistent with lymphocytic (Hashimoto) thyroiditis, Bethesda Reporting Category II. The isthmus nodule was interpreted as being suspicious for papillary thyroid carcinoma associated with lymphocytic (Hashimoto) thyroiditis, Bethesda Reporting Category V. Lesion coexistence was suspected. The ensuing recommendation referred to the algorithm as published through the Bethesda reporting system, being that of thyroidectomy. Histopathologic Findings: Total thyroidectomy was performed. Tissue sections from the thyroid isthmus nodule revealed PTC, while the non-neoplastic thyroid tissues showed HT with multi-focal hyperplasia (particularly in the right lobe). Neither definite extrathyroidal tumor extension nor lymphovascular invasion was noted; but deposits of metastatic carcinoma were identified in 5 of 14 dissected lymph nodes. Surgical biopsy confirmed the coexistence of PTC and HT in the isthmus nodule; demonstrating two distinct, characteristic lesions resting adjacently separated by bands of connective tissue (Figs. 11, 12 and 13). Notation: Post-surgery nuclear I131 scanning showed complete ablation of the thyroid tumor.

Conclusions

The HT cytomorphologic template includes polymorphous lymphocytes, plasma cells, robust Hurthle cells, and follicular cells that may occasional have intranuclear invaginations and grooves. In this case, FNA cytopathology conveyed diagnoses of lymphocytic (Hashimoto) thyroiditis for the left, right and isthmus nodules sampled based on the characteristic cytomorphology noted in the smears. The isthmus nodule diagnosis also emphasized suspected coexistence of well-differentiated PTC mainly based on the scattered groups of atypical follicular cells with abnormal nuclear shapes and sizes, chromatin features, and pseudo-papillary 3D architecture with depth of focus. We favored a conservative diagnosis for PTC bearing in mind the following considerations: the possible inflammatory effects of florid HT upon epithelial cells; the possibility of diffuse nodular follicular hyperplasia; and the rarity of intranuclear invaginations and grooves observed. The overall cytomorphologic findings in the smears from the isthmus nodule closely resembled the histologic patterns of well-defined PTC and HT separated by connective tissue. Multi-focal follicular hyperplasia was also identified in the left, right and isthmus thyroid nodules in this case. The lesions were coexisting but physically isolated, and the ultrasonographic detection of heterogeneous echo-texture may reflect this phenomenon. This case emphasizes the importance of adequate FNA technique to ensure the various aspects of thyroid nodules are sampled and proportionally represented through FNA processing and, particularly, for nodules with heterogeneity upon imaging. Also, screening, interpretive and cytopreparatory expertise with optimal fixation and staining are equally critical. HT is also termed chronic lymphocytic or autoimmune thyroiditis, first described by Hakaru Hashimoto. HT is believed to result from a combination of genetic susceptibilities and environmental factors. It is estimated that HT occurs 10–15 times more frequently in women compared to men, with a global incidence of approximately 0.3–1.5 cases per 1,000 individuals. HT is associated with circulating antibodies to thyroid antigens and hypothyroidism due to follicular cell depletion secondary to the effects of chronic inflammation. The possible association between HT and well-differentiated PTC has been described since 1955. Women with HT and any solitary ‘cold’ thyroid nodule upon radio-nuclear scanning should receive ultrasound-guided FNA due to the increased risk of coexisting thyroid cancer. Meta-analyses demonstrate a near three fold risk for patients with HT to also harbor PTC relative to patients without HT. Epidemiologic studies suggest that the detection of PTC in patients with histologically confirmed HT may reach 39%, and that in the presence of HT, the outcome and prognosis of PTC is more favorable relative to patients with PTC only. These data have been used to suggest a ‘protective’ phenomenon resulting from autoimmune targeting of follicular epithelial cells. However the increased risk of tumor suggests that the proliferative changes of the thyroid epithelium are a risk for tumor development, although this may have a better prognosis, again possibly due to local growth factors in the regenerating thyroid. This also suggests HT is a promotor of tumors rather than being protective, though the tumors that develop are less aggressive. An alternative hypothesis is that the developing tumor promotes or initiates an inflammatory response. Tumor infiltrating lymphocytes and immunity are very topical with numerous recent reviews in a wide range of tumors, but in this case it is curious that the tumor showed considerably fewer lymphocytes than the background thyroid as if the tumor expressed fewer immune targets to the lymphocytes and showing some immune avoidance. The role of the immune system and thyroid cancer is complex. The prevalence of coexisting HT and PTC varies globally and this is thought to reflect ethnic, geographic and cytopathologic reporting differences. However, the sensitivity of FNA to detect PTC in patients with HT exceeds 90%. FNA of thyroid in the pediatric population may prove challenging depending on the nature of the lesions sampled, the likelihood of excessive hemorrhage and the possible need for patient sedation. Thyroid nodules in childhood and adolescence are relatively rare: prevalence varies between 0.2–1.44%, and are 5–10 times less common than in adults. However, the mean malignancy risk in pediatric patients with thyroid nodules exceeds 26% with over 90% of cancers being the PTC type. Therefore any thyroid nodule discovered in this age group ought to be investigated with a high degree of suspicion and an aggressive FNA approach. The prevalence of HT in childhood is estimated to be 1.3–9.6%; whereas the prevalence of HT with PTC ranges between 1–30% reflecting geographic and ethnic diversity. As also noted for the adult population, coexisting PTC with HT in childhood is associated with favorable prognoses arguably due to the ‘protective’ immune phenomenon controlling the proliferation of malignant cells. Patient management meta-analytical studies demonstrate favorable outcomes and prognoses in patients with coexisting PTC and HT. For such patients at presentation, the typical clinical parameters include: a female gender prevalence, multifocal nodularity, no extrathyroidal extension, no lymph node metastases and longer recurrence-free survival rates. Nevertheless, the biological coexistence of HT and PTC remains controversial. Some researchers have suggested that HT induces PTC, but conversely, that lymphocytic infiltration in HT may result from an autoimmune reaction against tumor-specific antigens from pre-existing PTC. Recent work reports a higher prevalence of activating point mutations in BRAF V600E in cases of PTC with an associated poor prognosis. Relevant studies on Korean patients demonstrate the presence of BRAF V600E mutation to be linked with a higher likelihood of lymph node metastases and a lower frequency of HT. Therefore, these findings support the hypothesis that HT may pose a ‘protective’ immune response in patients with concomitant PTC.

Autism Spectrum Disorder (ASD) is a lifelong neurodevelopmental disorder characterized by deficits in social communication and interaction, repetitive and restrictive behavior, extensive clinical and etiologic heterogeneity. ASD underlying genetic basis, range from effects of single genes to that of multiple genes and chromosomal regions, hallmarking the multifactorial and complex etiology. While a genetic etiology is identifiable in about one quarter of the cases, in the remaining three quarters it remains elusive. A family with two males affected by ASD was studied to identify the genetic basis of ASD in the family. Two siblings, a brother and his sister, each had a male child with ASD through marriage to their respective unrelated spouses. The first family [F1] has one affected male child; the second [F2] has one affected male and one unaffected female children. The father of F1 and the mother of F2 are brother and sister. No aneuploidy or deletion/duplication defects were detected by Whole-Genome SNP CNV analyses. WGNGS for all members, comparative genome analyses and data mining are as follows: [Only exonic variants considered, prioritized according to increasing population frequency (0–1%), damaging effects according to mutation prediction models and will be presented in table format]. 1. The two ASD events are unrelated and due to individual de-novo variants. Trio analyses for identified 36 de novo variants in F1 and 32 in F2. Variants in KCNH1, ANKRD36C, FRG2C, LOC12989375 were in common 2. The two ASD events are due to heterozygous variants inherited from the related parents. In F1, 370 father-to-son heterozygous variants, in F2, 398 mother-to-son heterozygous variants identified with 132 in common. 3. The related parents are protected from the damaging effects of ASD predisposing variants transmitted to their respective children by protective variants that were not transmitted, hence ASD in their offspring. Analyses were for heterozygous variants shared among the related parents but not transmitted to their affected children. 4. The non-related parents contributed rare predisposing variants, which in synergy with the inherited variants from the related parents exceed a disease onset threshold in the affected offspring. For F1 mother-to-son heterozygous variant transmission; For F2 father-to-son heterozygous variant transmission examined. Complex comparative genome analyses are required to decipher the genetic basis of ASD in families with multiple affected individuals. WGNGS, analyzed for the exome data, is a powerful tool for studying the genetic causes of ASD producing superior results than exome data in which other enrichment technologies are used. WGNGS, even when analyzed only for the exome data, provides clues to novel ASD risk genes, that can be pursued clinically, bioinformatically and functionally.

To answer questions from parents of children autism spectrum more than 300 children from various Arab countries as well as Arab expatriates residing in the foreign and Arab countries. We found that 90% of children have been exposed to neglect inadvertently leaving them in front of children's channels in the first year for a long time or screens devices in the first year or second. The most of them left by parents to watch the channels that repeat content and songs. We also found more cases that led to leaving the child of the TV is:

1. Working mother

2. Expatriate mother or traveling

3. Pregnant another child early

4. Busy father

5. Ignorance of the seriousness of the TV.

6. Addiction mother on social networking sites such as Face book.

We also found the answer from the mothers of the children and some of the videos that the child's behavior after the birth and to the pre-watch TV normally be in terms of sounds, movement and visual communication, hearing, especially in the first 6 months.

Symptoms of TV autism spectrum

- Lack of eye contact - Lack of attention to the appeal - Link TV or screens - Age-stop linguistic and stop mental age where he does not fit the true age - Non-participation of children playing - Trouble sleeping and eating Repetitive movements - problems in some senses such as smell - touch - taste - and the sensitivity of sound.

We have reached the symptoms depend on several factors and varies from child, including:

1- In any month of the child's age began to watch TV for a long time in the first year it led to a mental age and freeze them know mental age of a child.

2- How much the period that lasted for Child Show TV screens or devices and the way parents interact with the child and that will affect the senses and by how the brain works and the impact of television addiction children how we came to cause these symptoms TV and how it works to freeze the mind and the senses.

The idea of therapy

By how the brain works for the Very young child's mind when they are born to be his mind Loader with operational program acquired genes. This operational program provides for the is

1- To pay attention to what is repeated.

2- Pay attention to visual and auditory stimuli.

3- The basis of learning is repetition and stimulate the senses Comes from the interaction, in order for this program works must interact with the child to stimulate the child's mind and its cells nerve The most important year of the child is the first year then the second and child neglect in the first two years whether to let him watch TV or maid or not to interact with it leads to stop some senses and stop the mental and linguistic age of the child.

The child is born and all the senses memory empty and the sense of each storage and retrieval program it must be filled to the age of 18 months and the formation of memory for each sense. Repetition is the basis of learning, the basis for the work of the senses is the interaction what happens when you watch the child to repeat content channels and songs and cartoons

How the impact of the TV screens in the senses and the mind happens

1- The sense of sight To operate the sense of sight must be watched repeated things and stores it in memory of sight colors, sizes and shape as well as the normal speed stored in memory in order to be considered and very young children learn best by relating to real live people. In the case that the child was TV it stores the two-dimensional images without the size without touching without smell. What happens in a child's memory - will not be storage size and shape and the smell will not prolong the consideration of the fact things Because he used to watch Photos shown great speed and two-dimensional resulting in lack of eye contact of the child Things really are not two-dimensional

2- Example hearing Natural that a child hears different intensity, size and level far and close sounds and different directions And heard his name many times and pay attention to the call and stores voice his name and repetition soon be named in memory of hearing When you call it retrieves the sound of the voice memory and quickly analyze the mind and pay attention to the call quickly In the case of watching TV heard voices and one level - from one direction - will store specific sounds like what you watch from songs and animation films - Due to the channels repeating the content of the songs and songs will be stored these sounds in the audio memory and where the child spends a long time without having heard the sound of his name or Call it will become the voice of his name store down memory because the foundation is repetition will lose the sound of his name and therefore will not respond to the call of his parents, although he pay attention to the voices of the TV - also cause sensitivity of the hearing because the ear will be used on a one-level sounds also happen speech problems Because child labor channels to repeat songs, therefore the child tends to happen almost addictive frequencies songs to find that the child does not respond to the call of his father, but as soon as the employment of children are attracted to channel quickly even if the child was in another room

3- The lack of movement and sitting in front of the TV for a long time Problem will happen in the system estimate distances, balance and touch

4- Eating natural person is an important topic when watching TV in a mind will not eat cares tastings - this is what happens to the child while watching TV will be attentive to the TV will weaken the sense of taste and also in the future and also will not be digested without eating has swallowed digest TV addiction will lead to feelings and not to freeze the growth of social relations and do not care even in the absence of the mother in some cases.

5- Speech and becomes a child receives only laziness happens to think and speak weaken the speech where language acquisition results from the interaction with others and not to watch TV and weaken the sense of touch, smell, at least in this moment of interaction depends mental age As the American Academy of Pediatrics recommended preventing children less than two years of watching TV and that our recommended weaning in two years and we recommend that leaving the child less than two years to television is crime.

We demand from channels that repeat content and songs put a warning on the channel to prevent viewing of less than two years for the prevention of the spread of autism spectrum Recommendation prevent children less than two years to watch TV once, especially in the first year of interaction with the child as much as possible with sound and movement and smile and touch. There are some people messages, most of which prove that the child is exposed to neglect is intentional to leave him in front of children's channels, especially with a nanny does not speak baby language was the mother is busy with work or pregnancy in the most important months needed for the child to interact and evolve and also busy mothers to the Internet and social networking sites and it is clear that children channels a major reason for the increased prevalence of autism spectrum leaving the child who is less than two years, is to watch TV screens is a crime.

Can restart the mind and the senses again by reverse engineering and without medication starts to work the mind and the senses notice of appeal identifying himself and prolong the matter and get ready to learn and make up for lost and begin the appearance of improvement in the first week.

Channels affecting children and that repeated content

Channels of the most influential in the children come to the fore Channel Toyyour aljannah TV by 70% and it shares Space Toon – BRAAEAM TV - Tom and Jerry.

Message from one of the mothers of children born harmony boy and girl Posted says that the child has a lack of eyes contact - do not pay attention to the appeal - does not realize what around him - not following orders - the child likes to eat only cake and yogurt An inquiry was made of them Does the child and the girl child and how much TV they been seen an hour and why his sister was not affected The answer is as follows: From the age of 6 months, the child watches TV more than 7 hours per day Is his sister was watching TV a period equal to the period of her brother or less? The answer is that the mother of his twin sister was watching TV a period of not less concerned with road a lot - and always come out with me and leave the other child sits at home with his grandmother.

Background

Endothelial cells line blood vessels and the heart where they release cardio-protective hormones that prevent thrombosis. Available replacements to treat heart valve diseases are limited by the lack of the endothelial cell layer, making them susceptible to calcification and thrombosis, which limits utility and increases the need for multiple replacements[1]. A suggested solution is to adapt tissue engineering techniques to formulate intelligent instructive scaffolds decorated with specific molecules to enhance the adhesion and population of circulating progenitor endothelial cells from the blood.

Aim

In this study, we aim to modulate nanofibrous scaffolds fabricated from the biodegradable polyster polycaprolactone (PCL) to provide a viable environment for endothelial cells from blood progenitors (blood outgrowth endothelial cells; BOECs) to adhere, proliferate and function successfully. To date we have (i) compared the behaviour of BOECs to endothelial cells isolated from human heart valves (hVECs) under shear conditions, (ii) tested the compatibility of PCL with BOECs by assessing cell viability and inflammatory responses on modified PCL films, and their ability to populate structured PCL scaffolds.

Methods

BOECs were isolated from the blood of healthy volunteers by selective plating of peripheral blood mononuclear cells (PBMCs), and phenotyping was assured by measuring the expression of endothelial cell markers using fluorescent activated cell sorting (FACS). hVECs were isolated from human aortic valves by collagenase digestion. Shear stress was studied using a cone and plate model. PCL films were prepared by solvent evaporation method, and sterilized with ethanol for 30 min, or modified by plasma oxidation at 30 w and 0.1 m bar for 30 min. PCL Films were coated with extracellular matrix proteins before cell seeding. After 72 hr of culture, cell viability was measured using alamar blue and cell secretory function measured by the release of CXCL8, endothelin-1 (ET-1) and prostacyclin by ELISA. Finally, the ability of these cells to populate 3D structured nanofibrous PCL scaffolds fabricated by jet spraying technique2 was determined by confocal microscopy of phalloidin stained cells.

Results

BOECs colonies appeared at between 7 and 21 days of culture. FACS analysis of expanded cells showed positive expression of the typical endothelial cell markers CD31, CD90, VE Cadherin, CD44, and CD105. Also, BOECs stained negative for the (non-endothelial) exclusion markers CD14, CD45, and CD133. In shear stress studies, BOECs and hVECs aligned similarly to ventricular flow (ie. directional shear stress). Both cell types displayed similar viability responses when grown on PCL which was ∼40% less than achieved when cells were grown on control glass slides. Modifying PCL with plasma oxidation or extracellular matrix coating did not improve viability of either cell type. Both BOECs and hVECs released CXCL8 and ET-1 when grown on control glass slides which was not increased in cells cultured on PCL. In addition, both cell types released the cardio-protective hormone prostacyclin when grown on glass or PCL, suggesting that they would provide a viable anti-thrombotic surface. Regarding to 3D structures, BOECs were able to populate both modified and unmodified aligned nanofibrous PCL scaffolds, and appeared to align with the direction of the fibres.

Conclusions

BOECs expressed the requisite endothelial cell markers and, as we have shown previously, responded appropriately to shear and released CXCL8, ET-1 and prostacyclin 3; suggesting that BOECs are suitable seeding cells for tissue engineering. For the endothelialization of tissue engineered heart valves, the target cells that BOECs need to mimic are hVECs. Our preliminary studies show that BOECs and hVECs profile similarly in population of PCL, alignment with shear stress and with endothelial cell hormone release. In addition, BOECs are able to align with the direction of PCL fibres, mimicking the native valve endothelial cells that were previously reported to align with the direction of the collagen fibers[4]. These results are promising and indicate the potential of BOECs as a cell source to populate PCL nanofibrous scaffolds designed to replace heart valves. Nevertheless, our data shows that standard PCL platforms are not optimal in terms of cell viability. This may be improved by biofunctionalizing PCL with specific molecules to support BOECs adhesion and proliferation. For that, we are currently studying the potential of linking the BOECs specific peptide (TPS) to PCL.

References

[1] Kasimir MT, Weigel G, Sharma J, Rieder E, Seebacher G, Wolner E, et al. The decellularized porcine heart valve matrix in tissue engineering: platelet adhesion and activation. Thromb Haemost 2005, 94(3): 562–567.

[2] Sohier J, Carubelli I, Sarathchandra P, Latif N, Chester AH, Yacoub MH. The potential of anisotropic matrices as substrate for heart valve engineering. Biomaterials. 2014; 35(6):1833–44.

[3] Reed DM, Foldes G, Kirkby NS, Ahmetaj-Shala B, Mataragka S, Mohamed NA, Francis C, Gara E, Harding SE, Mitchell JA. Morphology and vasoactive hormone profiles from endothelial cells derived from stem cells of different sources.Biochem Biophys Res Commun. 2014 12;455(3-4):172–7

[4] Deck JD. Endothelial cell orientation on aortic valve leaflets. Cardiovasc Res. 1986; 20:760–767.

Klinefelter Syndrome (KS) was first described 73 years ago. Surprisingly KS yet remains underdiagnosed in the population. Lack of awareness about symptoms and the guidelines of KS diagnosis among paediatricians and General Practitioners (GP) could be the main reason. Failing to perform a thorough physical examination, not considering or unable to get cytogenetic confirmation on suspected patients are causative factors for delayed diagnosis. Hence long standing comordities such as learning disabilities, psychosocial problems and Attention Deficit Disorder in children remain under-recognized behind an undiagnosed clinical condition. The benefits of early diagnosis at prepubertal age are much higher with the available technologies such as sperm retrieval from testes biopsy and intracytoplasmic sperm injection. This could drastically improve fertility and quality of life (QoL). As prenatal diagnosis is not promising, population screening is suggested to be the way of ensuring timely diagnosis. We reviewed KS patients in Oman presented over a decade at our tertiary care centre and compared our finding with review of literature available. We realized that lack of awareness, cultural reasons and early marriage among men is some of causes for late diagnosis. Hence we propose that prepubertal diagnosis should surely be the focus to improve fertility and QoL in KS patients. This is the first report of KS from the Omani population.

Klinefelter syndrome (KS) is a well-known sex chromosomal disorder in men, affecting 0.1–0.2% of the male population. About 80–90% of KS patients show 47,XXY and the remaining lesser percentage of them present in either mosaic (47,XXY/46,XY) forms or with additional sex chromosomes (48,XXXY,48,XXYY,49,XXXXY) or structurally abnormal X chromosome acquired through non-disjunction during maternal or paternal gametogenesis. KS results from a nondisjunction event in the father, in nearly half of the cases. The prevalence of KS is much higher even among men with infertility (azoospermia) ranging from ∼3%–13%. It remains underdiagnosed with significant number of patients being unidentified, while only a 10% of prepubertal KS being diagnosed. Moreover in adulthood it was estimated that only one fourth (25%) of affected males are diagnosed, mostly due to their investigation for infertility.

Comorbidities of this syndrome are more prominent than symptoms of hypogonadism and hence the underlying disorder remains undiagnosed while the comorbidity gets treated. KS is characterized by a genetic, whole gonadal dysfunction affecting germ cells from early fetal life. Signs may vary depending upon gonadal dysfunction and sex hormone deficiency during early life (young adults) to infertility in an adult male without any other signs of hypogonadism. During pre-pubertal years, the condition is underdiagnosed because of low androgen levels and non-production of sperms. Any mild developmental disorders or cryptorchidism should be an alerting signs. There are no symptoms that reliably indicate KS in prepubertal patients. Hence, suspected KS can be diagnosed by thorough physical examination (testosterone deficiency and testicular size) or chance observation during treatment of concomitant disease. The goal should be to achieve timely diagnosis. Isidori et al, (2008) claims ultrasound can suggest KS as early as puberty stage, before any signs are noticed on clinical examination.

Prenatal diagnosis of KS is a chance finding with no specific indication of ultrasound marker feature or serum screening marker. Hence, no effective way is currently available to screen the population prenatally, apart from cytogenetic(karyotype) analysis. Thus, it is only possible when amniocenteses are performed due to advanced maternal age and are likely to have the opportunity of ‘chance-diagnosis’. Although maternal age is a well-known risk factor for meiotic non-disjunction (especially for Down Syndrome); Bojesen et al found a 4-fold increase in the prevalence of KS cases with maternal age being greater than 40 years when compared to those below 24 years.

KS is also the most frequent genetic cause of azoospermia. A significant proportion of men with KF remain undiagnosed, probably because of the wide phenotypic variability and lack of knowledge of the symptoms among health professionals. From the data based on patient registries in Denmark, it is suspected that, only 25% of all KS patients are diagnosed in their lifetime. Therfore, awareness regarding KS is important among health care professionals and General Practitioners (GP). Careful clinical examination to rule out KS, followed by cytogenetic confirmation helps in early detection. This might enable early treatment which in turn might improve fertility and their quality of life (QoL). The incidence of KS may vary across different populations, however studies suggest a higher prevalence in Australian and Asian population. To date, there are no reports on KS published from Omani population.

Therefore, we have made an observation/assessment of age at diagnosis of KS, and the reasons for delayed presentation/diagnosis, from patients encountered at our centre, over the past decade. Based on our experience and the review of data available from other population, we hereby present our data from Omani population studied at our tertiary care centre. As prenatal diagnosis is not promising in detecting KS foetus, population screening is likely to the best option of ensuing timely diagnosis for these patients. We also suggest early diagnosis could possibly improve fertility and QoL among KS patients.

Background

Vascular calcification and increased mortality is now well linked in hemodialysis (HD) patients. Among the inhibitors and promoters of calcium-phosphate and bone metabolism, we studied Sclerostin, a glycoprotein mainly expressed by osteocytes, involved in regulating bone formation by inhibiting Wnt–β-catenin signaling. Studies demonstrated, in experimental CDK animal model, a positive association between Sclerostin production and dietary phosphate intake.

Materials and Methods

We investigated the relation between Sclerostin levels (SL) and dietary habits in 49 Caucasian HD patients (age 36–90, M/F 31/18, dialysis vintage 1–144 months). Nutrient intake was analyzed with 24 h-recall method. Serum Sclerostin was measured by ELISA method. Statistically analysis was performed by SPSS software.

Results

Mean calcium and phosphate intake were 705 ± 584 and 1196 ± 498 mg/day, respectively. Serum SL (3356 ± 2118 pg/ml) were increased in HD patients. Positive correlation was found between serum SL and calcium (r = 0.33, p = 0.025) and phosphate intake (r = 0.34, p = 0.021). Patients with SL higher were older (p = 0.003), showed higher protein (p = 0.012), calcium (p = 0.017) and phosphate (p = 0.004) intake. Multiple linear regression testing Sclerostin as dependent variable (dialysis vintage, age, body-weight, serum calcium and phosphate, serum PTH, calcium and phosphate intake as independent variables) found that dietary phosphate intake was the only significant determinant of SL (r = 0.356, B = 1.5, p = 0.004). In Patients with calcium carbonate or acetate therapy (n = 31), serum SL was positively correlated with dietary calcium (r = 0.433, p = 0.017) and phosphate intake (r = 0.377, p = 0.04).

Conclusions

The dietary phosphate and calcium intake may influence serum Sclerostin levels and potentially affect bone remodeling or soft tissue calcification in HD patients.

Introduction

Cirrhosis is an abnormal liver condition, mainly caused by viral hepatitis B or C, fatty liver diseases and alcoholism. Ascites is common complication of cirrhosis, associated with poor quality of life, abnormal cognitive functions, increased work disability and increased risk of infection, consequently development of hepatic encephalopathy1. Studies have suggested that inflammation caused by secondary infection with hyper ammonia act as synergistic factor responsible for hepatic encephalopathy in cirrhotic patients2. Magnetic resonance imaging (MRI) is the most commonly used method to observe brain abnormalities in in cirrhotic patients. These patients showed hyperintensities in basal ganglion on T1-weighted MRI and abnormal brain metabolites such as increased Glx, decreased myo-inositol and choline level on MR spectroscopy (MRS), decreased magnetization transfer ratio, and increased mean diffusivity on diffusion tensor imaging MRI 3,4,5,6,7,8. Though different neuroimaging studies investigated structural, diffusion, functional and metabolic brain changes in adult cirrhotic, the regional changes in gray matter and structural brain connectivity are not yet studied in pediatric cirrhotic patients. In this study, we evaluated the gray matter changes and global and regional topological properties of structural brain networks in pediatric cirrhotic compared to pediatric controls.

Materials and methods

Institutional regulatory board and ethics committee approved the current study protocol. 22 pediatric cirrhotic (mean age 11.6 ± 3.4 years, no prior HE), and 17 age and sex matched heathy controls were included in this study. Written informed consent was obtained from each individual prior study. Cirrhosis was diagnosed by the presence of a combination of high serum-ascites albumin gradient ascites, splenomegaly, large varices without EHPVO, irregular liver surface, portal vein ≥  13 mm and collaterals. Magnetic resonance imaging (MRI) was performed at 3-T clinical MR Scanner (GE Healthcare Technologies, Milwaukee, WI, United States) using a standard quadrature head coil. Conventional T2-, T1-weighted imaging and high-resolution T1-weighted structural imaging using a fast spoiled gradient echo BRAVO pulse sequence (TR =  8.4 ms; TE =  3.32 ms; inversion time =  400 ms; FA =  13°; matrix size =  512′512; FOV =  240′240 mm²; slice-thickness =  1.0 mm), were performed on each subject. T2-, T1-weighted images were examined for any gross brain pathology, such as cysts, tumors, or any other mass lesions, and presence of such anomaly was used as an exclusion criteria. We used high-resolution T1-weighted structural images for measuring regional gray matter changes and construction of structural network. Brain imaging data were processed using the statistical parametric mapping package (SPM8, http://www.fil.ion.ucl.ac.uk/spm/), MRIcroN, and MATLAB-based (The Math Works Inc, Natick, MA) custom software. High-resolution T1-weighted images from all subjects were visually examined for the presence of tumors and cysts. High-resolution T1-weighted images corrected for any bias and inhomogeneity-corrected images were partitioned into gray, white, and cerebrospinal fluid tissue types using a unified segmentation approach9,10. Gray matter tissues maps were normalized to the Montreal Neurological Institute (MNI) space and were modulated and smoothed using a Gaussian filter (FWHM, 10 mm). For the strctural networks construction we used graph theory based analysis using GAT software by using gray matter maps as described in details elsewhere11. In brief we generated 90 cortical and subcortical regions of interest (ROIs), excluding the cerebellum, from the Automated Anatomical Labeling (AAL) atlas using the WFU PickAtlas Toolbox. The extracted residual volumes of all 90 anatomical ROIs were used for construction of structural correlation networks.

Statistical analysis

All statistical computations were performed using the Statistical Package for Social Sciences (SPSS) version 16.0 (SPSS Inc., Chicago, USA). The normalized and smoothed gray matter tissue probability maps were compared between groups using analysis of covariance (ANCOVA; uncorrected threshold, p =  0.01; extended threshold, 100 voxels), with age and gender included as covariates. A p value of less than 0.05 was considered to be statistically significant.

Results

Glass brain images in Fig. 1 are showing significantly lower gray matter volumes (GMV) in multiple brain sites with few brain areas showing significantly higher GMV in pediatric cirrhotic compared to those of controls (Fig. 1). The correlation matrix of the cirrhotic group showed overall lower correlation strength than the control group (Fig. 2). In pediatric cirrhotic reduced brain network characteristic across a range of network densities was observed compared to control (Fig. 3). Pediatric cirrhotic also showed altered structural connectivity networks and hubs (Fig. 4).

Discussion

Cirrhotic patients showed altered gray matter volumes suggesting the brain tissue injury and decreased regional connectivity (clustering coefficient), while reduced global network organization (small worldness) and integration (hubs) suggesting decreased robustness and efficiency of the brain network. These results contribute to novel insights regarding the neurobiological mechanisms underlying cognitive deficits in these patients. The pathophysiological mechanism of brain tissue injury may include hyper ammonia secondary to inflammation resulting neuronal tissue injury2. This structural analysis using voxel based and graph theory might provide a more appropriate paradigm for understanding complicated neurobiological mechanism of cirrhotic patients, and may help to improve the clinical managements of these patients 12.

References

1. Ferenci P, Lockwood A, Mullen K, et al. Hepatic encephalopathy: definition, nomenclature, diagnosis, and quantification-final report of the working party at the 11th World Congresses of Gastroenterology, Vienna, 1998.Hepatology 2002;35:716–21.

2. Butterworth RF. Pathogenesis of hepatic encephalopathy in cirrhosis: the concept of synergism revisited. Metab Brain Dis. 2015.

3. Lai PH, Chen C, Liang HL, et al. Hyperintense basal ganglia on T1-weighted MR imaging. AJR Am J Roentgenol 1999;172:1109–15.

4. Geissler A, Lock G, Fründ R, et al. Cerebral abnormalities in patients with cirrhosis detected by proton magnetic resonance spectroscopy and magnetic resonance imaging. Hepatology 1997;25:48–54.

5. Rovira A, Grivé E, Pedraza S, et al. Magnetization transfer ratio values and proton MR spectroscopy of normal-appearing cerebral white matter in patients with liver cirrhosis. AJNR Am J Neuroradiol 2001;22:1137–42.

6. Laubenberger J, Häussinger D, Bayer S, et al. Proton magnetic resonance spectroscopy of the brain in symptomatic and asymptomatic patients with liver cirrhosis. Gastroenterology 1997;112:1610–16.

7. Miese F, Kircheis G, Wittsack HJ, et al. 1H-MR spectroscopy, magnetization transfer, and diffusion-weighted imaging in alcoholic and nonalcoholic patients with cirrhosis with hepatic encephalopathy. AJNR Am J Neuroradiol 2006;27:1019–26.

8. Kale RA, Gupta RK, Saraswat VA, et al. Demonstration of interstitial cerebral edema with diffusion tensor MR imaging in type C hepatic encephalopathy. Hepatology 2006;43:698–706.

9. Ashburner J., Friston K. Multimodal image coregistration and partitioning—a unified framework. Neuroimage. 1997;6:209–217

10. Friston K.J., Holmes A., Worsley K.J., Poline J.B., Frith C.D., Frackowiak R.S. Statistical parametric maps in functional imaging: a general linear approach. Hum. Brain Mapp.1995;2:189–210.

11. Hosseini SM, Hoeft F, Kesler SR GAT: a graph-theoretical analysis toolbox for analyzing between-group differences in large-scale structural and functional brain networks. PLoS One. 2012;7:e40709.

12. Petrella JR: Use of graph theory to evaluate brain networks: a clinical tool for a small world? Radiology 2011, 259:317–320.

Critically ill patients in the intensive care units (ICUs) are often in acutely disturbed state of mind characterized by restlessness, illusions, and nervousness. Such patients, for instance, those who are mechanically ventilated may incur difficulties during treatment procedures such as endotracheal tube intubation/extubation. Apart from critical illness, treatment induced delirium may cause them to dislodge themselves from life-saving equipment and thus hinder cooperative and safe treatment in the ICU. Hence, it is often recommended to moderately sedate such patients for several days to reduce patient anxiety, facilitate sleep, aid treatment and thus endure patient safety. However, most anesthetics affect cardiac and respiratory functions. Hence, it is important to monitor and control the infusion of anesthetics to meet sedation requirements while keeping patient vital parameters within safe limits. The critical task of anesthesia administration also necessitates that drug dosing be optimal, patient specific, and robust.

Towards this end, we propose to use a reinforcement learning based approach to develop a closed-loop anesthesia controller that accounts for hemodynamic parameter variations. Main advantage of the proposed approach is that it does not require a model, it involves optimization, and is robust to interpatient variabilities. We formulate the problem of deriving control laws that track a desired trajectory as a sequential decision making problem represented by a finite Markov decision process (MDP) and then use reinforcement learning-based approach to solve the MDPs for goal oriented decision making. Specifically, we use reinforcement learning approaches, such as Q-learning, to develop a closed-loop anesthesia controller using the bispectral index (BIS) as a control variable while concurrently accounting for the mean arterial pressure (MAP). Moreover, the proposed method monitors and controls the infusion of anesthetics by minimizing a weighted combination of the error of the BIS and MAP signals. Account for two variables by considering the error reduces the computational complexity of the reinforcement learning algorithm and consequently the controller processing time.

We present simulation results and statistical results using the 30 simulated patients. For our simulations, the pharmacokinetic and the pharmacodynamic values of the simulated patients are chosen randomly from a predefined range. To quantify the performance of the trained agent in the closed-loop anesthesia control, we use the median performance error (MDPE), median absolute performance error (MDAPE), root mean square error (RMSE), and interquartile range (IQ). In order to further investigate the effect of simultaneous regulation of the BIS and MAP parameters on the sedation level (BIS) of a patient, we also conducted three different in silico case studies. In the first case study, a hemodynamic disturbance is considered in which the MAP is altered by d units. This case study considers the effect of other factors such as hemorrhage on MAP as an exogenous disturbance. In the second case study, the MAP is set to a constant value irrespective of propofol infusion, which corresponds to patients that remain intubated in the ICU with post-aortic aneurysm repair or septic patients with respiratory failure. In the third case study, a disturbance due to administration of a synergetic drug such as remifentanil is considered during the administration of propofol. This case study considers the effect of drug interaction on the closed-loop control of hypnotic agent administration.

In order to provide a protective host response to a vast array of invading pathological microorganisms the ability of naïve CD4⁺T cells to differentiate into discrete effector subsets, each able to mediate specific aspects of immunity is a central tenet of the adaptive immune response. T helper (Th) 1 cells mediate protection against intracellular pathogens, such as bacterial and viral infections, whereas Th2 cells mediate protective responses against extracellular pathogens such as helminthic parasites. Additionally, naive T cells may be induced to differentiate into T regulatory (Treg) cells, with Treg cells serving as an immunological checkpoint to dampen the immune response and protect against inappropriate activation of the immune system. As in the case of autoimmune diseases where aberrant Th1 cell differentiation occurs in response to self antigens or in the case of asthma and allergic responses where aberrant Th2 cell differentiation occurs in response to non-self environmental antigens. In order to activate naïve T cells and induce them to differentiate to combat a specific invading pathogen dendritic cells (DC), which are present throughout the body serving as cellular sentinels constantly surveying there surrounding for evidence of infection must be activated. Activation of DC occurs through ligand mediated activation of pathogen associated molecular pattern receptors or danger associated molecular pattern receptors. Allowing for the engagement of specific downstream patterns of effector molecule regulation that play an instructive role in the decision making process which occurs during the activation, division and differentiation of naïve T cells. Current dogma suggests that the generation of differentiated effector CD4⁺T cells takes place over a 3–4 day period following the initial engagement of the T cell receptor (TCR). Activation of CD4⁺T cells is thought to occur in three distinct functional phases, priming, proliferation and differentiation. Through the use of an in vitro culture system that allows for precise control of the factors which regulate the activation of naïve CD4⁺T cells we initially assessed the requirements for cytokine signaling vs. strength of TCR signaling during differentiation. Here, we determined that Th2 differentiation can be driven following activation with a weak TCR stimuli in the absence of additional cytokine inputs, indicating that Th2 differentiation occurs through a default endogenous pathway following activation. Whereas, Th1 differentiation required both a strong TCR signal and the presence of an instructive cytokine, in this case IFNg in order for differentiation to occur. We additionally investigated the kinetics of upregulation of the master transcription factors that regulate commitment to a specific T-helper phenotype. CD4⁺T cells acquire the disposition to commit to either Th1, Th2 or Treg lineages very soon after activation >24 hr and prior to the induction of division, as evidenced by increased expression of the master transcription factor proteins Tbet, GATA3 or Foxp3. Further, we show that entrance into cellular division is not required for the induction of a full program of differentiation to occur. By interrupting the activation of naïve CD4⁺T cells at different time points following stimulation through the use of anti-MHCII antibody. We were able to probe the effects that both alteration of TCR signal strength and alteration of TCR signal length have on the induction of differentiation as compared to division. This approach allowed us to demonstrate that TCR signaling is responsible for two distinct activation programs, whereby the strength of signal that a naïve T cell receives working in concert with or without cytokine at an early phase can induce Th1 or Th2 differentiation respectively. Whereas the length of the TCR signal can be construed as a secondary component of activation which controls the ability of CD4⁺T cells to enter into division. Here, we determined that at low concentrations of antigen TCR signaling must occur for 2 hr. Demonstrating that the signal delivered through the TCR represents not only an essential component for inducing an immune response through the initial activation of naïve CD4⁺T cells, but also acts as a rheostat that is able to determine both the strength and the length of TCR signal received. In turn controlling cellular decision making by dictating the outcome of differentiation and whether a cell will enter into cycle. As such these results have important implications for the rational design of vaccination strategies, in order to modulate components of TCR signaling cascade to direct an optimal response against the target vaccine antigens.

Background

Melanoma is an aggressive neoplasm characterized by a complex etiology. Several molecular alterations occur during melanoma progression. The most commonly mutated pathway is the mitogen-activated protein kinases (MAPK)/ERK cascade. The activation of the MAPK/ERK signaling occurs either through gain-of-function mutations in BRAF and NRAS gene or through autocrine growth factor stimulation. Documented mutations have been found in the kinase domain of BRAF gene encoded by exon 11 and 15 with a frequency of 50–70%. The majority of these mutations affect one critical amino acid, resulting in the V600E substitution which account for more than 90% of all BRAF mutations. Given the high incidence of BRAF V600E mutation in melanoma, patient management is based on the use of specific inhibitors when patients carry BRAF V600E mutation.

By comparing RNA-seq and DNA Sanger sequencing data, we found that among 15 melanoma cell lines 3 were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that these cell lines express very low level of BRAF RNA and the expression may be in favor of the WT allele.

Given the low BRAF V600E RNA expression, we tested in this study whether the three discordant cell lines may respond differently to BRAF-specific inhibitors compared to the concordant BRAF WT and BRAF V600E control cell lines.

Methods

The three discordant cell lines, one BRAF V600E and one BRAF WT control cell lines were treated with three BRAF inhibitors, including two BRAF V600E specific (vemurafenib and PLX4720) and one aspecific (sorafenib).

Measurement of cell proliferation was performed by MTT assay. Quantitative real time PCR and Western Blot were also performed to detect BRAF V600E RNA and protein expression and to assess MAPK pathway activation.

Results

The three discordant cell lines showed BRAF V600E expression both at the RNA and protein level and MAPK pathway activation although at a lower level as compared to the BRAF V600E control cell line. The proliferation rate of the discordant cell lines decreased after treatment with vemurafenib and PLX4720 but was not affected by treatment with sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the BRAF V600E control.

All together these data suggest that the cell lines carrying BRAF V600E mutations at the DNA level may respond differently to BRAF targeted treatment and the differential responsiveness may be related to a lower BRAF V600E RNA and protein expression.

Background

Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occur for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in about 5–20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes).

Methods

Genotyping was performed by Illumina HumanOmni1 beadchip. For the RNA-seq experiment, library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. Genotype calling and subsequent quality filtering was performed in Illumina's Genome Studio software. IMPUTE2 (University of Oxford) was used for imputation of the genotyped data. RNA-Seq analysis was performed using the Broad Best Practice Workflows for RNA-Seq with the exception that a genotype was created for every available base pair in order to compare to the genotyped array data. In house custom perl scripts were then created to compare the genotyping data to the processed RNA-Seq data. The genotype and the B-allele frequency were calculated the latter creating a range of expression to evaluate.

By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE.

Results

MAE was detected in all 15 cell lines although to a different rate (spanning from approximately 17% to 75% MAE). When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances and isodisomy in some instances. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma.

Conclusion

The biological implications of MAE are yet to be realized. Nevertheless our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.

Background

Obesity related diseases including type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease have become major health problems. Inappropriate insulin production and dyslipidemia are commonly associated with obesity. It is multifactorial and heterogeneous in origin. While 60–80% of obese subjects are insulin resistant (IR) and rapidly develop metabolic diseases, called pathologically obese (PO), a proportion retain sensitivity to the hormone and remain relatively healthy (MHO), either because the progression to disease is slower in these individuals or they have developed pathways that renders them immune. Stratification of this disease, depending on the range of associated pathologies, would help identify mediators, design targeted therapies, in the understanding of mechanisms for this apparent protection. Also some ethnicities, such as South Asians and Arabs, appear susceptible to both obesity and its associated pathologies, perhaps largely determined by lifestyle factors, such as diet and exercise. However weight loss, mainly surgical, is proving to be the most successful means of reducing body weight and improving insulin sensitivity.

Therefore the aims of this study were to compare morbidly obese patients of Caucasian and Arab origin prior to and after weight loss to identify biomarkers of insulin sensitivity and inflammation in Arabs and Caucasians.

Methods

Subjects: Morbidly obese patients of Arab and Caucasian origins awaiting bariatric surgery (gastric bypass, gastric sleeve, or gastric band) were recruited from the pre-operative clinics: Al-Emadi Hospital, Hamad Medical Corporation, (Doha, Qatar) and Whittington Hospital (North London Obesity Surgery Service, Whittington Hospital, London, UK). Morbid obesity was defined as BMI ≥ 40 kg.m^− 2 or BMI ≥ 35 kg.m^− 2 with significant co-morbidities. All studies were approved by the relevant National Ethics Committees.

The studies included both males and females over the age of 18 years. Patients with coronary artery disease, uncontrolled hypertension, malignancy or terminal illness, connective tissue disease or other inflammatory conditions likely to affect cytokine levels, immuno-compromised subjects and those with substance abuse or other causes for poor compliance were excluded.

Anthropometric measurements were recorded: age (years), weight (kg), and height (m) systolic and diastolic blood pressure (mmHg). BMI was calculated (kg.m^− 2). Patient information including demographic data (date of birth, gender, ethnicity), surgery type (gastric bypass, gastric band, gastric sleeve), co-morbidities, current medication, weight loss history, smoking habits and alcohol consumption were recorded from hospital notes.

Samples: Blood samples (EDTA, NaF, no anti-coagulent), following an overnight fast, were drawn from an ante-cubital vein on the day of the operation, immediately after anesthesia. Samples were centrifuged (3000 rpm, 15 minutes, 25°C), and the plasma or serum collected and stored at − 80 °C prior to assay.

Assays: Blood samples were used to determine glucose (hexokinase), lipids (Total Cholesterol, LDL and HDL: Roche) and insulin (ELISA, Mercodia). Adipokines (leptin, adiponectin, interleukin-6, and MCP1) were assayed by ELISA (R&D Systems, Oxon, UK). Insulin resistance was calculated using the homeostatic model assessment where HOMA =  (glucose in mmol/L ×  insulin in miU/ml)/22.5.

The criteria for classification: Subjects were considered MHO if free of T2DM, dyslipidaemia, and cardiovascular disease, and exhibited systolic blood pressure less than 140 mmHg, diastolic blood pressure less than 85 mmHg, fasting plasma glucose less than 6.8 mmol/l and insulin less than 6.5 miU/ml.

Statistics: Data were entered in SPSS version 22.0 for statistical analysis. Parametric tests were used for normally distributed data and non-parametric analysis for skewed data.

Results

Effect of ethnicity on cardiometabolic risk factors.

Despite the Arab cohort being significantly younger, they were hyperglycemic hyperinsulemic and hyperleptinaemic, compared to the Caucasians. This population also had elevated β-cell function and insulin resistance, while insulin sensitivity was lower. However there was no significant difference in blood pressure. Total-, LDL- and HDL-cholesterol were higher, but triglycerides lower, in Arabs. Leptin, a marker of adipose tissue mass and adipocyte hypertrophy, was elevated in Arabs. Furthermore the proinflammatory adipokines (MCP-1, IL-6) were higher and the anti-inflammatory adipokines (adiponectin) lower in this population.

Weight loss

In Caucasians, there was an increase in both HDL- and LDL-cholesterol in the PO group, and in serum adiponectin in both MHO and PO patients, following surgery. However, the insulin levels and HOMA-IR index decreased significantly in the PO group. Fasting plasma glucose did not change after weight loss in either group, whereas the total cholesterol increased in both groups significantly. In Arabs, three months after surgery, both MHO and PO subjects showed significant reduction in BMI, which was accompanied by lower systemic insulin, HOMA-IR and leptin. There was no change in total-, LDL- and HDL-cholesterol, whereas triglycerides were reduced after the weight loss. The inflammatory biomarkers CRP and IL-6 were unchanged.

Conclusion

Despite both populations being equally obese the Arabs had greater prevalence of risk factors for cardio metabolic complications, with fewer having a metabolically healthy phenotype. In non-diabetic Arabs, compared to Caucasians, insulin resistance and inflammation appeared to be predominant lesions. Interestingly higher leptin levels in the BMI-matched Arabs points to adipocyte hypertrophy and adipose tissue dysfunction as causal factors in these lesions. The young age at which Arabs develop obesity perhaps explains the greater susceptibility to its pathological consequences.

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ influx pathway essential for many physiological functions and failure to maintain normal calcium homeostasis is one of the leading causes of cellular dysfunction in a wide variety of pathological conditions. Orai1, a key regulator of SOCE, constitutively recycles at steady state in the frog oocyte and internalizes into intracellular vesicular compartments during meiosis, leading to inactivation of SOCE. Such mechanism provides proper regulation of Ca²⁺ signaling in preparation for fertilization and embryonic development. Previous data showed a role for Orai1 C-terminus in its internalization during meiosis. However, the minimal region required for Orai1 internalization at steady state and during meiosis is not known.

We began the study of the molecular determinants of Orai1 trafficking in Xenopus oocytes by comparing the localization of multiple GFP-Orai1 C-terminal mutants 1-266, 1-275, and 1-285 intracellularly. Orai1 mutants 1-275 and 1-285 were both internalized during meiosis behaving similarly to WT, however, Orai1 1-266 was significantly enriched at the plasma membrane and did not internalize during oocyte maturation. To further map the region required for Orai1 internalization, we will generate specific mutants in the region 267-275 and evaluate the contribution of these signals in Orai1 internalization during meiosis. Mutants showing defect in internalization will be tested for phenotype rescue by co-injecting Orai1 with different potential candidates. These proteins include: 1) Rab5, a member of Rab family of GTPases has been shown to play a role as a regulator of intracellular trafficking during endocytosis. Previous data from our group has shown that Rab5 colocalizes with Orai1 in both oocytes and eggs. 2) Caveolin-1, a coat protein mediating caveolin-dependent endocytosis. 3) Flotilin-1, a membrane-associated protein involved in flotilin-dependent endocytosis.

As a second step in exploring the molecular and biochemical mechanisms underlying Orai1 internalization during meiosis, we will identify Orai1 associated proteins by co-immunoprecipitation of Orai1 complexes followed by quantitative proteomic analysis using dimethyl labeling coupled with tandem liquid chromatography-mass spectrometry (LC-MS). Candidates that are enriched in wild-type Orai1 immunoprecipitation but missing in internalization-deficient Orai1 mutants are suggested to play role in Orai1 endocytosis.

In conclusion, our results here suggest that residues 266–275 at C-terminus of Orai1 are essential for its internalization during meiosis. Using specific mutant(s) within this region, we will test the rescue of mutant(s) phenotype with potential candidates, and by quantitative proteomic analysis identify proteins associated with Orai1 and potentially important in its internalization.

Background:

Colorectal cancer (CRC) is the third most common cancer in Qatar and a major health concern for the Qatari population. Qatar has the highest rate of colon cancer compared to other countries in the eastern Mediterranean, West Asia and North Africa. Colon cancer is the most common cancer among Qatar's male population and according to the world age standard rates, around 20.8% of male Qataris have this cancer. Although progress in diagnosis and treatment has helped to extend and save the lives of many colorectal cancer patients, it still remains as one of the most prevalent human cancers. Many drugs such as 5-fluorouracil (5-FU) are being used to treat colorectal cancer, but patient(s) response to these drugs vary widely in terms of efficacy and toxicity. Moreover, it is observed that in colon cancer patients the tumor starts to develop resistance against these drugs over the course of treatment. The harmful side effects exhibited by the drugs used in cancer therapy as well as the increasing frequency of resistance to drugs have become the most challenging issues in the treatment of colorectal cancer. Hence, there exists an immediate need to discover better targeted and reliable drugs that could act as therapeutic agents which can prevent colon cancer progression and control distant metastasis as well as cure the disease with minimal side effects. Chemotherapeutic agents obtained from natural sources (plants) holds promising potential and have gained significant recognition in the field of cancer therapy. Pristimerin is a triterpenoid quinine methide present in various plant species of Cleastraceae and Hippocrateaceae families. Pristimerin has been shown to inhibit the proliferation of glioma, leukemia, myeloma, breast, lung, prostate and pancreatic cancer cell lines. Recent studies shows that pristimerin is a potent inhibitor of NF-κB. Induction of apoptosis by pristimerin was found to involve activation of caspases, mitochondrial dysfunction and inhibition of Akt signaling pathways. Pristimerin was reported to induce apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-κB signaling and depleting Bcr-Abl.

Material and Methods

Reagents

Antibodies against Caspase 3, caspase-9, cleaved caspase-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyadenosine 5’-diphosphate ribose polymerase (PARP) antibody was purchased from Cell Signaling Technologies (Beverly, MA). BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit with BD GolgiPlug, 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, MitoSOX Red Mitochondrial Superoxide Indicator, SYTOX Blue Nucleic Acid Stain and Hoechst 33342 solution were obtained from Molecular Probes, Life Technologies (CA, USA). Pristimerin, Cell Counting Kit-8 (CCK-8), DAPI, Glutathione-Reduced (GSH) and N-Acetyl-L-Cysteine (NAC) were obtained from Sigma-Aldrich (MO, USA). Human Apoptosis Antibody Array and Phospho-Kinase Antibody Array were purchased from R&D systems (MN, USA). Cell Death Detection ELISAPLUS kit was purchased from Roche Diagnostics (Mannheim, Germany).

Methods

Cell culture: Human colorectal cancer cell line HCT116 was cultured in DMEM, OXCO1 and SW48 cells were cultured in RPMI 1640 medium. Both culture medium were supplemented with 10% Heat Inactivated fetal bovine serum (FBS), 100 U/ml Penicillin and 100 U/ml Streptomycin. Cells were cultured at 37°C under a humidified 95%:5% (v/v) mixture of air and CO2.

Pristimerin was dissolved in dimethylsulfoxide as a 10 mM stock solution and stored at 4°C for the in vitro experiments. Further dilution was done in cell culture medium as required.

Cell Viability: Effect on the viability of CRC cell lines, HCT116, OXCO1 and SW48 were determined following treatment with various doses of Pristimerin for 24, 48 and 72 hours using WST-8 kit.

Apoptosis: HCT116/OXCO1 cells were treated with various doses of Pristimerin for 24 hours. After incubation, 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 using BD LSRFortessa analyzer (BD Biosciences, USA).

H2AX, active caspase-3 and cleaved PARP were quantified by flow cytometry. After treatment with Pristimerin, HCT116 cells were fixed and permeabilized using BD Cytofix/Cytoperm Plus Fixation and Permeabilization Solution Kit, as per protocol from the manufacturer. 0.5 ×  10⁶ cells in Stain Buffer (FBS) were stained with 3 μL each of H2AX (pS139)-Alexa Fluor 647, Rabbit Anti- Active Caspase-3- BV605 and PARP Cleaved Form-AF700 antibodies for 30 minutes at room temperature. The cells were washed with Stain Buffer (FBS) and then analyzed by flow cytometry.

Cell cycle analysis: 0.5 ×  10⁶ cells (HCT116/OXCO1) were briefly stained with Hoechst 33342 solution (10 μg/mL) and then analyzed by flow cytometry.

Mitochondrial membrane potential: 0.5 ×  10⁶ cells (HCT116/OXCO1) were briefly stained with JC-1 stain for 15 minutes at 37°C as per instructions from the kit manufacturer. The cells were washed twice with 1x assay buffer and then analyzed by flow cytometry.

ROS Production: CellROX Deep Red Oxidative Stress Reagent is a fluorogenic probe designed to reliably measure reactive oxygen species (ROS) in live cells. The signals from CellROX Deep Red Reagent is localized in the cytoplasm. The production of superoxide by mitochondria was quantitated using the MitoSOX Red reagent. It is rapidly oxidized by superoxide but not by other reactive oxygen species and reactive nitrogen species. HCT116/OXCO1 cells were treated with Pristimerin (0, 1, 2.5, 5 μM) for 24 h and finally analyzed by flow cytometry for quantification of ROS and superoxide.

HCT116 cells were preincubated with NAC (2.5 mM) or GSH (5 mM) for 30 minutes before addition of Pristimerin (5 μM) in studies to confirm the role of ROS in induction of apoptosis.

Western blot: Following treatment with various doses/combinations of Pristimerin for 24 hours, HCT116 cells were lysed with RIPA buffer and proteins were isolated. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies. Target proteins were detected using an enhanced chemiluminescence (Bio-Rad ChemiDoc MP imaging system).

Statistics: Two-tailed Student's t tests was performed using the Origin Pro software to compare means of different treatment groups and a P value below 0.05 was considered statistically significant.

Results

The results show that Pristimerin causes a dose dependent inhibition of cell proliferation in various CRC cell lines, HCT116, OXCO1 and SW48. The inhibition of proliferation correlated with the induction of apoptosis in HCT116 and OXCO1 cell lines. Treatment with Pristimerin leads to activation of Bid, loss of mitochondrial membrane potential, as determined by JC1 staining, activation of caspase-9, subsequent activation of caspase-3 followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage and DNA double strand breaks (H2AX staining). Pristimerin was found to increase the oxidative stress in CRC cells in a dose dependent manner. Pretreatment of HCT116 cells with NAC/GSH, was found to inhibit Pristimerin mediated induction of apoptosis, confirming the role of ROS.

Conclusion

Altogether, these data confirm the role of ROS in the inhibition of cell proliferation and the induction of apoptosis in CRC cell lines by Pristimerin, and thus provide a strong rationale for pursuing the detailed mechanism of action and confirming the anti cancer activity using in vivo models and future clinical studies.

Keywords

Colorectal cancer, Pristimerin, oxidative stress, apoptosis.

Background

Imported malaria has been a great challenge for public health in the State of Qatar due to the large number of immigrant workers come from the Indian subcontinent and Sub-Saharan Africa. Antimalarial drug resistance has emerged as one of the greatest challenges facing malaria control today. Chloroquine (CQ) resistance in Plasmodium falciparum (Pf) is associated with genetic polymorphisms in Pf CQR-transporter (Pfcrt) and Pf multi-drug resistance (Pfmdr-1). Monitoring parasite haplotypes that predict susceptibility to major anti-malarials can guide treatment policies. CQ is used in State of Qatar mainly for treatment of uncomplicated Pf infection and incidence of CQ resistant Pf in Qatar is yet unknown. Thus, present study is conducted to determine the polymorphic regions of CQ drug resistance genes (Pfmdr1-codon86 and Pfcrt-codon76) in imported malaria cases in the State of Qatar.

Method

During September 2013 to September 2015, a total of 325 (79 Pf and 246 P. vivax) microscopically positive uncomplicated malaria samples were collected from Emergency Room, Al-Khor General Hospital and Hamad General Hospital, HMC, Qatar and confirmed by molecular assay. Nested-PCR and Restriction Fragment Length Polymorphism (RFLP) were used to detect alleles of pfcrt gene (K76T) and pfmdr1 gene (N86Y).

Result

Out of 79 uncomplicated malaria cases, the majority of patients were from East and West Africa followed by Indian Sub-continent and Central Africa. Molecular genotyping at codon 86 of the pfmdr1 gene showed that 60.7% harboured wild/susceptible allele (N86), 26.6% mutant/resistant (Y86) and 11.4% had mixed alleles (N86Y). However, the prevalence of Pfcrt mutant allele (T76), wild type (K76) and mixed alleles (T76K) was 72.1%, 22.8% and 5% respectively.

Conclusion

Molecular surveillance strategy based on imported malaria cases can be used to detect and track drug-resistant malaria. The data presented here might be helpful for enrichment of molecular surveillance of antimalarial resistance and will be useful for developing and updating antimalarial guidance for non-immune imported cases in State of Qatar.

Introduction

The levels of chronic disease amongst the Qatari population have increased dramatically in recent years. Whilst these diseases are highly prevalent in Qatar, awareness surrounding the recognition of symptoms and the disease itself are limited. Sourcing accurate information about health conditions is crucial. It is currently unknown how Qatari people source information concerning health problems for themselves as well as others. This information, however, is essential for our understanding so that strategies can be derived to assist the population concerning the various health problems that are encountered. We explored how and where the Qatari population seek information regarding health problems.

Methods

Ethical approval was gained from Hamad Medical Corporation/Weill Cornell Medical College in Qatar Joint Institutional Review Board (14-00017). Adult Qataris 18–85 y were approached at different sites, including educational establishments and shopping malls, and asked to complete an anonymous questionnaire to ascertain basic information concerning demographics, health status, and utilisation of health care services during the past year and sources of health information that individuals access. The data were analysed using SPSS version 23.

Results

A total of 394 questionnaires were completed, with 62% respondents being women. More men rated their health as very good compared to women (60.1% and 53.1%, respectively). However, this was not statistically significant X2(3, 387) =  5.7, p =  0.319.

Overall, more people in Qatar used the Internet as a source of health information (71.1%) than in previous studies in the USA (23.8–53.5%)(1,2). This difference between US and Qatar percentages can be explained by the fast diffusion of Internet use and overall wide spread of technology usage among Qataris (3).

More women (78.7%) than men (60.8%) used the Internet as a source of seeking information about health (X2 (1, 72) =  14.8, p

Background

Despite being a major cellular component of various engineered skin substituent, underlying mechanism of successful fibroblast transplantation in wound healing is not clear. Here we show that substances derived from starved fibroblast accelerate wound healing process in rat.

Material and methods

Starved human fibroblast cell culture supernatant (SFS) was prepared and tested for its wound healing capacity on rat skin. Twelve Wistar adult male rats were randomized into four different groups of three. On the back of each rat two wounds were created with area of 452 mm². Each wounds were treated daily with one milliliter of SFS or cell culture medium (DMEM). The size of the wounds was measured daily until crust formation. The animals were scarified on 4th, 8th, 11th and 15th day of experiment and skin was removed for H&E and trichrome staining. Infiltration of fibroblast and inflammatory cells and collagen formation were analyzed.

Results

The diameter of the wounds treated the SFS solution was significantly decreased within the first week of treatment, compared with control wound receiving DMEM only (p

Background

Diabetes is a chronic illness in which the body cannot manage the levels of sugar, and it affects an estimated 387 million people worldwide. According to World Health Organization it is the 7th leading cause of death in the world. Frequent monitoring (3 to 10 times) of blood glucose is important for diabetes patients to prevent serious health problems, but existing commercial products of blood glucose measurement often cause discomfort and pain. In these products, a lancet is used for pricking the skin to obtain a blood drop and then this blood drop is placed on the test strip for quantification of blood glucose level with a meter. This inconvenient, painful and invasive technology is the main obstacle to achieve high quality health monitoring. Providing a pain-free and noninvasive diabetes monitoring solution at an affordable cost is the major challenge to replace the existing solutions.

Objective

Based on the recent experimental findings about breath acetone as a potential correlated biomarker for changes in blood glucose, we introduce an electronic nose based breath analyzer solution for identification/ quantification of exhaled acetone and hence, blood glucose level.

Method

The experimental setup used to acquire the signatures of the acetone with the electronic nose, containing an array of four low-cost gas sensors, is shown in Fig. 1. Mass flow controllers (MFCs) are used to control the concentration of acetone in the gas chamber from 0.25 to 2.5 parts per million (ppm), which corresponds to high point for breath acetone concentration. The sensor array, placed in a gas chamber, is periodically exposed to air for 750 seconds and to acetone for 500 seconds to extract meaningful information or feature vector at each concentration in the target range. The feature vector is formed by taking the ratio (a.k.a. sensitivity) of the change in each sensor resistance during the gas exposure stage with its baseline resistance (resistance at the end of air exposure). The resultant feature vector is further used for acetone identification/quantification. For acetone identification, we form sensitivity codes by arranging their sensitivities in an ascending order and introduce hardware friendly rank-order classifier. After its identification, support vector regression is used for its quantification by utilizing feature vectors.

Results

Acetone data at 10 different concentrations in the target range of 0.25 to 2.5 ppm is acquired to evaluate the performance of our approach. Figure 2 shows the typical response of the four sensors in the array corresponding to air (from 0 to 750 seconds) and acetone (from 751 to 1250 seconds). On testing with resultant feature vectors from the experimental data, rank-order classifier identifies acetone signature with 100% accuracy, and then support vector regression (SVR) quantifies acetone with a mean square error of 0.0059. Predicted concentration with SVR against true concentration in the target range is shown in Fig. 3.

Conclusion

We have introduced a breath-based acetone monitoring solution which will not only be eagerly acceptable by the diabetes patients due to its noninvasive and pain-free nature but it will also facilitate high quality health monitoring by acquiring a large number of breath samples at an affordable cost. Test results of the proposed electronic nose system illustrate good response of the sensors to acetone induced by breadth.

Background

With the advance of semiconductor technologies, bio-technological application-specific integrated circuits (ASICs) have become a major trend of the industry. Examples include Microelectrode measurement array systems for in-vitro and in-vivo physiological research at the cellular level [1] [2]. Using DNA microarrays can lead to a high throughput, which finds wide applications in genome research and drug development [3].

Objective

This paper presents a DNA detection CMOS micro-array, which can help in detection of presence of specific DNA sequences. The array is comprised of 16 × 16 sensor sites (interdigitated electrodes), each with a dedicated readout circuit, which enables fast parallel measurements. The use of CMOS processes results in much more cost effective solution as opposed to the optical systems that are currently used in DNA detection. The proposed IC also utilizes a minimal number of electrical signals, and can be easily interfaced to the outside world.

Method

Figure 1 shows the proposed sensor and readout. The single stranded DNA probe molecules can be immobilized on the sensor sites, after target molecules are added to the chip, which causes hybridization in case of a match between the receptor and probe (Fig. 2). After the addition of a suitable substrate, a RedOx reaction can be initiated on the matching sites, by the application of a suitable potential [3]. The resultant current is then fed to a Current Mode Sigma Delta ADC for digital conversion.

Typical values of the current from the redox reaction range from a few pA to the nA range, which entails the use of a high resolution ADC. Sigma Delta ADCs are capable of achieving such resolution at the expense of smaller conversion frequency. However, even a conversion time of a few seconds (which is sufficient to obtain > 15 bit resolution) should be enough in this particular case.

A typical Sigma Delta Modulator is shown in Fig. 3. The input current from the Electrode array is integrated in a current mode integrator, which is enclosed in a feedback loop. The reference current (IREF) can be generated using an on-chip bandgap reference circuit [5].

Conclusion

The use of CMOS arrays and readout circuits for biotechnological applications is a very promising field. On chip A/D conversion provides a compact alternative to bulky expensive DNA detectors. Both the array as well as the sigma delta ADC consume very low power, and the whole system can be designed to achieve sub mW power consumption.

Moreover, the integration of frontend and detection on a single substrate with minimal post-processing presents a highly cost effective solution to the optical systems widely in use. CMOS sensors can also provide higher sensitivity and reliability than their optical counterparts.

References

[1] J. Guo, J. Yuan, J. Huang, J. K. Y. Law, C.K. Yeung, and M. Chan, “29.2nV/rt Hz -59.6dB THD dual-band micro-electrode array signal acquisition IC”, IEEE J. Solid-State Circuits (JSSC), Vol. 47, pp. 1209–1220, May, 2012.

[2] J. Guo, J. Yuan, and M. Chan, “Modeling of the cell-electrode interface noise for microelectrode array measurement”, IEEE Trans. Biomedical Circuits and Systems (TBCAS), Vol. 6, pp. 605–613, Nov. 2012.

[3] Stagni, C.; et al”CMOS DNA Sensor Array With Integrated A/D Conversion Based on Label-Free Capacitance Measurement,” in Solid-State Circuits, IEEE Journal of, vol.41, no.12, pp.2956–2964, Dec. 2006.

[4] Schienle, M.; Paulus, C.; Frey, A.; Hofmann, F.; Holzapfl, B.; Schindler-Bauer, P.; Thewes, R., “A fully electronic DNA sensor with 128 positions and in-pixel A/D conversion,” in Solid-State Circuits, IEEE Journal of, vol.39, no.12, pp.2438–2445, Dec. 2004.

[5] Bo Wang; Man Kay Law; Bermak, A., “A Precision CMOS Voltage Reference Exploiting Silicon Bandgap Narrowing Effect,” in Electron Devices, IEEE Transactions on, vol.62, no.7, pp.2128–2135, July 2015

Rationale

Epilepsy is one of the most prevalent neurologic conditions. It is estimated to affect 70 million people worldwide. Epilepsy is an important cause of disability and mortality. It is associated with social stigma and significant economic costs. Although epilepsy is a disease with a worldwide distribution, its prevalence varies between different countries. Very little is known about the epidemiology of epilepsy in Qatar. Qatar's population is a mixture of native citizens and immigrants. We aim at describing the features of epilepsy in Qatar as such information is virtually lacking from the current literature.

Methods

A database was created in 2014 to summarize information retrospectively collected on patients with epilepsy seen through the national health system (HMC) adult neurology clinic. For each subject, in addition to the typical demographic variables, we identified the age at onset, seizure types, epilepsy syndrome, etiology, treatment and outcome. Brain imaging and EEG results were also tabulated. All these variables were analyzed using the statistical package for social science (IBM-SPSS, version 20).

Results

Of 504 patients included in the database, 467 with sufficient information were analyzed. Sixty percent were men. The mean age at the last clinic visit was 35. Native Qataris represented 38.5%, Asian subjects 33%, and Middle Eastern/North African (MENA) origin accounted for 25% of the studied population. Generalized tonic-clonic seizures were the most common seizure type, noted in 89% of subjects. Epilepsy was classified as focal in 65.5% of the cases, and generalized in 23%. EEGs were abnormal in 55.5 %, showing epileptiform discharges in 49% of subjects. Imaging studies revealed epileptogenic pathologies in 40% of reports. Common causes of epilepsy were: vascular (11%), hippocampal sclerosis (8%), infectious (6%) and trauma (6%). Sixty six percent of patients were receiving a single antiepileptic drug, and 53% were seizure free at the last follow-up. Overall, the most commonly prescribed drug was Leviteracetam (41%) followed by Valproic Acid (25%) and Carbamazepine (22%). On current therapy, 54% of patients were seizure-free, 41% had a partial response and five percent were refractory. When the patients were divided by geographical background, some differences were noted. Remote infections caused the epilepsy in 15% of Asian patients (with neurocysticercosis accounting for 10%), but only in 1% of Qatari and 3% of MENA subjects (with no reported neurocysticercosis) (p

Background and Objective

Often times, clinicians use a three-dimensional set of medical images to diagnose and plan treatments, which typically include visual identification of structures such as bones and tissues [1]. This can be a challenging task as anatomical structures of interest can contain significant noise, and easily blend with neighboring tissues. We propose to tackle 2 cases: (a) treatment planning of pelvic fractures where a small size ring formed by the fused bones of the ischium, ilium, and pubis attached to the sacrum contains vital structures (including major blood vessels, nerves, digestive and reproductive organs) and should be carefully delineated, and (b) liver cancer treatment where malignant tissue has to be carefully removed. The pixel intensity of tumor is similar to those of healthy tissue and proper delineation is of utmost important before proceeding to plan therapy.

To address the aforementioned challenges, in this work we present a soft-clustering technique using Enhanced Fuzzy C-Means (EFCM) along with a bilateral filter to detect the region of interest. The key feature of the proposed algorithm combines domain and range filtering allowing the filter to maintain balance between preservation of relevant details and the degree of noise reduction. The approach allows traditional Fuzzy C-Means not only to exploit useful spatial information, but also to dynamically minimize clustering errors caused by common noise in medical images.

Methodology

A three-step workflow is used to process the medical images:

Step 1: After MR/CT images are acquired; clinician initially draws a rough outline around the region of interest (where the fracture or tumor is present) on the two-dimensional image slices. The manual input reduces the computational time to determine the desired tissue cluster by providing the region of interest instead of scanning/processing the entire image.

Step 2: In this step, a bilateral filter is used to remove noise while preserving details of the edges [2]. Linear filters, such as Gaussian, compute a weighted average of pixel values in the neighborhood. The weights decrease with distance from the neighborhood center. This works well for images where local neighboring pixels have similar values (slow spatial variation). As the noise that corrupts these neighboring pixels is mutually less correlated than the signal, the noise is averaged away while signal is preserved. However, the assumption of slow spatial variations fails at edges, which are consequently blurred by linear low-pass filtering. In this context, we use a non-linear/bilateral filter that combines both domain and range filtering. In smooth regions, the pixel values within a local neighborhood are similar to each other, and the normalized similarity function is close to one. Consequently, the bilateral filter acts essentially as a standard domain filter, averages away the weakly correlated differences between pixel values caused by noise, and preserves edge details.

Step 3: As a last step, EFCM clustering algorithm is applied to the noise-filtered image. Fuzzy C-Means clustering works by assigning membership to each data point with respect to the cluster centers [3]. A distance is computed between the cluster center and the data point. The membership of the data towards a particular cluster center varies linearly as per the distance. Closer the data to a cluster center, higher is its membership. The summation of membership of each data point across different clusters is equal to one. An objective function based on the Euclidean metric is then used to update the membership and cluster centers iteratively. However, the parameter estimation resulting from the described objective function may not be robust in a noisy environment. Therefore in this work, we develop an algorithm that uses a modified Euclidean term (described in Table I) that is robust against noise and allows meaningful clustering of compact pixels for image analysis by the clinicians (Fig. 1).

Results and Conclusion

The method proposed in this work was evaluated using two datasets: (a) CT images of pelvic fracture (two subjects) publicly available online for research purposes, on OsiriX website (http://www.osirix-viewer.com/datasets). The image acquisition details are as followed: Slice Thickness: 2 mm, Pixel Spacing: 0.29 mm ×  0.29 mm, Bit-depth: 12, and Acquisition Matrix: 512 × 512. (b) CT images containing liver with tumor from five anonymized subjects, obtained from Hamad Medical Corporation, Doha, Qatar. The image acquisition details are as followed: Slice Thickness: 3 mm, Pixel Spacing: 0.32 mm ×  0.32 mm, Bit-depth: 16, and Acquisition Matrix: 512 × 512.

The algorithms were implemented on MATLAB R2013a running on a workstation with 16 GB RAM and 2.8 GHz Intel processor. The average time required to perform segmentation was recorded as 8 ±  1.5 minutes. However the computational time could be further reduced, as implementation was done without optimization of the internal function calls. An initial assessment of the experimental results has shown satisfactory outcomes in both the cases to detect pelvic fracture and liver tumor (Fig. 1). The use of traditional linear filters on the datasets has failed to identify clusters with similar pixel intensity values. The use of bilateral filter with Euclidean modification proposed in this work has lead to desired soft clustering identifying the required anatomical structures in the images.

In future, we plan to optimize and validate the method extensively on different tissue-types using multiple imaging modalities.

References

[1] Vona G. et al., “Impact of cyto-morphological detection of circulating tumor cells in patients with liver cancer,” Hepatology, 39, 792–797, 2004.

[2] Sugimoto K. et al., “Compressive Bilateral Filtering,” IEEE Trans. on Image Processing, 24, 3357–3369, 2015.

[3] Havens T. C., “Fuzzy c-Means Algorithms for Very Large Data,” IEEE Trans. on Fuzzy Systems, 20, 1130–1146, 2012.

In the last decade, starting from family-based association analysis and progressing to population-based association analysis have helped in understanding etiology of many rare and complex disorders. As of Nov. 2013, National Human Genome Research institute (NHGRI) Catalog of published Genome-Wide Association Studies (GWAS) contained 1,751 manually curated publications with 11,912 SNP-trait associations. Despite large number of studies reporting different loci for complex traits, the aggregate variance explained by these loci is relatively low and more efforts are needed to look for loci with larger variance. These loci with larger variance are difficult to find as they tend to be at low frequency in general population. Previously, 1000 Genome project have shown common variants ( ≥  10) were found in all the populations, but 17 of low frequency variants (0.5–5) were seen in single ancestry group and 53 of rare variants ( <  0.5) were present In a single population. Therefore, more number of sequencing studies is required especially on understudied populations which are not part of big sequencing projects like 1000 genome project or UK10K to unearth low frequency and rare variants which aid in our understanding of missing heritability in etiology of common disorders.

The Arabian Peninsula located at cross roads between Africa and Eurasia is not well represented in global sequencing projects. This limits our understanding of human genetic diversity as these populations are recipients of constant gene flow over generations from Africa and Eurasia. The State of Qatar located on northeastern coast of the Arabian Peninsula with alarmingly high rate of obesity and diabetes among nationals has started new initiatives to understand genetic causes for these common disorders. But, to further extrapolate information from these genetic studies to molecular disease mechanisms and generating significant biomarkers for clinical settings a comprehensive data resource is required integrating existing information from informatics and experimental approaches. This central resource requires annotations about the variant, the gene, epigenetics, gene expression and protein expression. Many resources like UCSC, Ensembl and NCBI provides these annotations but with limited capabilities in analyzing large number of variants. Variant centric resources which provide capabilities for annotation of large number of variants limit themselves to a specific category of annotation like amino acid changes, associations to traits or regulatory effects. Further, these large resources and variant centric resources share a common drawback towards difficulty in retrieving population specific annotations especially for populations which are not part of global sequencing projects. The annotation data from these understudied populations get lost in wealth of data. Therefore, population specific annotation resources are needed integrating annotations from various sources which can be used to compare population specific differences. For e.g. Genome browser part of Singapore Genome Variation Project (SGVP) integrates annotations from sources like dbSNP, NHGRI GWAS catalogue, Reactome into Malay population specific linkage disequilibrium (LD) data. In this work, we present a Qatar specific webserver for visualization and annotation of genetic variants implicated in association studies conducted in Qatari population. The webserver seamlessly integrates genomic annotations obtained from public databases with Qatar specific pre-computed genomic characteristics like Linkage Disequilibrium (LD), Recombination Rates and Allele Frequencies using already published genomic data from Qatar. It also provides user-friendly starting points for annotating single and list of genetic variants, LD blocks or genetic regions of interest. In conclusion, this webserver combines various annotation sources with genomic information from Qatari population with varied uses in field of genomic research.

A self-calibrated gas sensing system for breath analysis is introduced. The system is composed of an array of high sensitivity gas sensor combined with temperature, humidity and flow sensor for calibration. The system is able to diagnose common diseases and help people stop them at early stages.

1. Introduction

Breath analysis is a new method for point-of-care health monitoring and diagnosis. Compared to traditional diagnostic techniques like blood test, urine test, biopsy, endoscopy and imaging, it has at least two advantages: complete non-invasiveness and limitless repeatability. Exhaled breath contains thousands of volatile compounds that can serve as biomarkers of metabolism processes [1]. By analyzing the pattern of the exhaled compounds, various diseases, including airway inflammation, renal failure and lung cancer, can be detected at early stages [1].

Gas chromatography (GC), mass spectrometry (MS) or the combination of the two (GC-MS) are the most frequently adopted methods in breath analysis given their high reliability and sensitivity. Nevertheless, these techniques require bulky equipment not suitable for point-of-care application. Gas sensor array based electronic nose (eNose) is a promising alternative to GC and MS. It has the advantage of portability and low cost which makes it suitable for home-based healthcare solutions. However, the response of gas sensor array also varies with different temperature, humidity and flow rate. To address this problem, in-system calibration techniques needs to be adopted.

2. Implementation

2.1 Diagram of Self-calibrated Gas Sensing System

As shown in Fig. 1, the proposed gas sensing system is composed of three major parts, which are the multi-sensor stage, the analog front end (AFE) and the digital processing unit. The sensor part includes a gas sensor array and three other sensors for calibration, which are flow, temperature and humidity sensor, respectively. The AFE part consists of two individual blocks. One is the frequency shift detection circuit for Film Bulk Acoustic Resonator (FBAR) gas sensor array and the other is a voltage detection circuit shared by the flow, temperature and humidity sensors. The digital processing unit takes charge of the sensor data sampling, processing and transmission.

Figure 1: Diagram of Self-calibrated Gas Sensing System

2.2 FBAR Gas Sensor Array and AFE

2.2.1 FBAR Gas Sensor Array

The gas sensor array is the core of the system and directly determines the overall performance of the eNose. Here, FBAR gas sensor is chosen as the element of the array to achieve high sensitivity. The selectivity of the FBAR-based sensor is drastically affected by the sensing material. By depositing different sensing materials or changing the properties of the sensing material on each individual sensor in the array, a unique pattern response to a specific gas mixtures can be generated by the array. The gas composition and concentration of the mixture can be acquired from the sensor array. For breath analysis, a set of specific sensing materials targeted at sensing biomarkers of common diseases are selected. For example, palladium is sensitive to hydrogen which is related to indigestion [1].

2.2.2 Analog Front End of FBAR

The response of FBAR gas sensor is illustrated in a frequency shift caused by mass loading effect when exposed to target gases. Two different methods can be used to detect the output signal. One is to count the high frequency signal directly with a proper ratio pre-scaling. The other method is to count the intermediate frequency (IF) signal after moving the original signal to the IF band. Because the design targets at mobile application, power consumption of the system should be one of the major considerations when designing the system. As direct counting at high frequency consumes a great deal of power, the second scheme is preferred.

Figure 3 shows a brief diagram of the frequency shift detection circuit for the FBAR gas sensor. Through RF multiplexer, each FBAR is alternatively connected to the first stage of the circuit to build an oscillator. Another reference FBAR without sensing material is used to build the reference oscillator. By mixing the sinusoidal signal of the two oscillators and low pass filtering the mixed signal, an intermediate frequency (IF) signal contains the frequency shift information of the sensor is acquired. At last, the frequency of the IF signal is read out using a simple counter.

Figure 3: Frequency shift detection circuit for FBAR gas sensor

2.3 Sensors Calibration and AFE

The response of the gas sensor array can drift due to the variation of flow rate, temperature and humidity. Calibration of the drift can be achieved by subtracting the influence of those variations. To obtain the calibration information, flow, temperature and humidity sensors need to be implemented on the same die. A BJT temperature sensor with ± 0.4 accuracy, a capacitive humidity sensor with ± 4% RH accuracy and a thermal mass flow meter was adopted. With signal conditioning interfaces, all the three sensors share the same 16 bit, 90 Hz Σ-Δ ADC.

2.4 Digital Processing Unit

The digital processing unit is designed to control the sampling rate and transmits the sensor data off-chip through a I2C transceiver. The processor also performs data fusion as multiple sensing data are obtained in the proposed system. Data processing and wireless communication are accomplished by the host controller.

3. Conclusion

A self-calibrated gas sensing system with high sensitivity and good reliability is introduced to achieve diagnosis of multiple common diseases like diabetes, airway inflammation and indigestion. The system is power efficient and small enough to be integrated in mobile devices like smart phone and tablets. The system features a self-calibration methodology through the use of multi-sensing platform namely: temperature, humidity and flow.

References

[1] De Lacy Costello, B., et al. “A review of the volatiles from the healthy human body.” J Breath Res 8 (2014): 014001.

[2] Wilson, Alphus D., and Manuela Baietto. “Advances in electronic-nose technologies developed for biomedical applications.” Sensors 11.1 (2011): 1105-1176.

[3] Benetti, M., et al. “Microbalance chemical sensor based on thin-film bulk acoustic wave resonators.” Applied Physics Letters 87.17 (2005): 173504.

Circular RNA (circRNA) represent a large class of noncoding RNAs that were previously considered as possible artifacts of abnormal RNA splicing, however, recent studies has shown that circRNA play an important role in regulating gene expression in mammals. Unlike linear RNAs, the downstream 5’ (splice donor) and upstream 3’ (splice acceptor) join together to form a closed continuous loop and this is one of the ways circRNA are formed. These exons are referred to as scrambled exons or shuffled exons and the process is known as backsplicing. circRNA can also be formed from lariat introns which escape debranching. Epithelial - mesenchymal transition (EMT) is a reversible process in which cells loses their epithelial phenotype and obtain mesenchymal phenotype. Moreover, EMT decreases expression of epithelial marker genes such as E-cadherin and increases expression of mesenchymal marker genes such as Mucin, N-cadherin, MMP2, Snail, Twist, VIM, FN, ITGA, FOX, TGFβ. Thus, EMT can play a major role in facilitating the migration, invasion and progression of cancerous cells in human body tissue. Several studies showed the regulatory role of linear forms of RNA transcripts (mRNA) on EMT, this study aims to highlight the regulatory role of circRNA in EMT.

Two epithelial ovarian cancer cell lines (CaOV3 and SKOV3) were treated with EMT inducing media supplement. RNA was isolated using all prep DNA/RNA kit. iScript cDNA synthesis kit was used for cDNA synthesis and reverse transcription. SYBR select master mix kit was used for PCR amplification. Product size was checked on 2.2% agarose gel (flash gel DNA cassettes-Lonza). CircRNAs with clear prominent bands were selected for gene expression analysis using eleven EMT signature genes by real time PCR. Migration scratch assay was used to test the ability of cells to migrate when subjected to EMT inducing media. Cells were seeded in a 6 well plate, and when they reached 80% confluency, a scratch was made using 1 ml tip. EMT media was added, and cells were kept in serum free media for 12 hours interval. Distance migrated by cells was measured in both treated and untreated wells using a Zeiss microscope. Extracted RNA from treated and untreated cells with commercially available kits was prepared for Illumina paired-end sequencing. Illumina deep sequencing using HiSeq 2500 yielded an average of 30 million read pairs per library of 100 bp read length. Using an in-house developed computational pipeline we identified and characterized the circRNA expression in EMT versus non-induced cells and compared it with the linear (mRNA) expression.

Real time PCR assay with eleven EMT signature genes showed a complete epithelial to mesenchymal transition after EMT supplement media was induced in both cell ovarian cancer cell lines (CaOV3 and SKOV3). Scratch assay showed cells which were subjected to EMT media migrated to the middle of the well and almost closed the scratch gap. However, cells in untreated wells neither showed motility nor migration. RNA-sequencing data showed that a large number of candidate circRNAs and mRNAs are differentially expressed between Epithelial and Mesenchymal states and belong to well characterized EMT markers like E-cadherin, N-Cadherin, Fibronectin, Snail and Vimentin genes. We further report that compared to mRNAs, circRNAs show a stronger differential expression trend for EMT related biochemical pathways like tight junctions,gap junctions and adherens junctions indicating a regulatory role for circRNAs in Epithelial to Mesenchymal transition.

In conclusion, our results clearly demonstrates the potential role circRNA has on EMT induced cancer cells. In future, further analysis will be done to investigate the regulatory potential of the circRNA forms by using knockdown based assay.

Neuroblastoma, a type of solid malignant tumour diagnosed during infancy represents around 10% of all paediatric cancers, marking it as the second most common paediatric cancer. It is identified as a highly heterogeneous tumour that varies from persistent progression to a spontaneous progression. Development of neuroblast masses occur mostly in abdomen (65%), chest (20%), neck (5%) or pelvis (5%) and is classified to four major stages as L1 (very low), L2 (low), M (intermediate) and MS (risk) group of patients. Intracellular calcium ([Ca²⁺]i), the secondary messenger, plays a vital role in regulating cellular processes and hence maintaining the cellular calcium homeostasis is critical. Levels of [Ca²⁺]i in cancer cells is eminent as it modulates the proliferative or apoptotic pathway of the cell, bestowing the effect of anti-cancer drugs on [Ca²⁺]i. Here we focus on the effect of [Ca²⁺]i in the development and treatment of neuroblastoma. In resting cells, the concentration of [Ca²⁺]i ranges between 10 and 100 nM, reaching up to a 100 fold increase upon the Ca²⁺ entry into the cells from the extracellular space or its release from the internal Ca²⁺ stores (endoplasmic reticulum and mitochondria). [Ca²⁺]i homeostasis is maintained in the cell by a Ca²⁺ influx and efflux mechanism, in which the inositol-1,4,5 triphosphate receptor (InsP3R) and the ryanodine receptor (RYR) play an important role in regulating the influx mechanism. Signalling pathways involved in association to neuroblastoma includes growth factors like Epidermal Growth factor (EGF), Insulin-like Growth Factor (IGF), Nerve Growth factor (NGF), Platelet-derived Growth Factor (PDGF) and Vascular Endothelial Growth Factor (VEGF). Activation of these growth factor signalling pathways, activates a cascade of downstream signalling molecules including PI3K/AKT, ALK and FAK as intermediate kinases to MYCN, NF-KB and p53 as important transcription factors involved in the development of neuroblastoma. [Ca²⁺]i and PI3K/AKT pathway interactions lead to a loop of continuous activation of this cascade, while the activation of ALK and FAK leads to the activation of calmodulins and CaM dependent protein kinase kinase. Several studies confers the role of [Ca²⁺]i in inducing differentiation, proliferation and apoptosis in neuroblastoma. This poster explains in detail on the intracellular pathways that regulate differentiation, proliferation and apoptosis in neuroblastoma and the mechanism by which the [Ca²⁺]i homeostasis is maintained in the cells. Calcium-Sensing Receptor (CaSR), a G-protein coupled receptor is a vital mediator protein that sustains the cellular responses and determines the cell fate in response to the external Ca²⁺ concentration between 0.05-5 mM from a proliferative stage to a stage of quiescence and differentiation. CaSR activation is often associated with an up regulated expression of parathyroid hormone related peptide (PTHrP), which has a role in inducing hyperglycemia in cancer cells. It is conferred that these CaSR exerts tumour suppressor functions in neuroblastoma and is found in differentiated favourable neuroblastoma tumours. Evidences suggest that in neuroblastoma cells, common chemotherapeutic drugs like arsenic trioxide (As2O3), cisplatin (CDDP) and trimethytin chloride (TMT) that are used in treatment of cancer interferes with the [Ca²⁺]i homeostasis and induce apoptosis. It suggests a promising role for targeting [Ca²⁺]i for the development of a new drug. Thus, in a nut shell, we elucidate the intracellular mechanisms that are associated with the development of neuroblastoma with the key focus on the [Ca²⁺]i along with possibility of development of a new drug target in the treatment of neuroblastoma.

Background

Technological breakthroughs witnessed over the past decade have led to an explosive increase in molecular profiling capabilities. This has ushered a new “data-rich era” for biomedical researchers. Indeed the recent availability of vast compendia of biomedical “Big Data” offers unique opportunities to devise novel approaches to knowledge discovery. We have launched an innovative “Collective Data to Knowledge” (CD2K) platform at the Sidra Medical and Research Center, which provides a hands-on accelerated training path for young biomedical researchers. The originality of this approach stems from the fact that it does not rely on de novo generation of data but leverages instead large dataset collections available in public repositories for the discovery of novel scientific knowledge. It does however recapitulates all other steps involved on the path to transformation of data into novel biomedical knowledge, including knowledge gap assessment and prioritization, hypothesis generation and testing, and generation of reports for publication in peer reviewed journals. Furthermore, besides providing accelerated hands-on training it also potentially constitutes a highly efficient approach to the generation of intellectual capital in Qatar.

Methods

The approach that we have devised relies on a wide range of available bioinformatics tools and resources.

a) Data integration and dissemination: For data dissemination we rely on a custom web application – the gene expression browser – that is used for integration of heterogeneous data (e.g. molecular profiles, together with clinical information, sample information and results from ancillary assays) [1]. It provides user with seamless access to large and complex datasets that can be viewed in an interactive format. This tool has now been deployed at Sidra Medical and Research Center and is used to create curated themed dataset collections that will be described in peer-reviewed communications (manuscripts in preparation).

b) Knowledge gap assessment: Knowledge gaps are identified via profiling of the biomedical literature for sets of differentially expressed genes. For instance knowledge gaps may be revealed among the hundreds of genes identified via transcriptome profiling as being induced by TNF-α, a host-derived pro-inflammatory cytokine. Among those genes many will be associated in the literature with pro-inflammatory responses, but a number of them will be shown via literature profiling not to be associated with inflammation, thus constituting a knowledge gap in which lies the opportunity for discovery.

c) Hypothesis generation and In Silico validation: the identification of knowledge gaps is the first step towards generation of novel knowledge. Next we rely on tens of thousands of publically available datasets to validate and extend the initial finding, often leading to formulation of novel hypotheses that can be immediately tested by accessing other relevant datasets.

d) Information extraction: We also devised standardized approaches for extracting and structuring information. These methods are used when profiling the literature and dataset collections in view of preparing the background and result sections of the reports. This principled approach helps trainees with manuscript preparation, which often constitutes a hurdle for scientists at an early in their careers.

e) Knowledge dissemination: trainees are then encouraged to submit their work in peer-reviewed scientific journals. It is the opportunity for them to learn about this essential process that is one of the cornerstones of the scientific discovery process.

Results

Workshops carried out in Qatar and in several countries around the world have been instrumental in the development of a CD2K training curriculum. In addition, a proof of principle of the effectiveness of the CD2K platform has been established with identification in a short amount of time of new discoveries with potential for high impact.

1) CD2K Training Workshops

We have conducted hands-on training workshops this year for 6 organizations. Each workshop spanned between 1 to 3 days and involving overall more than 100 participants.

An introductory CD2K training workshop was organized at the Sidra Medical and Research Center. Training material was further developed by conducting CD2K workshops in a wide range of settings: in academic research institutes, in the United States at the Jackson Laboratory for Genomics Medicine and in Singapore at the A-star Institute; in a University in Thailand (Chulalongkorn University, Bangkok); in a research hospital in France (Hopital Europeen, Marseille); in a large pharmaceutical company in the United States (MedImmune, a subsidiary of AstraZenca, in Gaithersburg, Maryland).

These workshops were instrumental to the establishment of a robust training curriculum; consisting in the following learning objectives:

a) Collective biomedical data profiling

b) Literature profiling

c) Identification and prioritization of knowledge gaps

d) Hypothesis generation and in silico validation

e) Information extraction

f) Knowledge dissemination

2) CD2K Proof of principle

A post-doctoral research fellow has been assigned to the piloting the CD2K platform at Sidra, with the objective of identifying and prioritizing potential knowledge gaps and submitting reports for publication in peer reviewed journals within 12 months for three novel findings with high potential for translation. At the time of submission of this abstract 10 months within this pilot 2 manuscripts have been submitted with a third one being finalized. The first two articles have appeared online pre-peer review in March and September of this year in the journal “Faculty of 1000 Research”:

a) The first article reports the identification of “ADAM metallopeptidase 9” (ADAM9) as a candidate biomarker for the specific assessment of tissue damage caused by infection, independently of pathogen-driven inflammatory processes [2]. This work revealed a new potential role for ADAM9 in immunological homeostasis and pathogenesis. The abundance of ADAM9 transcripts in the blood was increased in patients with acute infection but changed very little after in vitro exposure to a wide range of pathogen-associated molecular patterns (PAMPs). Furthermore it was found to increase significantly in subjects as a result of tissue injury or tissue remodeling, in absence of infectious processes. Therefore this marker could potentially be used as a triage tool for patients presenting with symptoms of infection in the emergency room that may or may not require hospitalization

b) The second article reported the identification of blood molecular signatures that correlate with protection, or lack thereof, conferred by the RTS,S malaria vaccine [3]. This finding is important because this vaccine, which was licensed this year by European regulatory authorities, only protects about 40% of vaccinated individuals. Understanding the mechanisms that undermine the efficacy of this vaccine could lead to the development of a universally protective prophylactic modality for a disease that affects about 200 millions people and causes 500,000 deaths each year worldwide.

c) A third report that is being finalized investigates the role of Aquaporin 9 (AQP9), a water-selective membrane channel protein. This molecule is regulated during infection and appears to play a role in maintaining elevated metabolism associated with inflammatory responses. It may also inadvertently promote pathogen growth with adverse consequences in conditions such as pregnancy where elevated baseline metabolic states may contribute to enhance disease severity. Finally our observations also suggest a role for AQP9 in mediating pathogen clearance via phagocytosis.

Conclusions

The approach that we have devised recapitulates all the steps involved in the scientific discovery process, from data interpretation to knowledge dissemination. It allows screening, identification and prioritization of potential knowledge gaps, followed by in sillico validation and hypothesis generation and testing, finally resulting in preparation and publication of reports in a peer-reviewed journal. Its effectiveness as a training platform stems from the fact that it does not rely on de novo generation of data for discovery and validation. It leverages instead the vast amounts of available biomedical data, which will allow for accelerated and highly efficient hands-on training of aspiring biomedical researchers.

References

[1] Speake C, Presnell S, Domico K, Zeitner B, Bjork A, Anderson D et al. An interactive web application for the dissemination of human systems immunology data. J Transl Med. 2015;13:196. doi:10.1186/s12967-015-0541-x

[2] Rinchai D, Kewcharoenwong C, Kessler B et al. Abundance of ADAM9 transcripts increases in the blood in response to tissue damage [version 1; referees: 3 approved with reservations] F1000Research 2015, 4:89 (doi: 10.12688/f1000research.6241.1)

[3] Rinchai D, Presnell S and Chaussabel D. Blood Interferon Signatures Putatively Link Lack of Protection Conferred by the RTS,S Recombinant Malaria Vaccine to an Antigen-specific IgE Response [version 1; referees: awaiting peer review] F1000Research 2015, 4:919 (doi: 10.12688/f1000research.7093.1)

Understanding population level food consumption, which has considerable influence on population health, is a major challenge. For instance, obesity, which is particularly pressing in the Gulf region, is largely driven by changes in food consumption. Many other widespread diseases, such as diabetes, heart disease, and high blood pressure, are directly or indirectly affected by eating habits. Understanding food consumption generally involves surveys and self reporting which introduce certain biases, latency and substantial cost. Social media offers new possibilities to passively monitor and study eating behaviors, and track them in real-time across regions and time.

Predicting population level statistics (e.g. tracking seasonal epidemics like Flu) can be obtained through social media (e.g. shared tags and texts) which provides large scale, non-intrusive, and location-aware (regional) data in real time. Instagram is a hugely popular image sharing application, particularly in the Gulf region. Although users often annotate their social media posts with hashtags, a lot more information remains “hidden” in the actual image, requiring novel processing methods. Noting that “a picture is worth a thousand words”, we make use of this visual information through state-of-the-art deep learning models, particularly concentrated on food-related images.

We propose a method for tracking regional and temporal food habits though social media images. Concerning technical aspects, we have two major contributions: (a) learning visual concepts from social media images with extremely noisy labels, and (b) predicting regional statistics through visual information analysis.

The recent developments in scene and image categorization, particularly the advances in deep learning, show that with large quantities of data (i.e. big data) we can achieve great performance, approaching the level of human annotations. Here we show that even with extremely incomplete labels (i.e. only a limited set of tags exist for images taken from instagram) a robust auto-tagging system can be learned using deep convolutional neural networks, particularly with the help of millions of images. We mainly focus on food images and food related tags collected from instagram. We explored two major deep learning architectures for food label prediction: the Alexnet [1] (see Fig. 1) and VGG network[2].

We explored both training from scratch (i.e. learning the system only using instagram images), and finetuning an existing convolutional network (i.e. build upon an already trained system for object categorization using large-scale imagenet database). Although both models have very strong prediction capabilities, VGG network gives better predictions overall. Figure 1 shows some example images auto-tagged with food labels using our system.

We employ our food label predictor for tracking regional and temporal food habits. These population level statistics not only inform us on healthy eating habits but also provide us cues for predicting consequences of these behaviors such as conditions and diseases. We show that certain public health statistics (e.g. alcohol consumption) can be reliably predicted by analyzing social media images through our deep learning models.

The data collection and fusion is another major issue, particularly required for real-time analysis. We developed a system that can reliably identify all the geo-location tags (e.g. places that are used while tagging the location of the images - houses, cafes, etc.) by performing a large scale grid search using geographic tessellation models. We also developed a distributed system that can keep track of the shared images in the identified locations in real time. In depth analysis are performed over these collected images.

One of the potential outcomes of our project is a system that can visualize regional and temporal food habits which can be used as a tool for predicting the future habits and understanding main dynamics that affect the food consumption. The system can track the food habits across regions, cultures and time (e.g. daily, seasonal, yearly). For instance it will be possible to see how fast food is gaining or losing prominence compared to healthier food, and in which regions it happens more drastically. We can track Christmas dinner changes over time, or what different regions of the world ate for Christmas. We can display what type of dishes are mostly consumed in the month of Ramadan, and if food image sharing is changed during the day and the night. Many other potential applications can be build upon the proposed system.

References

[1] Krizhevsky, A., Sutskever, I., and Hinton, G. E. ImageNet classification with deep convolutional neural networks. In NIPS, pp. 1106–1114, 2012.

[2] K. Simonyan and A. Zisserman. Very deep convolutional networks for large-scale image recognition. CoRR, abs/1409.1556, 2014.

Background

Respiratory viruses have a predictable seasonality, which varies regionally. The reason for such seasonality is not well known yet, but atmospheric factors such as high humidity and temperature may assist virus survival in small particle droplets or aerosols, and on infected surfaces.

Objective

The goal of the study was to determine the seasonal variation in respiratory syncytial virus (RSV) infection in a desert climate.

Methods

A retrospective and cross sectional study was performed at Hamad Medical Corporation (HMC), the only tertiary and academic medical center in the State of Qatar. The study included infants and young children ages 0 to 24 months that were admitted to our pediatric ward with diagnosis of acute bronchiolitis from the period of January 2010 to December 2012. The following information were collected: gestational age, gender, respiratory virus real time polymerase chain reaction (RVRT-PCR) conducted on nasopharyngeal secretions, and hospital length of stay (LOS).

Results

835 infants and young children met the study criteria with mean age at diagnosis of 3.61 ± 3.56 months ranging from 0.33 to 24 months. RVRT-PCR was performed on 769 (92.0%) of the participants. RSV was positive in 352 (45.7%) children admitted with clinical bronchiolitis. In addition, no viruses were identified in 142 (18.4%), and respiratory viruses other RSV were found in 275 (35.7F%) of children. Our investigation shows that there has been a steady and periodic seasonal variation in the RSV rate over the study period. A seasonal trend for the RSV (detected by RVRT-PCR) rate was evident (Fig. 1), showing annual peaks in the months of October, November, December, and January, with a significant test for seasonality (test statistics [T] =  3.15, P = 0.009).

Conclusions

In countries with desert hot weather, bronchiolitis might affect infants and children throughout the year. Our results suggest that the combination of uninterrupted RSV seasonality can provide factual guidance for healthcare planning and application of RSV prevention scheme, such as extending the palivizumab vaccine series.

Figure 1: Sequence chart for RSV rate of infection during various months in children admitted with acute clinical bronchiolitis

Summary

In this paper, we present a novel interpolation-based Stokes image reconstruction scheme for the division-of-focal-plane (DoFP) polarization image sensors. Different from the previous implementations, our proposed method first demosaics the raw image by mainstream interpolation algorithms then converts the up-sampled images to Stokes images with much richer polarization-related physical information. This not only leads to significant resolution improvement to the captured raw image, but also greatly reduces the caused pixel mean square error (MSE). Experimental data from the test images have validated the effectiveness of this proposed scheme.

Motivation

Solid-state image sensors, which are capable of extracting the incident light's polarization information in addition to intensity and color (i.e. wavelength), take great advantages in a wide range of applications [1]. By looking through a layer of patterned micrometer-scale pixelated polarizing elements, a set of mosaicked polarization raw sub-images can be generated simultaneously, namely DoFP polarization imaging. As shown in Fig. 1 (a), similar to the widely-exploited Bayer pattern of color imaging, the mosaicked polarization raw sub-images down-sample each polarization channel by 75%, leading to significant spatial resolution loss. Meanwhile, in order to make the raw sub-images physically meaningful, they are typically translated to Stokes sub-images, which correspond to first three Stokes parameters representing the unpolarized and linearly polarized components of the incident light. In the previously reported implementations, the neighboring four sub-pixels (i.e. I0, I90, I45, I135) are directly substituted to the Stokes parameters' classical expressions. As a result, a 75% down-sampled Stokes images are acquired. In order to compensate this resolution loss, we propose a new Stokes image reconstruction scheme: before the Stokes image conversion, interpolate the mosaicked polarization raw sub-images with mainstream algorithms, including linear, cubic and spline.

Results

Figure 1 (c) illustrates the proposed detailed image reconstruction flow. In addition, we have also compared our proposed method to the traditional implementation of directly applying the same interpolation algorithm to the aforesaid 75% down-sampled Stokes images [Fig. 1 (b)]. For the extracted down-sampled Stokes images in the previous implementations, even with the same interpolation algorithms applied, our proposed method still outperforms by almost 10 folds in term of Stokes parameter S2's MSE (Fig. 2), which well-balances the spatial resolution compensation and the Stokes image generation by minimizing the caused overall MSE. Figure 3 illustrates the real images with different Stokes image reconstruction schemes applied.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grant No. 61504087), the Kongque Technology Innovation Foundation of Shenzhen (Grant No. KQCX20120807153227588), the Fundamental Research Foundation of Shenzhen (Grant No. JCYJ20140418095735624, and JCYJ20150324141711677).

Reference

[1] M. Kulkarni, V. Gruev, “Integrated spectral-polarization imaging sensor with aluminum nanowire polarization filters”, Optics Express, vol. 20, no. 21, pp. 22997–23012, 2012.

1. Background

Stroke, i.e. loss of brain function(s) due to disturbance in the blood supply to the brain, is the main cause of adult disability (e.g. paralysis) in the world, leaving more than half of the patients dependent on daily assistance. In Qatar, stroke is a major health problem with an estimated incidence of 238/100,000 per year for the population over 45 years old [1]. Stroke patients are often hospitalized and/or subjected to intensive rehabilitation programs for long periods of time, and their quality of life is severely affected socially and economically. Around 10% of the hospital beds in Qatar are occupied by stroke patients [1]. Thus, without major advances to improve prevention, treatment and rehabilitation of stroke, the social and economic costs of this disease will increase dramatically.

There are pathological and physiological changes on the cellular and molecular levels associated with stroke. The objective of this work is to determine the molecular and structural changes occurring in the tissue of rat's brain. Vibrational spectroscopy, i.e. Fourier transform infrared (FTIR) imaging spectroscopy, was used as rapid and objective diagnostic platform to investigate the pathological and pathological changes in the rat's brain sections three weeks after stroke. FTIR spectroscopy was also used to differentiate between the biochemical makeup of the white and grey matters of a healthy control brain samples. Also, in the current study, scanning electron (SEM), energy dispersive X-ray spectroscopy (EDX), and atomic force microscopic (AFM) techniques were assessed to study the structural changes in the rat's brain tissues after experiencing an induced stroke.

2. Experimental

2.1. Sample preparation

Rats were anesthetized using 2–3% isoflurane. Experimental stroke was induced in rats by 90-min occlusion of the right middle cerebral artery with an intraluminal filament. Rats were euthanized with a lethal dose of sodium pentobarbital and transcardially perfused with 4% paraformaldehyde. Rat's brains were extracted, embedded in paraffin and then serially sliced, using semi-automated rotary microtome, into 5 μm thickness sections for the FTIR imaging and AFM analysis and 35 μm thickness for the SEM and EDX analysis. The brain sections were mounted on MirrIR CFR, Low-e microscope slides for the FTIR imaging analysis, and on aluminum metal for the SEM analysis and EDX analysis. The paraffin was removed from the samples by using xylene and isopropanol.

2.2. Instrumentation

2.2.1. FTIR Imaging Measurements

The FTIR images were obtained using FTIR spectrometer (Agilent Technology) at a reflection mode within the range of 4000–700 cm^–1. Spectral images were analyzed using Metlab software (The Mathworks Inc.). Origin 2015 software was used for graph drawing. Principal component analysis (PCA) was performed to study the spectral data variations between the FTIR spectra and images.

2.2.2. Scanning Electron Microscopy (SEM)

Rat's brain sections of 35 μm thickness were mounted on aluminum slides for SEM analysis. All the samples were viewed with a FEI Quanta 200, USA scanning electron microscope at 10 kV. SEM micrographs of the brain stroke and healthy rat's sections were compared. Elemental distribution in both healthy and induced stroke brain sections were investigated by using energy dispersive X-ray spectroscopy (EDX) equipped with SEM. The spectra provided a semi-quantitative view of the elemental composition of both weight and atomic percent.

2.2.3. Atomic Force Microscopy (AFM)

Bruker atomic force microscopy (AFM) was used for imaging and quantitatively determining the local elastic properties of healthy and induced stroke rat's brain sections. A controllable and constant force was applied at each data point and using the resulting force-distant curve for the formation the AFM images. Brain sections were scanned at 10 μm by 10 μm. About 100 force-distance curve were collected for each healthy and induced stroke brain sections and two random scan lines of force-distance curves was recorded.

3. Results and Discussions

The FTIR spectroscopy results indicated that the white matter is richer in lipid content than the grey matter as shown in Figs. 1 and 2. The infrared spectrum images showed a decrease in the lipid content of the white matter associated with the induced stroke brain sections. FTIR bands assigned to the bio-chemical makeup such as proteins, lipids and ester varied in positions, line-shape, and intensity between control and induced stroke brain samples. The spectral images showed that there is a configuration changes is associated with the lipid bands in the rat's brain white matter that experienced stroke.

The FTIR spectral images of the white matter in the induced stroke brain sections indicated that amide I and ester bands experienced a bio-chemical changes as shown in Fig. 3 and 4. Figure 5 shows the second derivative of the collected FTIR spectra from induced stroke brain sections. In Fig. 5, there are spectral differences that assigned to ester and protein regions. Figure 6a represents the loading spectra of the first three principal component analysis (PC1, PC2 and PC3). The variations principally were located in the regions of amide I band at (∼1695-1637 cm^–1) and small variation in the amide II band at (1543 cm^–1). Figure 6b represents the loading spectra of the PC4, PC5 and PC6. The variations principally were located in the protein region, mainly amide I band at (∼1695-1637 cm^–1) and ester band at about 1730 cm^–1. The use of FTIR imaging and chemometric analyses such as principal component analysis (PCA) of spectral data allows to investigate and differentiate spectral images pattern collected from control and stroke rat's brain samples.

The scanning electron microscope results showed that lesion region in the induced stroke brain sections are enriched by the selected elements such as Fe and Ca as shown in Fig. 7 (a & b). Scanning electron microscope (SEM) micrographs indicated that there is structure change in the induced stroke brain section. The structure of stroked brain sample in the nanometer scale appeared to be significantly rough compared to the control brain sample (Fig. 8 a & b).

Atomic force microscope (AFM) images showed that the stroke brain section is swollen compared to healthy brain sections. The AFM images of the induced stroke brain sections appeared more stretched when compared to the control brain section image as shown in Fig. 9 (a & b). AFM results also showed that the force-distance curves in Fig. 10, recorded using control (healthy) brain sections (blue) and induced stroke brain sections (red). The force-distance showed that the AFM cantilelver deflection of the healthy brain samples is higher than the induced stroke brain section. This indicate that the healthy brain section are softer and elastic than the induced stroke brain sections.

4. Conclusion

FTIR imaging spectroscopy, scanning electron and atomic force microscopy techniques were able to analyze and differentiate between the healthy and induced stroke rat's brain sections on the molecular, structural and global levels making them valuable tools to investigate, diagnose and study the structural plasticity of the stroke induced brain. FTIR imaging spectroscopy in combination with multivariate analysis such as principal component analysis (PCA) is a non-destructive technique that proves to be rapid, accurate and straightforward to be performed. It constitutes a powerful approach to be used as a medical diagnosis tool to investigate the pathological changes associated with stroke in the brain tissues.

Keywords

Fourier Transform Infrared (FTIR) imaging spectroscopy, Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), Brain tissue, Stroke, Chemometric Analysis

Acknowledgments

This article was made possible by a NPRP award [5 - 381 - 3 - 10] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors. We would like to acknowledge the Center for Advanced Materials at Qatar University for performing the AFM analysis, and Central Laboratories Unit at Qatar University for performing the SEM and EDX analysis.

Reference

[1] Hamad, A., Sokrab, T.E., Momeni, S., Mesraoua, B., and Lingren, A., Stroke in Qatar: a one-year, hospital-based study. J Stroke Cerebrovasc Dis, 2001. 10(5): p. 236–41.

Thymidylate kinases (TMKs) play a central role in the production of nucleotide precursors that are required for the replication of DNA. Consequently, this enzyme is a potential drug target for the discovery of anti-bacterial, anti-fungal, and anti-parasitic drugs. In addition, TMKs are also involved in the activation of prodrugs. In particular, the anti-HIV drug AZT is activated by human TMK (huTMK) and the low efficiency of huTMK towards AZT is a significant problem in the use of AZT in the treatment of HIV. Finally, nucleotide precursors are required in large amounts by cancerous cells, thus the inhibition of huTMK by chemotherapeutic agents may enhanced the arsenal of drugs that are used to treat cancer.

Although there has been some effort to develop inhibitors of TMKs, these efforts have been hampered by the difficulty in performing high throughput screening using compound libraries. In addition, the characterization of TMK-drug complexes has been limited to X-ray diffraction studies which provide static information about the enzyme-drug complex. There have been no attempts to apply high-resolution multi-nuclear NMR techniques to determine the fundamental dynamic properties of these enzymes and how the structure and dynamics of the enzyme are altered by the binding of substrates or inhibitors.

As a preliminary step in characterizing these enzymes by NMR we have over-expressed TMKs from yeast, human, and two pathogens - Plasmodium falciparum and Candida albicans. Expression of these TMKs was optimized by the design of synthetic genes for expression in bacteria. In the case of the human enzyme, we are able to routinely produce 250 mg of the enzyme/L of culture. Preliminary NMR spectra of the yeast, human, and plasmodium enzyme show that the protein is a homo-dimer in solution, as anticipated from X-ray studies. The amide and methyl spectra are well resolved, indicating that resonance assignment by traditional TROSY based methods will be feasible for both the amides and the methyl resonances. In particular the high sensitivity and dispersion of the methyl spectra will facilitate characterization of the dynamic properties of these enzymes by carbon and deuterium relaxation. Ligand induced changes in the dynamics and structure of huTMK in solution will be characterized using NMR methods. These studies will provide additional insights into the inability to huTMK to effectively activate AZT. The entropic component of the thermodynamics of substrate binding to TMK from the parasite that causes malaria will also be characterized by determining dynamic changes by NMR methods.

The development of NMR methods to study these enzymes also provides a method for high throughput screening of compound libraries by detecting chemical shift changes in the NMR spectral of the enzyme due to binding of a potential lead compound.

Background

Lung cancer is the second most common cancer in both men and women and it is the leading cause of cancer deaths worldwide. Lung cancer can be divided into two broad categories: non-small cell lung cancer (NSCLC), which consists of about 85% of all lung cancers and small cell lung cancer (SCLC), which account for 15% of all lung cancers. The evolution of lung cancer is a multistep process involving genetic and epigenetic alterations. Standard treatment therapies such as radiation therapy, chemotherapy and surgery has reached a plateau phase. As a result, much work has centered on identifying the molecular targets involved in the tumor cell proliferation, survival and metastasis in effort to identify novel therapeutic approaches. p90 ribosomal S6 kinase (p90RSK) constitutes a family of serine/threonine kinases that have been shown to be involved in cell proliferation of various malignancies via direct or indirect effects on the cell-cycle machinery.

Objectives

To investigate the role of p90RSK in lung adenocarcinomas and whether the inhibition of p90RSK diminishes cancer progression. Moreover, we investigated the involvement of glycogen synthase kinase-3β (GSK-3β) and osteopontin (OPN) in the p90 RSK induced lung adenocarcinoma progression.

Methods

p90RSK, OPN, GSK-3β protein expression were examined in the A549 human lung adenocarcinoma cell line in the presence and absence of BI-D1870 (BID), a p90RSK inhibitor. Gene expression of anti-apoptotic and pro-apoptotic markers namely Bcl2 and Bax, respectively, were studied by reverse transcription polymerase chain reaction. In addition, the A549 lung adenocarcinoma cell line was characterized for cell proliferation using the MTT assay and cell migration using the scratch migration assay.

Results

Our study revealed that the treatment of the A549 lung adenocarcinoma cell line with BID resulted in a significant reduction in protein expression of p90RSK 1 (69.32 ±  12.41% of control; P <  0.05). The inhibition of p90RSK also showed a significant suppression of cell proliferation (54.3 ±  6.73% of control; P

Breast cancer is the most common type of tumor in women in the MENA region and it represents about 20% of the cancers diagnosed every year in Qatar. Although the implementation of cancer therapy has led to an improvement of patients' survival, metastatic breast cancer remains an incurable condition. Immunotherapy is emerging as an innovative therapeutic tool able to cure in some cases established metastatic tumors such as melanoma. There is a growing interest in exploring this fascinating approach in breast cancer. However, little is known about the immune biology of this aggressive disease. Studies from our and other groups have described intratumoral immune gene signatures associated with better responsiveness to immunotherapy and prolonged survival (Galon, Angell, Bedognetti and Marincola, Immunity 2013; PMID: 23890060). In general, these gene signatures imply the activation of interferon stimulated genes (e.g. IRF1 and STAT1), the recruitment of lymphocytes through CXCR3/CCR5 ligand chemokines, and the activation of immune-effector functions such as PRF1 and GZM1 (Wang, Bedognetti, Marincola, JCO 2013; PMID: 23715576). We refer to these genes as the Immunologic-Constant-of-Rejection (ICR) (Bedognetti, Wang, Sertoli, Marincola, Exp Rev Vacc, 2010; PMID: 20518712). The activation of the ICR pathways is accompanied by the counter-activation of immune-regulatory mechanisms such as the expression of PD-L1 and IDO1. Based on these observations, we hypothesize that tumors can be divided in two opposite phenotypes (Bedognetti, Hendrickx, Marincola, Miller, Curr Opin Onc 2015; PMID: 26418235). The first phenotype displays the activation of pro-inflammatory (ICR) and immune-regulatory mechanisms and is characterized by a favorable prognosis and responsiveness to immune manipulations. The second phenotype lacks these two characteristics and is associated by a poor prognosis and resistance to immune manipulations. Whether the development of such different immune phenotypes is influenced by the intrinsic genetics of the tumor cells is presently unclear. The understanding of genetic mechanisms associated with differential immune response can lead the development of targeted approaches. To answer this critical question we analyzed copy number variation, exome and RNA sequencing data from the The Cancer Genome Atlas (TCGA) breast cancer dataset. By using consensus clustering analysis based on RNA-seq data of more than 1000 breast cancer samples, we defined four immunophenotypes characterized by progressive expression of the ICR genes (ICR1