Oral Presentation Abstracts

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Oral Presentation Abstracts

If the presenting author is not the corresponding author, the email of the corresponding author will appear.

Paper reference: Paper 1.A The Ethics and Regulation of Human Genetic Screening Dr Peter R Brinsden MB BS FRCOG, Consultant Medical Director, Bourn Hall Clinic, Cambridge, UK ([email protected])

Abstract Ever since the first creation of human embryos in vitro and the birth of the World's first IVF baby in 1988, the assisted reproductive technologies (ARTs), originally developed to help infertile couples to have children, have since evolved to include techniques that allow the diagnosis of genetic diseases at the early embryonic stage of life. It is now possible to screen polar bodies, single blastomeres or trophectoderm cells from blastocysts for single or multiple gene defects. Screening for hundreds of defects is now practised routinely in many centres worldwide, including for such common disorders as beta-thalassemia and breast cancer predisposition to much more rare conditions like amyotrophic lateral sclerosis (ALS). Our first priority as ART specialists must be consideration of the welfare of any future child we hope to help an infertile couple to have. This includes not helping a couple to have the much wanted child which might have a high risk of a serious inherited abnormality and therefore also a high risk of having a severely diminuished quality of life as a child and adult. In achieving this aim, ART specialists, geneticists and genetic counsellors must be able and willing to cooperate freely and to use the technology ethically. We are now able to create 'saviour siblings' using PGD for HLA typing to create a child who may be able to assist his/her existing sibling who has a life threatening disorder. Severe disorders caused by mitochondrial DNA defects can be 'cured' by mtDNA transfer techniques currently available and being debated. Other related advances - many of them controversial - that have evolved from this technology include cloning (both reproductive and therapeutic), whole genome sequencing, gene therapy and stem cell therapy.

It is possible that in the future, as the technology becomes more available and cheaper, that every IVF embryo is screened by analysis of its whole genome before transfer to the uterus. However, problems will arise when conditions are detected that may be serious or lethal, but may not arise until adulthood. Whether or not that embryo should be transferred is controversial. There are many other issues that arise out of these technologies that will be reviewed in this presentation.

As important to the scientific mastering of these treatments and techniques is the careful consideration of the legal and particularly the ethical issues arising from these current available and potential future treatments. In this paper, the ethical and legal/regulatory issues of many of the most controversial and critical of these issues will be discussed, with a look at what the future might hold for the benefit of mankind.

Paper reference: Paper 1.1 Identification of Known and Novel Variants Associated with Paediatric Disorders using Whole Exome Sequencing and Array-CGH Dr. Arif Anwar, Sengenics, Malaysia ([email protected]) Abstract Here we present a novel pipeline for identification of clinically significant mutations using Array-Comparative Genomic Hybridization (a-CGH) combined with Whole Human Exome Sequencing. For the Exome Sequencing, variants in exons of more than 20,000 genes were screened in nearly 1000 samples. A proprietary methodology was developed to filter, optimize and categorize variants. Variants were filtered based on empirical rules including novelty, zygosity, effect on protein structure and pathway related attributes. A proprietary reference database specific to the Arab population was used to classify variants linked to more than 300 different diseases. Regions of loss of heterozygosity (LOH) were identified using a previously published method. Samples from parents and affected/unaffected siblings were sequenced for familial aggregation analysis to finalise diagnosis. Furthermore, structural variations such as genomic deletions and amplifications detected using a-CGH were verified using MLPA. Identification of clinically significant mutations using this comprehensive strategy resulted in a significant increase in diagnostic yield. Paper reference: Paper 1.2 From point-of-care to genomics research: how a new research centre will unleash the full potential of personalised medicine in rare diseases Dr. Joana Lamego (Lamego J), Instituto de Medicina Molecular (IMM), Portugal ([email protected]) Dr. Maria Carmo-Fonseca, Instituto de Medicina Molecular(IMM), Portugal Abstract Rare diseases (RD) are socially, medical, economic, and scientifically challenging. According to the latest statistics there are currently 7,000 types of RD affecting 350 million people in the world. Europe and USA have each 30 million people affected by RD. In the Middle East the latest estimations point to the existence of 2.8 million patients suffering from a RD in the population. Proper diagnosis and effective treatment of pa tients affected by rare diseases are rare. Personalised medicine is the best approach to deal with RD as it has the potential of allowing designing targeted therapies with increased probabilities of success based on the genomics of each individual and their affected and healthy familiars as well as paving the way for the discovery of biomarkers that may change the currently available unsatisfactory diagnostic tools. Through the joint efforts of a renowned biomedical research institute affiliated to the University of Lisbon Instituto de Medicina Molecular (IMM) and the Portuguese patient advocacy group for mental and rare deficiencies (Raríssimas) a new centre

for the study of rare diseases, CDRare, has been created aiming to become an international reference centre for knowledge creation on rare genetic disease disorders and its translation to the benefit of the patients, their families, and the society in general. CDRare resorts to state of the art scientific methods for the diagnosis prevention and the development of new options for the treatment of rare diseases such as whole exome sequencing, preimplantation genetic diagnosis and CRISPR technology. We will present the strategic path thrilled in the establishment of CDRare and the latest scientific results achieved by the diagnosis, prevention, and new treatment development units of the Centre. We will also present and discuss the advantages of potential win-win partnerships with Middle East stakeholders. Background Rare diseases (RD) are socially, medical, economic, and scientifically challenging According to the latest statistics there are currently 7,000 types of RD affecting 350 million people in the world. Europe and USA have each 30 million people affected by RD. In the Middle East the latest estimations point to the existence of 2.8 million patients suffering from a RD in the population. Proper diagnosis and effective treatment of patients affected by RD are rare. International collaboration in rare diseases may allow minimising the inherent difficulties in their study. Materials and Methods The Centre for Diagnosis and Research on Rare Diseases resorts to state of the art scientific methods for the diagnosis prevention and the development of new options for the treatment of RD Diagnosis is currently performed through whole exome sequencing (NextSeq Illumina) of patients and their affected and healthy familiars in a universe of 800 thousand Portuguese people affected by RD. Prevention is achieved through preimplantation genetic diagnosis (PGD). New options for the treatment of RD are being researched through the application of CRISPR technology. Results Instituto de Medicina Molecular (IMM) and the national patient advocacy group for mental rare deficiencies (Raríssimas) have joined efforts through an unprecedented effort of knowledge transfer to the society which translates in several achievements: an already established not for profit research centre, CDRare; successful engagement with international leading organisations; fundraising efforts that have allowed securing the needed funding; international projection of the Centre with a dedicated Scientific Officer dedicated to the search and establishment of partnerships worldwide. Conclusion Through the joint efforts of a renowned biomedical research institute Instituto de Medicina Molecular (IMM) and the national patient advocacy group for mental and rare deficiencies (Raríssimas) a new centre for the study of rare diseases, CDRare, has been envisioned and created. The road ahead involves the strengthening of the already established scientific collaborations as well as the inception of new partnerships with different stakeholders worldwide for the share of knowledge and the development of innovative projects.

Paper reference: Paper 1.3 Rapid diagnosis for unexplained anaemia using targeted massively parallel sequencing. *Dr. Noemi Roy (Noemi BA Roy), Weatherall Institute of Molecular Medicine, UK([email protected]). Dr. Chris Babbs, WIMM, UK Dr. Juliana Teo, Department of Haematology, Sydney Children’s Hospitals Network, Westmead, Australia, UK Dr. Julie Curtin, Department of Haematology, Sydney Children’s Hospitals Network, Westmead, Australia, UK Dr. Wale Atoyebi, Oxford University Hospitals NHS Trust, UK Dr. Deborah Hay, WIMM, UK Dr. Jennifer Eglinton, Oxford University Hospitals NHS Trust, UK Dr. Georgina Hall, Department of Paediatrics, Oxford University Hospitals NHS Trust , UK Dr. Veronica Buckle, WIMM, UK Dr. Irene Roberts, WIMM, UK Dr. David Roberts, NHS Blood and Transplant, NHSBT, UK Dr. Doug Higgs, WIMM, UK Dr. Shirley Henderson, Oxford University Hospitals NHS Trust, UK Dr. Anna Schuch, Oxford University Hospitals NHS Trust, UK Abstract Clinicians faced with unexplained congenital anaemia need to refer samples to multiple centres for investigations since specialist centres focus on the genes for particular subtypes of anaemia leading to delays in diagnosis and increased costs. Targeted next - generation resequencing ( NGS ) obviates these problems offering clinicians a ' onestop ' test screening rare and common mutations causing congenital anaemias ( Diamond - Blackfan anaemia dyskeratosis congenita Schwachman - Diamond syndrome sideroblastic anaemia congenital dyserythropoietic anaemia ( CDA ) and the enzyme deficiencies G6PD, PKLR and 5 ' pyrimidine nucleotidase. This panel covers 33 genes ( target region ~ 118kb ). Sequence capture and library preparation is carried out using TSCA ( Illumina ) and sequencing is performed using an Illumina MiSeq. Variant calling and filtering is carried out using Basespace Custom amplicon workflow and Variant Studio for annotation and filtering. Coverage is excellent with > 95% of known mutations covered by > 30 reads. Thirty mutations in known positive controls were correctly identified by the panel and validated by Sanger sequencing One SNP identified by Sanger sequencing was not detected. Validation determined a 100% specificity and 99.7% sensitivity. 57 diagnostic samples were analysed. Overall, a diagnosis was made in 33% of the cases, although this varied by phenotype (DBA 7/9, enzyme deficiency 1/1, CDA 6/10, sideroblastic anaemia 1/4, “unexplained anaemia” 4/33). We also detected novel mutations in RPL5 and CDAN1. Targeted NGS for congenital

anaemia using a bespoke panel of genes offers clinicians a fully validated diagnostic test for unexplained congenital anaemias. Background Clinicians faced with unexplained congenital anaemia need to refer samples to multiple centres for investigations since specialist centres focus on the genes for particular subtypes of anaemia leading to delays in diagnosis and increased costs. Targeted next - generation resequencing ( NGS ) obviates these problems offering clinicians a ' onestop ' test screening rare and common mutations causing congenital anaemias ( Diamond - Blackfan anaemia dyskeratosis congenita Schwachman - Diamond syndrome sideroblastic anaemia congenital dyserythropoietic anaemia ( CDA ) and the enzyme deficiencies G6PD PKLR 5' pyrimidine - nucleotidase Materials and Methods This panel covers 33 genes ( target region ~ 118kb ). Sequence capture and library preparation is carried out using TSCA ( Illumina ) and sequencing is performed using an Illumina MiSeq. Variant calling and filtering is carried out using Basespace Custom amplicon workflow and Variant Studio for annotation and filtering. Coverage is excellent with > 95% of known mutations covered by > 30 reads. Thirty mutations in known positive controls were correctly identified by the panel and validated by Sanger sequencing. One SNP identified by Sanger sequencing was not detected. Results The process, machine, pipeline and assay were further verified using the Illumina Infinium Human OmniExpress Exome v1.2 beadchips. Comparing the gVCF data with the microarray results showed 100% concordance of the calls. Combining the data from Sanger sequencing and microarray yields a 100% specificity and a 99.7% sensitivity. 57 diagnostic samples were analysed. Overall, a diagnosis was made in 33% of the cases, although this varied by phenotype (DBA 7/9, enzyme deficiency 1/1, CDA 6/10, sideroblastic anaemia 1/4, “unexplained anaemia” 4/33). We also detected novel mutations in RPL5 and CDAN1. Conclusion Targeted NGS for congenital anaemia using a bespoke panel of genes offers clinicians a fully validated diagnostic test for unexplained congenital anaemias. Patients found not to have mutations in the genes covered on the panel are considered for further genetic analysis such as exome sequencing on a research basis, with the aim of identifying novel genes involved in erythropoiesis Paper reference: Paper 1.4 New Solutions for Genetic Testing of Embryos *Dr. Alan R Thornhill, Illumina, UK ([email protected]) Abstract This presentation will describe the gold standard (array CGH – 24sure) and latest methods (including the Veriseq-PGS Next Generation Sequencing solution) for investigating aneuploidy in human embryos (preimplantation genetic screening - PGS) as well as a new, comprehensive test, karyomapping for detecting single gene disorders in embryos (preimplantation genetic

diagnosis - PGD) which utilises single nucleotide polymorphisms as markers to provide robust, reliable and highly accurate genome wide linkage analysis. Background Testing embryos to avoid genetic disease is not a new idea, but has roots in decades of previous work. Bob Edwards, who won the Nobel Prize for his work on in vitro fertilization (IVF), sexed rabbit embryos in the 1960s. The first clinical case of preimplantation genetic diagnosis (PGD) to avoid sex-linked disease was performed in 1989. This presentation will focus on comprehensive methodologies to (i) test for single gene disorders and (ii) screen for all chromosomes simultaneously. Materials and Methods Methods include ( i ) karyomapping ( a comprehensive PGD test utilizing the power of SNPs across the genome ) for the detection of single gene disorders in single embryonic cells using a linkage - based approach ( ii ) a commercially available array comparative genomic hybridization ( aCGH ) solution - sure which identifies chromosome gain loss in human embryonic samples for all chromosomes and ( iii ) Veriseq - PGS – a new commercially available solution for chromosome screening in embryos using Next Generation Sequencing technology Results With at least 50 SNP markers present in each region of interest, karyomapping is highly concordant with the current gold standard test for PGD and should deliver a more robust, reliable, and accurate test. 24sure is the ‘gold-standard’ for aneuploidy detection in human embryos with > 400,000 clinical samples analysed. RCTs demonstrated improved IVF outcomes with 24sure and further RCTs are ongoing. Extensive validation demonstrates the Next Generation Sequencing solution (VeriSeq-PGS) is highly concordant with 24sure. Conclusion Karyomapping is a comprehensive, off-the-shelf test for detecting inherited single-gene disorders in embryos. By leveraging the power of ~280,000 genome-wide single nucleotide polymorphisms, karyomapping should provide greater accuracy for PGD than current linkagebased tests with reduced patient waiting times. While 24Sure can improve IVF outcomes, the transition to sequencing based methods (eg VeriSeq PGS) will ultimately improve accuracy and sensitivity, (dynamic range), testing flexibility (inclusion of targeted sequences) and cost savings through multiplexing Paper reference: Paper 2.A Recent Advances in Hematological Malignancies * Maher A. Sughayer M.D., FCAP, King Hussein Cancer Center, Amman, Jordan ([email protected]). Abstract Among the most recent and exciting development in hematological malignancies is that which was seen in the field of the myeloproliferative neoplasms (MPN). The Philadelphia-negative myeloproliferative neoplasms (Ph-negative MPN) polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are characterized by the presence of a primary genetic lesion which is a gain-of-function mutation in the gene encoding a tyrosine

kinase, Janus kinase 2 (JAK2 ), the first signaling element common to several hematopoietic growth factor receptors. Most frequently these mutations are in the region of the gene encoding for the tyrosine kinase domain of the protein (JAK2V617F and JAK2 exon 12 mutations). The JAK2V617F mutation is present in almost all PV, around 50% of ET, and around 65% of PMF. Other less common mutations are found in the myeloproliferative leukaemia virus oncogene (MPL), LNK, TET2, ASXL1, EZH2, IDH1/2, CBL, IKZF1, which have been observed in 3-20% of MPN patients. However, just recently a novel and exciting finding was reported by 2 separate groups; Klampfl et al. and Nangalia et al. who used exome sequencing identified novel mutations in exon 9 of calreticulin (CALR) gene in the majority of ET and PMF patients who did not harbour JAK2 or MPL mutations. CALR mutations were not found in healthy controls, lymphoid neoplasia, acute leukaemias, or solid tumours, indicating their specificity for ET and PMF. The mutations were either insertions or deletions. The commonest (80-90%) of which were a 52 bp deletion (CALRdel52) and a 5 bp insertion (CALRins5). All of them encode for a mutant protein with a novel C-terminus lacking the KDEL signal localisation domain. The CALR gene is located on chromosome 19p13.2 and contains nine exons. The CALR mutated MPN appear to be a separate entity of MPN with a better prognosis.

Paper reference: Paper 2.1 Study of the Association of Genetic Polymorphisms in Biotransformation Genes (CYP1A1, GST, EPHX1) and Tumor-Suppressor Gene (p53 codon72) with the Susceptibility to Adult Acute Myeloid Leukemia *Dr. Abhishek Mittal, Faculty Of Medical Sciences,Delhi, India, India ([email protected]) Abstract INTRODUCTION Acute myeloid leukemia (AML) is the most common acute leukemia in adults with median incidence of 2.4 cases per 100,000 individuals. Biotransformation plays a crucial role in carcinogenic activity and many genetic polymorphisms in xenobiotic metabolizing enzymes have been associated with an increased risk of developing AML . Tumour Suppressor Gene acts as protective gene as it controls and monitor the cell growth and apoptosis. Genetic polymorphisms in drug metabolizing enzymes are extremely common but very few studies have investigated influence of GSTM1 and GSTT1-null polymorphisms , EPHX1and CYP polymorphisms on risk of developing adult AML. MATERIAL & METHODS The study was conducted on 70 patients (range 18-85 with median age 32 years). Primers for PCR were designed and standardized using gradient PCR and by taking annealing temperature ± 5°C of the Tm of primers. PCR and RFLP were used to study

polymorphisms in Biotransformation Genes and Tumor-Suppressor Gene. All statistical analyses were performed with SPSS. RESULT Frequency distribution of GSTM1 null genotype was 33% and 49% whereas 19% and 30% for GSTT1 null genotype in AML patients and controls respectively. GSTT1 null genotype conferred marginally significant 2.0 fold reduction in risk of AML relative to presence of the GSTM1 gene (OR=0.50, 95%CI: 0.25-0.98, p= 0.06). Frequency of EPHX1 Exon 3 and exon 4 variant genotypes were marginally different in cases versus controls. Protective effect of the Tyr113His genotype of EPHX1 gene against AML lacked a statistical significance. Frequency of p53 codon 72 polymorphism for Arg/Arg, Arg/Pro and Pro/Pro genotype was 31%,50%,19% respectively in cases while 25%, 57%, 18% respectively in controls. CONCLUSION Present study indicates lack of association of EPHX1, CYP1A1 and p53 with risk of adult AML. On the other hand GSTT1 deletion seems to play protective role.

Paper reference: Paper 2.2 Characterization and prevalence determination of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification (MLPA) in the North Moroccan population. *Dr. Achraf LAGHMICH, Faculty of Sciences and Techniques of Tangier, Morocco ([email protected]) Dr. Mohcine BENNANI MECHITA, Faculty of Sciences and Techniques of Tangier, Morocco Abstract Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. It is a hemolytic anemia with an autosomal recessive transmission. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. The exact determination of gene defect for thalassemia carriers is essential for planning future interventions. In this study, we conducted a molecular study of those suspected of carrying alpha-thalassemia mutated genes in order to detect potential deletional mutations in the alpha globin gene cluster. The multiplex ligation-dependent probe amplification (MLPA, P140B HBA kit) method was used to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 84 suspected individuals (29 males and 55 females; age: 4 months to 56 years). These individuals were selected from those referred to public hospitals in the north of Morocco with MCV < 80 fl, MCH < 27 pg, normal serum iron, and HbA2. Alpha globin gene deletions are detected in four patients: Two cases with the heterozygote form of -α3.7 deletion (-α3.7/αα), one case is homozygote for -α3.7 deletion (-α3.7/-α3.7) and the fourth one revealed the African polymorphism (G-->TCGGCCC at position 7238 and T-->G at 7174). The prevalence of alpha-thalassemia in the studied population is estimated to 1.23% with a predominance of the -α3.7 deletion.

Paper reference: 2.3

Anti-cancer, Anti-inflammatory and Anti-microbial activities of plant extracts used against hematological tumors in traditional medicine of Jordan. Dr. Areej M Assaf (Assaf A.M.), The University of Jordan, UAE, [email protected] Abstract Ethnopharmacological relevance Mercurialis annua L Bongardia chrysogonum L and Viscum cruciatum Sieb have been traditionally used by local herbalists in Jordan for the treatment of hematopoietic neoplasms Aim of the study To determine the anti - cancer anti - inflammatory and anti - microbial potentials of the three extracts against two of the most common hematopoietic malignancies in the Jordanian populations Burkitt ’ s lymphoma and Multiple myeloma Materials and methods The anti - cancer activity was tested against the two cell lines ( BJAB Burkitt ’ s lymphoma and U266 Multiple myeloma ) using the MTT and trypan blue assays The agar dilution assay was used to study the anti - microbial activity against Gram - positive bacteria Gram - negative bacteria anaerobic bacteria and yeast The pro - inflammatory cytokines interleukin ( IL ) - β, IL - and tumor necrosis factor -α ( TNF -α) were measured in the pretreated cell lines using ELISA assay to determine the anti - inflammatory activity of Viscum cruciatum Sieb against the two cell lines Results The results show no evidence of stimulation of tumor growth by any of the three extracts comprising cell lines from hematological malignancies but Viscum cruciatum Sieb showed a selective anticancer activity against BJAB cells with IC50 value of μ g mL The antimicrobial effect was only noticed with Viscum cruciatum extract by inhibiting S aureus C albicans and P acne but not P aeruginosa at MIC of and < mg mL respectively The highest activity was against the anaerobic bacteria Propionibacterium acne Viscum cruciatum Sieb extract showed an inhibitory effect on the pro - inflammatory cytokine IL - but it increased TNF -α and IL - β secretions in BJAB cells Whereas it had an inhibitory effect on TNF -α and IL - β cytokines while it

Paper reference: Paper 3.2 Identification of novel-melanoma associated microRNAs from transcriptomics of fish tumor models *Dr. Rasmi Rekha Mishra (Mishra RR), University of Wuerzburg, Germany – Deutschland ([email protected]) Authors Dr. Susanne Kneitz, University of Wuerzburg, Germany – Deutschland Dr. Marina Mione, Karlsruhe Institute of Technology, Germany – Deutschland Dr. Manfred Schartl, University of Wuerzburg, Germany – Deutschland, Abstract Malignant melanoma is one of the most deadly form of human skin cancer, with an almost 100% development of resistance to current therapeutic approaches at progression stages. In our laboratory, a transgenic small aquarium fish melanoma model was developed in Oryzias

latipes (medaka) by expressing the xmrk oncogene from platyfish/swordtail hybrids of the genus Xiphophorus under control of the medaka pigment cell specific mitf promoter leading to early onset of melanoma with almost 100% penetrance. Pigment cell tumor of different histotypes and different malignancy occurs depending on the genetic background into which the transgene was introduced. Similarly a transgenic zebrafish melanoma model was achieved by crossing fish from Et(kita:GalTA4,UAS:mCherry)hzm1 line with fish from the Tg(UAS:eGFP-HRAS_GV12)io6 line, where the oncogenic HRAS was expressed under kita promoter develops melanoma. To find out if microRNAs may play a role for transformation of low malignancy grade, slow-growing melanoma to highly metastatic tumors, we sequenced the whole small RNA transcriptome of from different tumor histotypes. We could observe that our fish melanoma models are heterogeneous and complex like human melanoma, comprising deregulated microRNAs and other small RNAs. Many known human melanoma-associated microRNAs and their target genes are deregulated similarly. Comparison with human melanoma revealed consistent deregulated pathways involved in cell proliferation, antiapoptosis and angiogenesis. Our results illustrate that using a comparative approach for analysing whole transcriptome data from fish models is effective to identify and validate new melanoma associated microRNAs involved in tumorigenesis. Background Malignant melanoma is the most aggressive and lethal form of human skin cancer with increasing incidence worldwide. Owing to its heterogeneous nature, genetic and epigenetic changes leading to the process of melanomagenesis are poorly understood. Growing evidences in recent years have demonstrated that microRNAs can function both as tumor-suppressors and oncogene. Deregulated expression of microRNAs has been observed in many human cancers including melanoma. We propose that certain microRNAs are crucial for transformation of low malignancy grade, slow-growing melanoma to highly metastatic tumors. Materials and Methods In our laboratory transgenic medaka fish model was developed, where fish expressing melanoma oncogene xmrk from Xiphophorus under mitf promoter develops various types of melanoma ranging from cutaneous to extracutaneous, highly invasive and metastatic to exophytic, non-invasive, and from slow to fast growing. A genetic melanoma model by interspecific crossing of Xiphophorus maculatus and Xiphophorus hellerii is established in our laboratory. In transgenic zebrafish melanoma model, expression of oncogenic HRAS under kita promoter develops melanoma by 1-3 month of age. Results We sequenced the whole microRNA population from different tumor histotypes from the three fish models. Expression levels of selected microRNAs and their target gene expression were validated in fish melanoma samples and human melanoma cells. Different tumor types exhibit specific and diagnostic expression signatures at transcriptional level. Comparison of fish and human melanoma revealed highly consistent deregulation of known melanoma associated microRNAs in addition to a few novel microRNAs. Functional clustering of the target genes of deregulated microRNAs revealed highly enriched pathways for cell proliferation, migration, and angiogenesis. Conclusion

Our results demonstrate the importance of comparative small RNA transcriptome analysis from fish melanoma models as a tool to identify and validate new melanoma associated microRNAs involved in tumorigenesis.

Paper reference: Paper 3.3 EGFR Expression and Genetic Variation in Six Types of Cancer in the Arabian Gulf Region *Prof. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain ([email protected]) Dr. Raed H Qaddourah, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain Abstract As understanding cancer molecular pathology has progressed novel therapies targeting specific markers shown to be involved in tumor progression have emerged Epidermal growth factor receptor is one of the first validated targets for cancer therapy and has high diagnosis and prognosis potential In this work we report data on the EGFR cell surface expression and genetic variability in a total of Arab cancer patients distributed into groups of cancer types We used automated IHC and a scoring method that matches CAP criteria for measuring Her2neu expression We analyzed the genetic variation of three markers in the EGFR gene two SNPs rs712829 (- G T ), located in a sp1 recognition site rs712830 (- C A ) and intron embedded CA repeat ( SSR1 ), known to impact EGFR cell surface expression level The study also included an ethnically matched control group Our data show that EGFR expression varied according to cancer types with lung cancer having the highest rate ( %) and the lowest in breast cancer ( %). EGFR expression level was correlated with high grade tumors in Lung and Bladder cancers Single marker genetic analysis showed that the SSR1 marker Long ( number CA repeat > ) alleles as well as variant A of SNP rs712830 (- C A ) are associated with lung adenocarcinoma with a ( p < and odd ratio = ). Furthermore analysis of the three markers as a mini haplotype using Haploview and the SHEsis software identified two haplotypes that are significantly associated with lung ( SSR1L - G - A p < Odd Ratio = ) and bladder ( SSR1L - G - C p < Odd Ratio = ) cancers Our data provide valuable insights into the genetic of EGFR in Arab cancer patients and highlight the discrepancies in EGFR expression

Paper reference: Paper 3.4 The effect of GM-CSF on murine embryo development, aneuploidy, DNA fragmentation and morphokinetic parameters analysed using EmbryoScope™ *Dr. Aisha Elaimi, King AbdulAziz University, University College London, UK ([email protected]) Dr. Joyce Harper, Institute for Women's Health, University College London, UK

Abstract We designed this study to investigate whether adding different concentrations of GM-CSF to culture media had an effect on murine preimplantation embryos in terms of their developmental potential, chromosomal constitution, DNA fragmentation and morphokinetic parameters analysed using EmbryoScope™. Methods: Two cell stage murine embryos were cultured to the blastocyst stage in the Embryoscope™ in the presence of GM-CSF: 0ng/ml (control), 1ng/ml, 2ng/ml, 5ng/ml and 10ng/ml. The development and timing of each division were monitored. FISH was performed using locus specific probes for chromosomes 2, 11 and 16. DNA fragmentation analysis was performed using the Promega DeadEnd™ Fluorometric TUNEL system. Results: There was no significant effect in either incubator at all GM -CSF concentrations except 10ng/ml (decreased development). No significant difference in the rate of DNA fragmentation or aneuploidy between the control and the GM -CSF groups. The duration of cleavage divisions from to cell and to cell were significantly shorter for embryos that reached blastocyst stage compared to embryos that arrested in development. There was no relationship between the timing of cleavage and the rate of aneuploidy or the rate of DNA fragmentation. Conclusion: Adding various concentrations of GM-CSF to the culture media did not have any significant positive impact on blastulation rate, aneuploidy or DNA fragmentation. The time lapse analysis of the length of the first cleavage divisions using the EmbryoScope™ showed no acceleration in the growth rate with GM-CSF Background Whilst some studies have shown that GM - CSF enhances blastocyst development others have shown no effect Two studies have shown that GM - CSF has no effect on aneuploidy in human and murine embryos In a culture media was launched containing ng ml of GM - CSF claiming it enhanced blastocyst development and reduced the rate of apoptosis Time - lapse imaging provides kinetic parameters to allow continuous analysis of embryo development including the timing of each cleavage division. Materials and Methods Two cell stage MF1-murine embryos were cultured to the blastocyst stage in the Embryoscope™ in the presence of GM-CSF: 0ng/ml (control), 1ng/ml, 2ng/ml, 5ng/ml and 10ng/ml. The development and timing of each division were monitored. FISH was performed using locus specific probes for chromosomes 2, 11 and 16. DNA fragmentation analysis was performed using the Promega DeadEnd™ Fluorometric TUNEL system. Results There was no significant effect in either incubator at all GM -CSF concentrations except 10ng/ml (decreased development ). No significant difference in the rate of DNA fragmentation or aneuploidy between the control and the GM -CSF groups. The duration of cleavage divisions from to cell and to cell were significantly shorter for embryos that reached blastocyst stage compared to embryos that arrested in development. There was no relationship between the timing of cleavage and the rate of aneuploidy or the rate of DNA fragmentation. Conclusion Adding various concentrations of GM-CSF to the culture media did not have any significant positive impact on blastulation rate, aneuploidy or DNA fragmentation. The time lapse analysis

of the length of the first cleavage divisions using the EmbryoScope™ showed no acceleration in the growth rate with GM-CSF Paper reference: Paper 4.A Anthropogenic contamination in the Red Sea and its potential impact on marine water microbial community *Ms. Peiying Hong, King Abdullah University of Science and Technology, KSA([email protected]) Good water quality in the Red Sea is imperative for the continued sustenance of key industries like desalination and aquaculture, to ensure minimal impacts on human and ecosystem health. This study aims to evaluate the impact anthropogenic contamination has on seawater microbiota at four sampling zones of near-shore water and from two swash zones of local beaches. Water quality parameters relating to nutrient and enterococci numbers were monitored. Occasional breaches in enterococci (> 35 MPN/100ml) and higher abundance of genera associated with opportunistic pathogens were detected in samples collected from swash zones. TOC, total organic carbon (1.48-2.18 mg/L) and TN, total nitrogen (0.15-0.27 mg/L) in these samples were significantly higher than that detected in the near-shore waters (1.13 mg/L for TOC and 0.09 mg/L for TN). Abundance of certain genera, for example Clostridia group, was observed to correlate with TOC and TN (Spearman’s rank > 0.503). Furthermore, Enterococcus faecalis and E. faecium isolated from beach sands showed resistance to up to 5 antibiotics. Operational taxonomic units associated with cyanobacteria, Prochlorococcus and Ostreococcus spp. were more prevalent in the near-shore waters collected in close proximity to sewage outfall. Microbial richness at these sites was on average 702 OTUs, and was lower than the average 1062 OTUs determined from other samples. Our findings suggest that anthropogenic contamination resulted in different extent of perturbations on marine water community in the Red Sea, and that changes in the abundance of bacterial markers can be used as signatures to assess future contamination events. Paper reference: Paper 4.1 Enhanced Oil Recovery by Biosurfactants *Dr. Pattanathu K.S.M. Rahman (Rahman PK), TeeGene Biotech, Teesside University, UK ([email protected]) Abstract Surfactants are one of the most versatile products used in the chemical industry. Biosurfactants are biological surface active compounds released by microorganisms and can reduce surface tension. These molecules have both hydrophilic and hydrophobic moieties in their structure. They can be produced by bacteria, fungi and yeasts. This gives us an eco-friendly way for producing surfactants without the use of chemicals. So biosurfactants will have a lower toxicity, better environmental compatibility and also they will biodegrade much more easily than

chemical surfactants. The use of biosurfactants in mobilizing oil trapped in reservoirs is an exciting prospect. Unlike current chemical enhanced oil recovery (EOR) techniques, biosurfactants are an environmentally friendly option and pose no threat to the environment. Due to their unique ampiphilic nature they have the ability to alter the wettability of the reservoir rock by absorbing at it surface. They also have the ability to reduce the interfacial tension (IFT) between two immiscible fluids, in this case, crude oil and water. Unlike conventional core flooding experiments, the experiments conducted in this investigation used calcite and mica powder to model carbonate and sandstone reservoirs respectably. The results showed that the wettability of rock surfaces altered significantly with the introduction of biosurfactant into the selected sea water medium. Parallel to rock wettability experiments, water-oil IFT was also investigated using varying volumes of biosurfactant. Results showed a dramatic decrease in IFT following biosurfactant addition.

Paper reference: Paper 4.2 Potency of Chitinolytic Bacteria as Biocontrol of Scopulariopsis sp. Isolated from Cricularia trifenestra Cocoon *Dr. Nisa Rachmania Mubarik, Bogor Agricultural University, Indonesia ([email protected]) Dr. Prima Prima Novitasari, Bogor Agricultural University, Indonesia Dr. Sri Sri Listiyowati, Bogor Agricultural University, Indonesia Abstract Potency of Chitinolytic Bacteria as Biocontrol of Scopulariopsis sp. Isolated from Cricula trifenestrata Cocoon Nisa Rachmania Mubarik*, Prima Novitasari, and Sri Listiyowati Department of Biology, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University, Jalan Agatis, IPB Dermaga, Bogor 16680, Indonesia *Corresponding author, Email: [email protected]; [email protected] Abstract Gold Silkworm’s cocoon is a coat of Cricula trifenestrata’s pupa. The fiber which is resulted from processing cocoon has high economic value in Indonesia because the specific of gold colour that doesn’t have any else cocoon. However, on the process often occurred many problems that could decrease quantity and quality of cocoon, such as attack from pathogenic fungi. For controlling of the pathogenic fungi has been used chemical agent, but using regularly caused some negative effects. As alternative using chitinolytic bacteria as biocontrol which could degrade the chitin at cell wall of fungi. Therefore the objectives of this research were to isolate of chitinolytic bacteria from cocoon of C. trifenestrata and to test their potential as biological control of the cocoon’s pathogenic fungi. 17 strains chitinolytic bacteria were isolated from pupa of Cricula trifenestrata. There were four isolates CH2, CH10, CS1, and CS4 showed high score of chitinolytic index. Bacterial isolate CH10 showed the highest chitinase activity and identified as Shewanella putrefaciens. The isolate and its crude chitinase could inhibit the growth of Scopulariopsis sp. which isolated from cocoon of Cricula trifenestrata pupa. Keyword: biocontrol, cocoon, chitinolytic bacteria Cricula trifenestrata, Scopulariopsis sp. Background

Gold Silkworm ’ s cocoon is a coat of Cricula trifenestrata ’ s pupa. The fiber which is resulted from processing cocoon has high economic value in Indonesia because the specific of gold colour that doesn ’ t have any else cocoon. However on the process often occurred many problems that could decrease quantity and quality of cocoon such as attack from pathogenic fungi. For controlling of the fungi has been used chemical agent but it caused some negative effects. As alternative using chitinolytic bacteria as biocontrol. Materials and Methods Materials: Cocoon of gold silkworm (Cricula trifenestra) Methods: (1) Isolation of pathogenic fungi from Cocoon of gold silkworm (Cricula trifenestra), (2) Isolation of chitinolytic bacteria from the cocoon, (3) Antagonistic activity of the chitinolytic bacteria toward pathogenic fungi, (4) Identification of pathogenic fungi and bacterial isolate potential Results The were 17 strains chitinolytic bacteria isolated from pupa of Cricula trifenestrata. There were four isolates CH2, CH10, CS1, and CS4 showed high score of chitinolytic index. One of bacterial isolate,CH10, showed the highest chitinase activity and identified as Shewanella putrefaciens. The isolate and its crude chitinase could inhibit the growth of Scopulariopsis sp. which isolated from cocoon of Cricula trifenestrata pupa Conclusion The chitinolytic bacteria isolated from gold silkworm' cocoon could inhibit the growth of pathogenic fungi, Scopulariopsis sp. The chitinase enzyme or chitinolytic bacteria showed the potency as biocontrol of the fungi. Paper reference: Paper 5.A Molecular Epidemiology Studies in Developing Countries: Challenges and Opportunities *Dr. Mohamed Abdel-Hamid Professor of Microbiology, Faculty of Medicine, Minia University Director of Viral Hepatitis Research Lab. (VHRL), National Hepatology and Tropical Medicine Research Institute, Egypt,([email protected]).

The Molecular Epidemiology is a science that utilizes molecular biology techniques to identify the causes and modifying factors of diseases in human populations. Today, immigration, international job opportunities, tourism, terrorism, local and regional conflicts spur all countries to advance interest, studies and knowledge in Molecular Epidemiology. Knowledge and practical experience obtained from studies promote and control the spread of diseases, vaccine development against diseases, and advance diagnosis processes such as the mapping of genotypes and phenotypes of diseases. Due to limited resources in the developing countries, especially in the field of medicine, the main focus remains on diagnosis and treatment of diseases and not on advancing research or basic science knowledge.

Molecular diagnostics techniques such as gene sequence and gene detection, Southern blot, Northern blot, tagged probes, reverse hybridization, PCR (Qualitative, Quantitative, Real Time, TMA, etc), genomic and proteomic analysis are very expensive to perform, require special diagnostic equipment, reagents and require the highest degree of Quality Control.. Most of the equipment used in these processes are closed systems, contain Copy Right protected software, and highly skilled personnel are require to successfully operate such advance systems. Funding to maintain such equipment, which is generally performed by experienced technician, is limited. The reagents and supplies needed (primers, probes, enzymes, etc) are not always available locally therefore they need to be special imported. Difficulties are many for scientists involved in research in developing countries however successful examples of scientists performing high quality research can be named. One of these sites is the Viral Hepatitis Research Laboratory (VHRL) in Egypt. The activities of the VHRL, the challenges faced and overcome by VHRL are discussed in the paper. Paper reference: Paper 5.1 Clustering of Cerebral Malaria in families with Familial Febrile Convulsion *Dr. Adil Mergani Babikir, Taif University, Serbia – Србија ([email protected]) Dr. Amar Hasan Khamis, University of Dammam, Saudi Arabia Dr. Siddig Ahmed Rahoud, Albaha University, Saudi Arabia Dr. Nasr Eldin Mohamed Elwali, Al Imam Muhammad Ibn Saud Islamic University, Saudi Arabia Abstract Abstract: A cohort of 409 Sudanese children (6.1 ± 3.3) years old {223(54.5%) males, 186(45.5%) females} were admitted in three hospitals located in Wad Medani Sinnar and Singa in central Sudan due to severe malaria after informed consent. Two hundred seventy‐four (67%) had cerebral malaria (CM) and were belong to four tribal stocks {Johayna 93(22.8%), Nigerian 70(17.3%), Gaalian 68(16.7%) and Nuba 44(11%). The highest incidence of CM was among age group (4-6 yrs.), 123(30%) were offspring of consanguineous families, 100 cases (24.4%) were clustered in three tribal stocks (Johayna, Nigerian and Gaalian), 60 of them (60%) were offspring of consanguineous families. Seventy-four (21.3%) out of 347 families had history of febrile convulsion (FC) either with one or more affected siblings. Families with history of both FC and CM were 6 times more at relative risk for developing CM than those with history of CM only (P=0.0001,OR=5.62,CI 95% 2.978-10.605). Children {63(15.4%)} with history of FC were five times higher risk for developing recurrent CM than others (P=0.003, OR=4.567, CI 95% 1.77211.768). As conclusion, children with history of FC had a significant clinical association of CM (P=0.0001). The development of CM may needs more other factors (intensity of transmission, prevalence of specific parasite strain and probably a host genetic modifier factor that play a crucial role in predisposing of a febrile convulsive child to CM. Key wards: Familial FC, Childhood CM, Clustering and Recurrence. Paper reference: Paper 5.2

Genetic diversity of Vibrio cholerae and detection of El Tor Variant strains during 2012 cholera outbreak *Dr. Bita Bakhshi (Bakhshi B), Tarbiat Modares University, Iran ([email protected]) Abstract Genetic diversity of Vibrio cholerae and detection of El Tor Variant strains during 2012 cholera outbreak Background:Genetic diversity of outbreak strains may lead to determine the source of cholera outbreaks. The 'El Tor Variant' has been emerged recently which is typically identified as El Tor strains of V. cholerae but has the classical type ctxB sequence and produces cholera toxin of classical type. The aim of this study was to characterize the ctxB allelic sequence diversity of V. cholerae isolates from an outbreak in Iran (2012) and investigate the biotype and PFGE genotype specification of isolates. Materials and Methods: Eleven Vibrio cholerae strains were isolated and confirmed by conventional biochemical tests and by PCR using specific primer. Serotyping was performed by polyclonal O1 and monospecific Ogawa and Inaba antisera. The ctxB sequence of all isolates was subjected to direct sequencing using specific primers and compared with the reference ctxB sequences for El Tor and classical biotypes in GenBank database. The PFGE genotype specification of isolates was determined and genetic relatedness among isolates and also with those previously reported from Iran was assessed. Results: All the ctxB+ isolates in this study (classical and El Tor biotypes) possessed the ctxB sequence of classical biotype allele providing evidences of El Tor variants. Eight out of 11 isolates (73%) showed identical pulsotypes. Each of the remaining 3 isolates showed distinct pulsotypes with more than three band differences. One pulsotype was corresponded to an isolate (9%) with classical biotype. Conclusion: The result demonstrated diversity in virulence gene content of strains with identical PFGE patterns and also provided evidences of the import of a V. cholerae strain with classical biotype from out of our country since no classical biotype strain has been previously (19982011) reported from Iran. Background Genetic diversity of outbreak strains may lead to determine the source of cholera outbreaks. The 'El Tor Variant' has been emerged recently which is typically identified as El Tor strains of V. cholerae but has the classical type ctxB sequence and produces cholera toxin of classical type. The aim of this study was to characterize the ctxB allelic sequence diversity of V. cholerae isolates from an outbreak in Iran (2012) and investigate the biotype and PFGE genotype specification of isolates. Materials and Methods Eleven Vibrio cholerae strains were isolated and confirmed by conventional biochemical tests and by PCR using specific primer. Serotyping was performed by polyclonal O1 and monospecific Ogawa and Inaba antisera. The ctxB sequence of all isolates was subjected to direct sequencing using specific primers and compared with the reference ctxB sequences for El Tor and classical biotypes in GenBank database. The PFGE genotype specification of isolates was determined and

genetic relatedness among isolates and also with those previously reported from Iran was assessed. Results All the ctxB+ isolates in this study (classical and El Tor biotypes) possessed the ctxB sequence of classical biotype allele providing evidences of El Tor variants. Eight out of 11 isolates (73%) showed identical pulsotypes. Each of the remaining 3 isolates showed distinct pulsotypes with more than three band differences. One pulsotype was corresponded to an isolate (9%) with classical biotype. Conclusion The result demonstrated diversity in virulence gene content of strains with identical PFGE patterns and also provided evidences of the import of a V. cholerae strain with classical biotype from out of our country since no classical biotype strain has been previously (1998-2011) reported from Iran. Paper reference: Paper 5.3 Association of APOA5 gene variant with the dyslipideamia in Pakistani Population *Dr. MUHAMMAD FIAZ, SHAHEED ZULFIQAR ALI BHUTTO MEDICAL UNIVERSITY ISLAMABAD PAKISTAN, Pakistan ([email protected]) Dr. GHAZALA KAUKAB RAJA, PMAS ARID AGRICULTURE UNIVERSITY RAWALPINDI, Pakistan Abstract Association of APOA5 gene variant with the dyslipideamia in Pakistani Population Muhammad Fiaz1, S.M Saqlan Naqvi1, Bernard M.Y. Cheung2, Ghazala Kaukab1 1. Department of Biochemistry, PMAS Arid agriculture University Rawalpindi, Pakistan. 2. Division of Clinical Pharmacology, Department of Medicine, University of Hong Kong, Hong Kong Abstract Background Single nucleotide polymorphisms (SNPs) in the apolipoprotein A5 gene (APOA5) have association with metabolic syndrome and hypertriglyceridemia in different ethnic populations. We studied the associations of SNP rs662799 of APOA5 gene with dyslipidemia in the Pakistani Population. Method We genotyped the tagging SNP rs662799 of APOA5 in 712 (half cases and half control) unrelated subjects. The dyslipidemia was diagnosed by performing level of the serum triglycerides and high density lipoproteins in both cases and controls. Blood samples were collected from Pakistan Institute of Medical Sciences Islamabad, Pakistan. The fasting lipid profile was performed according to manufacturer’s instructions on chemistry analyzer (Hitachi 912). The DNA was extracted using Phenol Chloroform method. Genotyping was performed using Microarray, iPlex Gold technology. Results The results were analyzed using Plink software and statistical tool, SPSS 16.0. The SNP rs662799 of APOA5 was found to be associated with the dyslipidemia (odds ratio = 1.49, p =0.0051).

Conclusion Our result showed that rs662799 polymorphism in APOA5 is associated with dyslipidemia among the Pakistani Population. This explains that carriers of risk allele of APOA5 are more at risk of cardiovascular diseases. Correspondence: Dr Ghazala Kaukab Raja Associate Professor Department of Biochemistry, PMAS Arid Agriculture University Rawalpindi, Pakistan. [email protected] Background Single nucleotide polymorphisms (SNPs) in the apolipoprotein A5 gene (APOA5) have association with metabolic syndrome and hypertriglyceridemia in different ethnic populations. We studied the associations of SNP rs662799 of APOA5 gene with dyslipidemia in the Pakistani Population. Materials and Methods Method We genotyped the tagging SNP rs662799 of APOA5 in 712 (half cases and half control) unrelated subjects. The dyslipidemia was diagnosed by performing level of the serum triglycerides and high density lipoproteins in both cases and controls. Blood samples were collected from Pakistan Institute of Medical Sciences Islamabad, Pakistan. The fasting lipid profile was performed according to manufacturer’s instructions on chemistry analyzer (Hitachi 912). The DNA was extracted using Phenol Chloroform method. Genotyping was performed using Microarray, iPlex Gold technology. Results Results The results were analyzed using Plink software and statistical tool, SPSS 16.0. The SNP rs662799 of APOA5 was found to be associated with the dyslipidemia (odds ratio = 1.49, p =0.0051). Conclusion Conclusion Our result showed that rs662799 polymorphism in APOA5 is associated with dyslipidemia among the Pakistani Population. This explains that carriers of risk allele of APOA5 are more at risk of cardiovascular diseases. Paper reference: Paper 6.A Unusual presentation of parasitic diseases. When a molecular test is needed? *Nadia El Dib Professor Of Parasitology, Faculty of Medicine, Cairo University, Egypt ([email protected]( Abstract The majority of symptoms attributable to parasitosis are not specifically diagnostic. Parasites may not produce clinically demonstrable symptoms and may alter the metabolic balance of the host causing unusual response similar to many other diseases. Scientific knowledge and clinical experience supported by laboratory tests are very crucial to reach the suitable diagnosis. Methods of diagnosis of parasitic infections developed in the last few decades adding more sophisticated techniques as the detection of the parasite DNA. The necessity to use a

sophisticated test to diagnose a parasitic disease is discussed, exhibiting some rare but interesting infections met with in the department of Parasitology, Kasr Al Ainy Medical School, Cairo University. A case of an 18-months-old baby with anemia and melena was diagnosed as ancylostomiasis will be discussed showing why infection may occur in this young age. A case with a nodule in the upper eyelid diagnosed as Dirofilariaiasis. A case previously diagnosed and treated as cutaneous leishmanisis, will be discussed showing how history of the case could be seriously misleading in the management of a case. A case with symptoms of deep venous thrombosis in the lower limb was properly diagnosed and given treatment as Tropical pulmonary eosinophilia. A case with a liver abcess, was misdiagnosed as parasitic infection of the liver. A case with history of treated TB, presented with haemoptysis and was diagnosed as Dissyminated strongyloidiasis. A case with bertiellosis passing parasite parts out of the anus, was diagnosed and treated. A case with chronic diarrhea, mal absorption, cachexia and hypoproteinaemia was diagnosed as intestinal capillariasis. The development of a PCR technique to diagnose the last case was important to overcome the difficulties in the diagnosis. Molecular diagnosis was important in some of the mentioned cases but was not necessary in others. Paper reference: Paper 6.1 Recombinant fusion protein comprised of the two schistosomal antigens, Sm14 and Sm29, provides significant protection against Schistosoma mansoni in murine infection model *Dr. Mohammed Bahey-El-Din (Bahey-El-Din M), Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt ([email protected]) Dr. Eglal I. Amer, Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Alexandria, Egypt Dr. Radwa E. Ewaisha, Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt Dr. Amal M. Khalil, Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt Dr. Hamida M. Aboushleib, Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt Dr. Shereen F. Mossallam, Department of Medical Parasitology, Faculty of Medicine, Alexandria University, Aleandria, Egypt Abstract Schistosoma mansoni infection represents a major cause of morbidity and mortality in many areas of the developing world. Effective vaccines against schistosmiasis are not available and disease management relies mainly on treatment with the anthelmintic drug praziquantel. Several promising schistosomal antigens have been evaluated for vaccine efficacy such as Sm14, Sm29 and tetraspanins. However, most investigators examine these promising antigens in animal models individually rather than in properly formulated and adjuvanted antigen combinations. In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29. The fusion protein, FSm14/29, was

administered to Swiss albino mice either unadjuvanted or adjuvanted with polyinosinicpolycytidylic acid adjuvant, poly (I:C). Upon challenge with S. mansoni cercariae, mice preimmunized with unadjuvanted or poly (I:C)-adjuvnated fusion protein showed reduction of adult worm burden of 44.7 and 48.4 %, respectively. In addition, significant reduction of tissue egg burdens and granulomata was observed in mice immunized with the fusion protein as compared to the control groups. Light microscope and scanning electron microscope (SEM) investigation of adult worms recovered from FSm14/29-immunized mice revealed appreciable morphological damage and tegumental deformities. Histopathological examination of liver sections of immunized mice demonstrated reduced granulomatous and inflammatory reactions. These findings highlight the importance of the fusion protein FSm14/29 as a potential bivalent vaccine candidate that is worthy of further investigation. Background Schistosoma mansoni infection represents a major cause of morbidity and mortality in many areas of the developing world. Effective vaccines against schistosmiasis are not available and disease management relies mainly on treatment with the anthelmintic drug praziquantel Several promising schistosomal antigens have been evaluated for vaccine efficacy such as Sm14, Sm29 and tetraspanins. In the present study, we made a recombinant fusion protein comprised of the promising schistosomal antigens Sm14 and Sm29. Materials and Methods RNA was extracted from S. mansoni adult worms and was subjected to RT-PCR to amplify Sm14 and Sm29 genes. The two genes were spliced using a restriction-ligation approach and were cloned in pQE31 plasmid in E. coli M15 (pREP4). Expressed fusion antigen was purified using metal affinity chromatography. Mice were immunized with the fusion antigen and challenged later with cercariae. Adult worm burden, granulomas and antibodies (IgG1 and IgG2a) were evaluated to test vaccine efficacy compared to controls. Results The recombinant fusion antigen FSm14/29 was successfully expressed and purified as determined by SDS - PAGE and Western blot. Mice immunized with the fusion antigen demonstrated significant reduction (48.4 %) of adult worm burden granuloma size (64 %), liver egg burden (82.8 %) and intestinal egg burden (72.8 %). Significant structural and morphological distortions were observed in adult worms recovered from infected immunized mice as determined by light microscopy and scanning electron microscopy. This indicated strong immune response in mice immunized with the fusion antigen. Conclusion Overall, we have demonstrated that vaccination with the fusion protein FSm14/29 offers significant protection against S. mansoni infection in Swiss albino mice. Immunization resulted in significant reduction of adult worm burden, tissue egg loads and liver granulomata formation. Deleterious structural changes were also observed in adult worms recovered from immunized mice. Optimization of experimental conditions and the adjuvant used should greatly improve the protection outcome and consolidate the concept of using multi-antigen fusion proteins as vaccine candidates against S. man Paper reference: Paper 6.2 Genetic and Other Factors Controlling the Effect of Praziquantel

*Dr. Siddig Ahmed Gibril Rahoud, Albaha University, KSA ([email protected]) Abstract ABSTRACT An association study of a cohort of Sudanese patients infected with Schistosoma mansoni [ ( %) males and ( %) females ] was conducted to evaluate the factors controlling the regression of liver fibrosis months after treatment with Praziquantel using ultrasound evaluation Periportal fibrosis ( PPF ) was regressed in ( %) patients while the disease progressed to higher grades in ( %) patients The grade of PPF did not change in ( %) patients The mean values of portal vein diameter splenic vein diameter and index liver size in subjects in whom PPF regressed after treatment were significantly lower than in subjects in whom the disease was progressed ( P = P = and P = respectively ). The progression of hepatic fibrosis in males ( %) was greater than that in females ( %). Patients with regression or progression phenotypes tend to cluster in certain families The possible genetic control of PPF has been evaluated by studying the role of four polymorphisms ( IFN - g rs2069705 ( C T ), IFN - g R1 rs11914 ( G T ), rs1327474 ( A G ), and IL - rs1800925 (- ) ( C T ) in the regression of PPF There was an association between IL - rs1800925 T allele and the low grades of PPF ( P = ). No significant association was found between three polymorphisms ( IFN - g rs2069705 ( C T ) P = rs1327474 ( A G ) P = and IFN - g R1 rs11914 ( G T ), and PPF as response to PZQ Our study indicated that regression progression and stabilization of PPF after Praziquantel therapy is controlled by gender age grade of fibrosis and possibly inherited factors Key words Periportal fibrosis ( PPF ), Regression Progression Praziquantel ( Paper reference: Paper 6.3 IL-13 Polymorphism (IL-13 rs1800925 (-1055) (C/T) is Associated with Severe Hepatic Fibrosis in Human Schistosomiasis *Dr. Siddig Ahmed Rahoud, Albaha University, Saudi Arabia ([email protected]) Dr. Adil Mergani Babikir, Taif University, Saudi Arabia Dr. Amar Hasan Khamis, University of Dammam, Saudi Arabia Dr. Nasr Eldin Mohamed Elwali, Al Imam Muhammad Ibn Saud Islamic University, Saudi Arabia Abstract IL-13 Polymorphism (IL-13 rs1800925 (-1055) (C/T) is Associated with Severe Hepatic Fibrosis in Human Schistosomiasis Siddig A.Rahoud, ¹ Adil Mergani,2 Ammar H. Khamis, 3 Osman K. Saeed, 4 Qurashi Mohamed-Ali, 5 Christophe C., 6 Alain J. Dessein, 6 and Nasr Eldin M. A. Elwali. 7ABSTRACT An association study of a cohort of 177 Sudanese patients infected with Schistosoma mansoni {82 (46%) males and 95 (54%) females} was conducted to investigate the correlation of four polymorphisms {IFN-13 rs1800925 (-1055) (C/T)} to the regression and progression of liver fibrosis 39 months after treatment with praziquantel (PZQ). Regression and progression phenotypes were evaluated by ultrasound. DNA from patients infected with S. mansoni was extracted, purified and amplified

by PCR. Allelic typing was done using RFLP, primer extension reaction, DHPLC, and allelic discrimination assays (TAQ-MAN). SDS (Sequence Detection Systems) software was used for genotyping. There was an association between IL-13 rs1800925 T allele and the low grades of periportal fibrosis (PPF) (P = 0.02). No significant association was found between three polymorphisms (IFN(G/T), and PPF as response to PZQ. We conclude that IL-13 rs1800925 T allele is protective against PPF (P = 0.02).Key words: Periportal fibrosis (PPF), Regression, Progression, Praziquantel (PZQ), Polymorphisms. Paper reference: Paper 7.A Transplanting the Un-transplantable: A National Living Donor Paired Exchange Program Noureddine Berka, PhD., D(ABHI), Clinical Associate Professor and Laboratory Director, Calgary Laboratory Services and University of Calgary, Alberta, Canada ([email protected]) Abstract Kidney transplantation has been recognized as the best treatment of end-stage renal disease, acting as a means of limiting patient exposure to the toxic effect of dialysis. There is a shortage in organs around the world, complicated by ABO and HLA incompatibility barriers of living donors. This situation has pushed desperate patients in the Middle East to pursue transplant tourism and has motivated transplant programs to explore alternative therapies to deal with this critical shortage of organs. For the last decade, development of desensitization protocols has made the presence of circulating donor HLA-specific antibodies and the use of ABO incompatible organs a viable possibility that has helped many patients to undergo transplantation but still not sufficient especially for highly sensitized patients. Recently, two new modalities have been introduced at a national level, the Living Donor Paired Exchange (LDPE) and the Highly Sensitized Patient (HSP) registries. LDPE allows patients with incompatible live donors to receive compatible or better-matched organs by exchanging donors. The HSP registry aims at finding donors from around the country that present with acceptable HLA mismatches. The success of these national registries relies heavily on the standardization of histocompatibility methods cross the country. We present here our laboratory’s experience in these national registries for the last five years. Paper reference: Paper 7.1 Exploring the Use of Novel Combination of Pre-Existing Markers for Rheumatoid Diseases *Dr. Amna Al-Hammadi (Al-Hammadi A), Sharjah Higher Colleges of Technology, UAE ([email protected]) Dr. Nishi Singh, Sharjah Higher Colleges of Technology, UAE Dr. Muhammad Zaman, Sharjah Higher Colleges of Technology, UAE Dr. Ban Altoumah, Sharjah Higher Colleges of Technology, UAE Dr. Nadia Nagui, Sharjah Higher Colleges of Technology, UAE

Abstract Key words: Rheumatic diseases, Rheumatoid factor (RF), Anti-Cyclic Citrullinated Peptide antibodies (anti-CCP), and C-Reactive Protein (CRP) Background Autoimmunity is the basic feature of the most rheumatoid diseases. The most common type of rheumatic diseases is RA. Laboratories play a significant role is diagnosing patients with rheumatic complaints. Various markers and immunoglobulins are used to investigate these diseases. RF, anti-CCP, ANA, CRP and ESR tests are all used in determining the diagnosis and monitoring the patient condition after therapy. Exploring the correlation among all these tests and comparing the findings with the previous studies is quite helpful that leads to optimal results. Materials and Methods Data of a total of 317 patients with rheumatologic signs and symptoms from Jan to April were retrospectively analysed. Inflammatory activity using ESR and CRP tests are evaluated also the presence of RF anti - CCP and ANA were analyzed The ESR was determined by Westergren tube method CRP and PF by immunoturbidimetric assays with different wavelengths Whereas anti CCP and ANA performed under ELISA ( IgG ) principle The data were analyzed by different softwares such as; EXCEL 2010 and SPSS version 19 and 21. Results The independent variable is abnormal RF which is correlated with anti-CCP, ANA, CRP and ESR. Females presented the majority of the samples than males. Among these 317 samples, only 46 samples were abnormal with RF. The correlation between abnormal RF towards anti-CCP was (r=0.868), ANA (r=0.144), CRP (r=0.245) and ESR (r=0.330) is different. Additionally, the correlation between ESR and CRP is (r=0.518). Conclusion The results showed that anti-CCP is highly correlating positively with RF, followed by ESR then CRP. There was a very little positive correlation between RF and ANA which statistically insignificant. The CRP and ESR has moderate positive correlation but they cannot replace each other. Further studies are recommended by including patient history, physical examination and radiological findings. Paper reference: Paper 7.2 An interspecies conserved motif of the mouse Immune System Released Activating Agent (ISRAA) induced potential proliferative effects on human cells *Dr. Safa Taha, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain ([email protected]) Dr. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain Dr. Moiz Bakhiet, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain Abstract

We have recently described ISRAA as a nervous system-induced factor inducing immune responses in mouse spleen, but the human ISRAA has not yet been identified. In this study, we examined the effects of the mouse protein on human Peripheral Blood Mononuclear Cells (PBMCs) to observe if a biological activity of this molecule is shared between different species. Mouse ISRAA showed dose-dependent dualistic effects on human cells since 5microg exhibitedpositive apoptosis while 50pg revealed significant proliferation (p0,05).The results suggest that the Asn118Asn polymorphism of the ERCC1 gene may not be linked with appearance and development of colorectal cancer in West Algerian population. Conclusion In Conclusion, this is the first study on ERCC1 gene polymorphism in our population suggesting that the Asn118Asn polymorphism of ERCC1gene may not be associated with the colorectal cancer risk in West Algerian population. Further research with a larger sample size is needed to reveal more information about this polymorphism and the appearance of colorectal cancer in our population.

Paper reference: Paper 9.1 A putative Novel Immune Signaling Pathway driven by the Nervous System revealed by computational analysis of the mouse ISRAA protein. *Dr. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain ([email protected]) Abstract We have previously described in mouse a novel immune system released activating agent ( Israa ) over - expressed by splenocytes following a nervous stimulation We showed that the Israa gene is embedded in intron of the Zmiz1 locus Israa encodes a AA protein that seems to be involved in T - cell signaling pathways No typical gene orthologs were found in other species and no significant homology with known proteins To investigate the precise biological function of ISRAA we carried out a computational approach to study its structure - activity relationship Functional motif scan showed the presence of an SFK specific SH2 binding domain surrounding Tyr ( YTEV ). We generated an abinitio model of ISRAA and carried out docking analysis with Fyn and Lck SH2 domains - ]. We used this model to design and produce recombinant wild type and mutant ( YTEV ) isoforms of ISRAA When stimulated by WT - recombinant ISRAA mouse splenocytes showed no significant proliferation by measuring radioactive - Thymidine incorporation However MTT assay revealed a raise in cellular activity which does not occur following stimulation by the YT - double mutant In vitro direct binding assay showed that WT - ISRAA interacts with active mouse rFyn while simultaneous mutation of Y102 and T103 abolished this binding The analysis of the phosphorylation status of SFKs ( Src Fyn Lck ) showed a decrease in phosphorylation of the activating Tyr - in the early stage of stimulation by WT ISRAA Meanwhile YT - Mutant showed a normal rate compared to untreated cells Considering that Src - Family Kinases ( SFKs ) Fyn and Lck play an essential role in the T - cell development homeostasis and activation our data strongly suggest that ISRAA Paper reference: Paper 9.2 NUTRITIONAL GENOMICS AND PERSONALIZED NUTRITION *Dr. Dina Abdulrahman Muharib, king saud medical city, Saudi Arabia ([email protected]) Abstract Nutritional Genomics is the study of how foods affect gene expression(nutrigenomics) and how individual genetic variation affects the way an individual responds to nutrients in food(nutrigenetic) . Genetic variation certainly has an important influence on human nutritional requirements, and the introduction of genomics has both highlighted the complexity of the interaction between genes and diet and offered opportunities to reevaluate the criteria used to determine RDAs and the contribution of genetic variation to optimal nutrition for individuals. As the interactions

between genetic variation and nutritional requirements become more fully understood, it will allow dietary recommendations to be individualized according to genotype to ultimately reduce our risk of degenerative diseases and increase health and well-being in old age

Paper reference: Paper 9.3 Hypoxia may have a teratogenic effect on sex differentiation and determination of aquatic animals *Dr. Hamed Kolangi Miandare, Assistant Professor at Gorgan University of Agricultural Sciences and Natural resources, Iran ([email protected]) Abstract The sex of a fish is not only determined by its genotype, but may also be affected by environmental factors which can alter sex differentiation. While the gender of most organisms is determined at the time of fertilization, but in the most of the fish species sex can be effected by the environment after fertilization. One of the key environmental factors is oxygen; the hypoxia-inducible factors (HIFs) are the indicators of oxygen condition. HIFs are key transcriptional regulators of hypoxic response in embryonic organisms. HIFs are “master regulator” that either directly or indirectly regulate transcription of more than one hundred Genes those are important in divers function, HIFs also down regulate some of genes involved in steroid genesis that alter the ratio of testosterone (T) to estradiol (E2). In this study hypoxiainducible factors gene expression (HIF-1α, HIF-2α) were evaluated during development of an ancient fish species, Beluga sturgeon (Huso huso) in a normal oxygen condition. The hypoxia genes are affective on sex determination and differentiation, hypoxia genes regulating steroid genesis during embryonic development, thereby disrupting the hormonal balance and sex differentiation, subsequently may be leading change in sex ratio in sturgeon. The result of this study was suggested to study sox family gene and hypoxia genes expression in same time to find the relation between of them in future studies. Paper reference: Paper 10.A Traumatic Brain Injury Biomarkers: A Neuroproteomics Approach *Kobeissy FH, Department of Psychiatry, Center for Neuroproteomics and Biomarkers, 4. Center for Traumatic Brain Injury Studies, Univ. of Florida, gainesville, FL, USA, ([email protected]). Abstract Traumatic brain injury has an incidence of approximately 2 million persons resulting in 500,000 hospitalizations and 100,000 deaths in the United States. Diagnostic and therapeutic treatments of TBI are presently limited to brain temperature and pressure control and periodic MRI scans. In this study, a neuroproteomics approach was utilized to study differential protein expression which can serve as potential biomarkers for TBI. Controlled cortical impact is used to

produce TBI in rats. Control and injured pooled samples (n=7) were analyzed and processed for proteomic analysis. Cation-anion column chromatography in tandem with 1d-gel electrophoresis (CAX/1D-PAGE) was used as a new multi-dimension protein separation approach for neuroproteomics analysis. Differential bands were excised and subsequent protein identification was performed by in-gel digestion followed by reverse phase capillary separation online with LCQ Mass Spectrometry. Results included 59 differential protein components of which 21 decreased and 38 increased in abundance after TBI. Proteins with decreased abundance included collapsin response mediator protein-2 (CRMP-2), GAPDH, MAP2A/2B and hexokinase. Conversely, C-reactive protein, transferrin and breakdown products of CRMP-2, synaptotagmin and αII-spectrin were found elevated after TBI. Differential changes in the above-mentioned proteins were confirmed by quantitative immunoblotting. Lastly, the identified TBI differential protein subsets were used to construct a neuroproteomic spectrum map illustrating additional protein interactions relevant to the TBI insult. Some of the differential proteins identified may serve as potential markers to potentially facilitate patient management by monitoring the severity, progression and treatment of injury. A number of these markers have been tested in clinical setups and showed some promising results.

Paper reference: Paper 10.1 Insight into the aberrant protein expression in murine cerebral cortex and hippocampus measured by differential expression proteomics *Dr. Jody Daniel Haddow (J. D. Haddow), UAE University, UAE ([email protected]) Dr. Christoph H Borchers, UVic - Genome BC Proteomics Centre, UAE Dr. Terry W Pearson, University of Victoria, UAE Abstract Down syndrome (DS) is caused by aberrant protein expression due to a trisomy of chromosome 21 (Ts21), and is the most common cause of genetic mental dysfunction. In this study a mouse model of Ts21, based on the physical co-localization of genetic loci on the same chromosome (Chr) of human chr21 and murine chr16, was used for studying the mechanisms involved in DS. Protein expression was measured in immortalized cell lines derived from normal and trisomy 16 (Ts16) fetal mouse hippocampus (H1b and HTk respectively) and normal and trisomic cerebral cortex (CNb and CTb respectively). iTRAQ labeling and tandem mass spectrometry were employed to analyze the trisomic cell lines for significant up- or down-regulation of proteins. CTb cells showed increased expression of 71 proteins and decreased expression of 56 proteins when compared to the normal CNb control cells. HTk cells showed increased expression of 97 proteins and decreased expression of 77 proteins when compared to the normal H1b control cells. Several of these differentially expressed proteins have previously been reported to show aberrant expression in DS and other neurological disorders such as Alzheimer’s disease, a condition closely associated with Down syndrome. Our global proteomics analyses identified aberrantly expressed proteins with various cellular functions associated with the trisomy model

and may help contribute to a more complete understanding of the individual proteins, mechanisms and protein networks involved in the Down syndrome phenotype. Background Down syndrome (DS) is the most common genetic condition associated with mental disability and is the most complex genetic anomaly that is still compatible with life. Approximately 95% individuals of with DS have an additional and therefore triplicate copy of chromosome 21, a disorder known as trisomy 21 (Ts21). DS occurs at a world-wide average of in 1 in 700-800 births. Ts21 individuals experience various complex phenotypes with effect on multiple organs, including the heart and brain, with the latter giving rise to compromised cognitive abilities. Materials and Methods Trisomic cell lines were grown in culture and the lysed and proteins. Sample protein concentrations were measured for each sample using a bicinchoninic acid kit and proteins were precipitated from each sample by addition of nine volumes of ice cold acetone. The proteins were digested with trypsin and then labeled with iTRAQ tags. The labeled peptides with then combined into a single replicate. The process was repeated from the start to generate 3 replicates. The samples were fractionated by SCX chromatography and then analyzed by LCMS/MS. Results CTb cells showed increased expression of 71 proteins and decreased expression of 56 proteins when compared to the normal CNb control cells. HTk cells showed increased expression of 97 proteins and decreased expression of 77 proteins when compared to the normal CNb control cells. Several of these differentially expressed proteins have previously been reported to show aberrant expression in DS and other neurological disorders such as Alzheimer’s disease, a condition closely associated with Down syndrome. Conclusion Based upon the trisomy 16 mouse model used in this study, aberrant protein expression in the Down Syndrome neuron appears to effect many aspects of cell biology including cytoskeletal development and synaptic plasticity. In addition the neuron protein translation machinery seems to be compromised indicating a delicate balance between ER stress and prevention of apoptotic pathways. The data presented here will allow future hypothesis-driven studies of individual neuronal proteins in an effort to elucidate protein targets which may be useful for the development of DS therapies. Paper reference: Paper 10.2 Synaptic scaling as a probable cause of increase in synaptic markers in young adult triple transgenic mouse model of Alzheimer’s disease (AD) *Dr. Narjes Baazaoui, The New York Institute for Basic Research and Cuny the graduate center, United States ([email protected]) Abstract Synaptic loss is strongly correlated with cognitive decline in the brains of Alzheimer’s disease (AD) patients. The triple transgenic mouse model (3xTg-AD) is known to develop neuropathological features of AD. Here we report the study of the spatial reference memory of

12 week old 3xTg-AD mice using Morris water maze task as well as the study of general behavior. We also studied immunohistochemically the neuronal and synaptic markers in three different groups of 3xtg-AD mice: 13, 14 and 15 weeks of age. The 3xtg-AD mice showed a clear impairment in reference memory in the Morris Water Maze Task at 12 weeks of age. The 12 week old 3xtg-AD mice instead of synaptic loss they showed increase in the expression of presynaptic as well postsynaptic markers and in β-ΙΙΙ tubulin. Indeed, synaptophysin significantly increased in CA1, CA3, dentate gyrus (DG) and parietal cortex at 14 weeks of age and in the DG at 15 weeks of age. GluR1 increased both in DG and CA3 regions at 14 weeks of age and in the DG at 15 weeks of age. β-ΙΙΙ tubulin increased both in parietal and frontal cortices at 14 weeks of age and in the CA3 region at 15 weeks of age indicative of increase in neuronal excitability. No significant difference in MAP2 was detected between 3xTg-AD and wild type at 14 weeks of age but there was a decrease in the DG of 3xTg-AD mice at 15 weeks of age. These findings for the first time raise the intriguing possibility that AD pathology causing mutated transgenes may initially cause an increase in synaptic markers as a compensatory mechanism for synaptic loss akin to synaptic scaling and this increase most likely represents dysfunctional synaptic connections. Background Synaptic loss is strongly correlated with cognitive decline in the brains of Alzheimer’s disease (AD) patients. The triple transgenic mouse model (3xTg-AD) is known to develop neuropathological features of AD Materials and Methods Here we report the study of the spatial reference memory of 12 week old 3xTg-AD mice using Morris water maze task as well as the study of general behavior. We also studied immunohistochemically the neuronal and synaptic markers in three different groups of 3xtg-AD mice: 13, 14 and 15 weeks of age Results The xtg - AD mice showed a clear impairment in reference memory in the Morris Water Maze Task at weeks of age The week old xtg - AD mice instead of synaptic loss they showed increase in the expression of presynaptic as well postsynaptic markers and in β-ΙΙΙ tubulin Indeed synaptophysin significantly increased in CA1 CA3 dentate gyrus ( DG ) and parietal cortex at weeks of age and in the DG at weeks of age GluR1 increased both in DG and CA3 regions at weeks of age and in Conclusion These findings for the first time raise the intriguing possibility that AD pathology causing mutated transgenes may initially cause an increase in synaptic markers as a compensatory mechanism for synaptic loss akin to synaptic scaling and this increase most likely represents dysfunctional synaptic connections. Paper reference: Paper 10.3 Orientation Selectivity in Primary Visual Cortex is modulated by inputs from the primary auditory cortex *Dr. Leena Ali Ibrahim Marosh(Leena A Ibrahim) , University of Southern California, United States ([email protected])

Abstract Primary cortical areas have been thought to be strictly uni-sensory; a visual stimulation will only elicit a response in the visual cortex. Recently however, some extracellular recordings revealed interactions between primary auditory and visual cortices, challenging the strict uni-sensory view. The circuitry underlying these interactions and their functional role remain unknown. Using viral tracing methods, we observed fibers originating in layer 5 of the primary auditory cortex (A1) that project to the superficial layers of the primary visual cortex (V1). To investigate the role of these projections, we expressed channelrhodopsin in layer 5 of the auditory cortex and explored how optogenetic activation of this projection influences visual processing in different layers of V1. Loose patch recordings revealed significant sharpening of orientation tuning of excitatory neurons in layer 2/3 when the mouse was presented with low contrast stimuli as opposed to high contrast stimuli. This phenomenon was also observed using calcium imaging when the mouse was given visual stimulation at different contrasts coupled with sound stimulation. Both sound stimulation and Channelrhodopsin activation caused a robust increase of firing rate in layer 1 neurons, as observed using both loose patch recordings and calcium imaging. In addition, a subtype of inhibitory neurons in layer 2/3 of V1, the somatostatin positive neurons, which have previously been shown to be orientation tuned, were inhibited with sound stimulation. Layer 1 neurons have previously been shown to connect with layer 2/3 excitatory as well as inhibitory neurons. The inhibition of orientation tuned somatostatin neurons by layer 1 relieves the inhibition on excitatory neurons in an orientation dependent manner, thereby causing an increase of the firing rate at the preferred orientation. The combined effect of disinhibition and layer 1 inhibition on layer 2/3 excitatory neurons sharpens their orientation tuning. Background Excitatory neurons in the primary visual cortex tend to respond to stimuli at a specific orientation a property termed Orientation Selectivity This property primarily emerges in the primary visual cortex which is thought to be strictly uni - sensory Recently however this strict uni - sensory view has been challenged and more evidence is emerging that supports multi sensory integration even at the primary cortical levels Using virus injections we observed neurons projecting directly from Layer5 of the primary auditory cortex to superficial layers of the primary visual cortex Materials and Methods AAV2 - ChR2 ( flox ) virus was injected in the primary auditory cortex of Rbp4 - Cre mice ( labels layer5 neurons ). Loose patch recording was done to measure the activity of neurons in the primary visual cortex in response to visual stimulation consisting of moving sinusoidal gratings at different contrasts with and without Chr2 activation or sound stimulation Calcium Imaging was done using genetically encoded calcium indicator GCaMP6s to record responses from different types of inhibitory neurons in Layer1 and Layer2/3 using specific Cre mouse lines Results Sound stimulation or ChR2 activation of primary auditory neurons increases the activity of inhibitory neurons in Layer 1 of primary visual cortex. Layer 1 neurons in turn inhibit layer 2/3 excitatory neurons and somatostatin positive inhibitory neurons, which are themselves thought to be orientation tuned. Because of this, the firing rate at the preferred orientation of

excitatory neurons (orientation at which there is maximum firing rate) increases while at all other orientations decreases, thereby sharpening the tuning of the excitatory neurons. Conclusion Sound stimulation or ChR2 mediated activation of the projections from layer 5 of the primary auditory cortex sharpens the orientation tuning of neurons in layer 2/3 of the primary visual cortex. Paper reference: Paper 11.1 Hypoxia regulated long non-coding RNAs in breast cancer: Novel insights of hypoxic noncoding transcriptome *Dr. Hani Choudhry, University of Oxford, UK ([email protected]) Abstract Transcriptional responses to hypoxia are central to the pathogenesis of many types of cancer. To date, pan-genomic analyses of these transcriptional responses have focussed on proteincoding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. We undertook an integrated pan-genomic analysis of normoxic and hypoxic MCF7 breast cancer cells, employing RNA-seq of polyA selected and ribosomally depleted transcripts together with ChIP-seq for the major hypoxia-inducible transcription factor (HIF) and for chromosomal markers of active transcription (RNApol2 and histone H3K4 methylation). We establish a computational pipeline for total RNA-seq analysis to detect non-coding transcripts including piwiRNA, miRNA, tRNA, sn/snoRNA, and lncRNA. Analyses have revealed that hypoxia profoundly regulated all RNA classes. We describe a number of hypoxia regulated non-annotated RNA species. Significant numbers of lncRNAs were upregulated in hypoxia and these were associated both with epigenetic marks of increased transcription and with HIF binding. The most hypoxia upregulated lncRNA was NEAT1, which is a direct transcriptional target of HIF. We demonstrate that hypoxic NEAT1 induction is common in breast cancer cell lines and xenografts. NEAT1 directly induces the formation of nuclear paraspeckles in hypoxia, contributes to tumourigenicity in cell proliferation and colony forming assays and reduces rates of apoptosis. Finally, in a large cohort of 2000 breast cancers, high levels of NEAT1 correlated with poor clinical outcome. Our findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts. These results will provide new insights on functional and clinical potential of hypoxia regulated lncRNAs which may act as novel therapeutic targets in future. Background Transcriptional responses to hypoxia are central to the pathogenesis of many types of cancer. To date, pan-genomic analyses of these transcriptional responses have focussed on proteincoding genes and microRNAs. However, the role of other classes of non-coding RNAs, in particular lncRNAs, in the hypoxia response is largely uncharacterised. Materials and Methods We undertook an integrated pan-genomic analysis of normoxic and hypoxic MCF7 breast cancer cells, employing RNA-seq of polyA selected and ribosomally depleted transcripts

together with ChIP-seq for the major hypoxia-inducible transcription factor (HIF) and for chromosomal markers of active transcription (RNApol2 and histone H3K4 methylation). Results We establish a computational pipeline for total RNA-seq analysis to detect non-coding transcripts. We describe a number of hypoxia regulated non-annotated RNA species. Significant numbers of lncRNAs were upregulated in hypoxia and these were associated with epigenetic marks of increased transcription. The most hypoxia upregulated lncRNA was NEAT1, which is a direct transcriptional target of HIF. We demonstrate that hypoxic NEAT1 induction is common in breast cancer cell lines and xenografts. NEAT1 induces the formation of nuclear paraspeckles in hypoxia, contributes to tumourigenicity. Conclusion Our findings extend the role of the hypoxic transcriptional response in cancer into the spectrum of non-coding transcripts. These results will provide new insights on functional and clinical potential of hypoxia regulated lncRNAs which may act as novel therapeutic targets in future.

Paper reference: Paper 11.2 In vitro cytotoxicity testing and molecular analysis of modulation of gene expression of proapoptotic gene p53 and anti-apoptotic gene BCl2 on cancer cell lines after treatment with green synthesized metal nanoparticles *Dr. Hussaina Banu Buhari Malkhan Ali, Manipal University, Dubai, UAE ([email protected]) Dr. Renuka Seenivasan, Manipal University, Dubai, UAE Dr. Raees Ismail, Manipal University, Dubai, UAE Dr. Nadia Saadatmand, Manipal University, Dubai, UAE Dr. Vinitha Singh, Manipal University, Dubai, UAE Dr. Faheem SM, Manipal University, Dubai, UAE Dr. Geetha Vasanthakumar, HITS lab, UAE Abstract Cancer is a chronic health condition and scientists are continuously looking for an efficient approach to treat cancers. Due to the adverse side effects of chemotherapy, current cancer treatment focusses on the use of anti-cancer compounds present in medicinal plants with almost no side effects. Nanotechnology is an interdisciplinary science that offers a platform for increasing the solubility of bioflavonoids and improving the bioavailability of bioflavonoids by overcoming drug efflux pumps. The current study focusses on the green synthesis of stable metal nanoparticles using natural extracts of medicinal desert plants rich in bioflavonoids, namely date palm pollen, ghaf and miswak. The bioflavonoids acts as reducing and stabilizing agents in the preparation of metal nanoparticles and these bioflavonoids, which are bound to the metal nanoparticles, are delivered to the cancer cells. In vitro cytotoxicity testing and molecular analysis of modulation of gene expression of pro-apoptotic gene p53 and anti-

apoptotic gene BCl2 on MCF-7 breast cancer cell line gives insight into the molecular alterations happening in the cancer cell in response to the nanoparticle treatment. The research comprises the following objectives: Green synthesis of stable metal nanoparticles using bioflavonoids from date palm pollen, ghaf and miswak extracts as reducing and stabilizing agents; Characterization of surface plasmon resonance, size, shape, elemental composition and stability using UV Visible spectroscopy, Scanning Electron Microscopy and Energy Dispersive Spectroscopy; In vitro studies to compare the bioavailability of flavonoids of natural extract and metal nanoparticle with bioactive groups on MCF-7 cell lines using MTT cytotoxicity assay; Analysis of morphological changes in cell after nanoparticle treatment using fluorescent microscopy; and Molecular analysis of modulation of gene expression of pro-apoptotic gene p53 and antiapoptotic gene BCl2 using immunoassays. Paper reference: Paper 12.1 Effectiveness of using Liquid Chromatography-Tandem Mass Spectrometry and Gas Chromatography in detecting Methymalonic Acidemia *Dr. Huda Salim Al Ketibia (Huda S. Al Ketibia), Sharjah Higher Colleges of Technology, UAE ([email protected]) Dr. Nishi Singh, Sharjah Higher Colleges of Technology, UAE Dr. Ban Altoumah, Sharjah Higher Colleges of Technology, UAE Dr. Muhammad Zaman, Sharjah Higher Colleges of Technology, UAE Dr. Nafisa M Tawfiq, Metabolic Genetics Unit, DHA, UAE Dr. Shagira A Alnuaimi, Metabolic Genetics Unit, DHA, UAE Abstract Methylmalonic acidemia (MMA) is an inherited disorder whereby the body is unable to process selected proteins and lipids properly. The disease can range from mild to life-threatening, and includes symptoms such as dehydration, vomiting, hypotonia lethargy and hepatomegaly. Debilitating long term effects include intellectual disability, chronic kidney disease and pancreatitis leading to death. Screening of MMA is generally performed by amino-acid /acetylcarnitine analysis on dried blood spot samples and organic acid analysis on urine using tandem masses analyzers such as MS/MS and GC/MS. At Dubai Latifa Hospital initial MMA screening is performed by Liquid Chromatography-Tandem Mass Spectrometry (LC/MS/MS), with initial positives tested further by Gas Chromatography (GC/MS). In our study we tested the effectiveness of tandem LC-MS/MS and GC/MS methodology by comparing analysis with LC-MS/MS alone. We analyzed three hundred and sixty six DBS samples received for IEM screening at Dubai Genetic Center for the year 2008 to 2010 for AA/AC analysis (primary C3 concentration for MMA detection) with LC-MS/MS and organic acid analysis (primary methylmalonate or methyl citrate levels) with GC/MS. Of these 366 samples, 20 tested positive of which only 4 confirmed for MMA with GC/MS. By contrast 7 cases showed normal level of C3 but were MMA confirmed or suspected positive. Statistically our results demonstrated 36.4% sensitivity and 95.5% specificity with 1.09% population prevalence. The

low sensitivity rate is of concern, considering false positives are clinically less detrimental to MMA patient outcomes than false negatives. Background Methylmalonic acidemia is an inherited disorder whereby the body is unable to process selected proteins and lipids properly The disease can range from mild to life - threatening and includes symptoms such as dehydration vomiting hypotonia lethargy and hepatomegaly Debilitating long term effects include intellectual disability chronic kidney disease and pancreatitis leading to death Screening of MMA is generally performed by amino - acid acetylcarnitine analysis on dried blood spot samples and organic acid analysis on urine using tandem masses analyzers such as MS MS and GC Materials and Methods In our study we tested the effectiveness of tandem LC-MS/MS and GC/MS methodology by comparing analysis with LC-MS/MS alone. Results We analysed three hundred and sixty six DBS samples received for IEM screening at Dubai Genetic Center for the year 2008 to 2010 for AA/AC analysis (primary C3 concentration for MMA detection) with LC-MS/MS and organic acid analysis (primary methylmalonate or methyl citrate levels) with GC/MS. Of these 366 samples, 20 tested positive of which only 4 confirmed for MMA with GC/MS. By contrast 7 cases showed normal level of C3 but were MMA confirmed or suspected positive. Conclusion Statistically our results demonstrated 36.4% sensitivity and 95.5% specificity with 1.09% population prevalence. The low sensitivity rate is of concern, considering false positives are clinically less detrimental to MMA patient outcomes than false negatives. Paper reference: Paper 12.2 A novel MT-COI m.6498C>A variation associated with the m.7444G>A mutation in the mitochondrial COI/tRNASer(UCN) genes in a patient with hearing impairment, diabetes and congenital visual loss *Dr. Emna MKAOUAR-REBAI (MKAOUAR-REBAI E), Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA, Tunisia ([email protected]) Dr. Imen Chamkha, Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA, Tunisia Dr. Thouraya Kammoun, Service de Pédiatrie, C.H.U. Hédi Chaker de Sfax, TUNISIA, Tunisia Dr. Hajer Aloulou, Service de Pédiatrie, C.H.U. Hédi Chaker de Sfax, Tunisia, Tunisia Dr. Mongia Hachicha, Service de Pédiatrie, C.H.U. Hédi Chaker de Sfax, Tunisia, Tunisia Abstract Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. Sensorineural hearing loss (SNHL) has been described in association to different mitochondrial multisystem syndromes, often involving the central nervous system, neuromuscular, or endocrine organs. In this study, we described a

Tunisian young girl with hearing impairment, congenital visual loss and maternally inherited diabetes. No mutation was found in the mitochondrial tRNALeu(UUR) and the 12S rRNA genes. However, we detected the m.7444G>A mutation in the mitochondrial COI/tRNASer(UCN) genes. This mutation eliminates the termination codon of the MT-CO1 gene and extends the COI polypeptide by three amino acids (Lys-Gln-Lys) to the C-terminal. The whole mitochondrial genome screening revealed the presence of a novel mutation m.6498C>A (L199I) in the mitochondrial DNA-encoded subunit I of the cytochrome c oxidase (COX). This “probably damaging” transversion affects a highly conserved domain and it was absent in 200 Tunisian controls. The studied patient was classified under the haplogroup H2a.

Paper reference: Paper 12.3 Cardiac Proteome alterations induced by Diabetic Oxidative Stress in OLETF Rats *Dr. Abdelbary Mohammed Prince Hussein (Abdelbary Prince (A. Prince)), Cairo University, Egypt ([email protected]) Abstract Diabetic cardiomyopathy has been documented as an underlying etiology of heart failure among diabetics. Although oxidative stress has been proposed to contribute to diabetic cardiomyopathy, much of the evidence lacks specificity. Furthermore, whether alterations occur at the cardiac proteome level in diabetic cardiac complications with attendant oxidative stress remains unknown. Therefore, we sought to identify cardiac protein changes in relation to myocardial oxidative stress that are specific to diabetic cardiomyopathy. Animal model of type 2 diabetes (OLETF rat) was used to investigate the alteration of myocardial proteome in diabetic cardiomyopathy. OLETF rats were examined for diabetic cardiomyopathy at 35 weeks of age by histopathogical and histochemical analyses. Myocardial oxidative stress was shown in diabetic rats, as indexed by significant increase in mitochondrial superoxide formation. In-depth mining of the diabetic myocardial proteome by proteomic analysis utilizing two-dimensional gel electrophoresis and mass spectrometry (2DE/MS) techniques revealed down-regulation of antioxidant and anti-apoptotic proteins and up-regulation of fatty acid oxidation related proteins in diabetic hearts. These results characterize a role of substrate switch to fatty acid utilization in alteration of metabolic pathways in diabetic cardiomyopathy. Background Cardiovascular disease is the leading cause of death in patients with diabetes which presents as diabetic cardiomyopathy. Ample evidence has demonstrated the existence of a unique diabetic cardiomyopathy independent of microvascular complications. Diagnosis of diabetes often occurs after a prediabetic state accompanied by an absence of pathognomic symptoms. In the present study, we performed proteomic analysis on total cardiac proteins from rat model for spontaneous type 2 diabetes mellitus to identify protein changes that may contribute to diabetic cardiomyopathy. Materials and Methods Methods Experimental design Sampling Biochemical analysis Determination of plasma glucose Determination of plasma immunoreactive insulin Determination of plasma free fatty acids ( FFA

) Cardiovascular studies Isolated rat heart preparations Triphenyltetrazolium chloride ( TTC ) staining Electron microscopy Histopathological examination Myocardial proteome analysis Whole tissue protein isolation Determination of the protein concentration Isoelectric focusing ( IEF ) Immobilized pH gradient ( IPG ) strip equilibration The two dimensional ( - D ) separation Silver staining Protein identification ( MALDI - TOF ) Mitochondrial studies Flow cytometric measurement of mitochondrial Results A total of protein spots representing proteins are differentially expressed in diabetic hearts of type The altered proteins include apoptosis - associated proteins chaperones transport regulators gene and cell cycle regulatators cell - signaling proteins thrombolysis related proteins regulators for oxidative stress mitochondrial proteins and cytoskeletal proteins Some of the altered proteins have been previously shown to be regulated during diabetes whereas roles for the other altered proteins have not previously been established suggesting that they may be involved in the novel mechanisms of diabetic cardiomyopathy Conclusion In combination with physiological analyses the application of proteomics is the strategy of choice to elucidate the mechanisms of diabetic cardiomyopathy and to identify protein - based drug targets disease biomarkers and therapeutic peptides and proteins thus providing the basis for pharmacological and gene therapies. In this study proteins involved in fatty acid metabolism were found to be up-regulated. In connection with this finding there was a decreased expression of antioxidant and anti-apoptotic proteins

Paper reference: Paper 13.1 Stem cells based tissue engineering for lung repair *Dr. Layla Alhasan, RMIT University, Australia ([email protected]) Dr. Peggy Chan, RMIT University, Australia Abstract Lung diseases caused significant mortality and morbidity worldwide. Lung transplantation is the only option for the patient with end stage of lung diseases such as chronic obstructive pulmonary diseases (COPD), however, there is limited donor lungs available for transplantation. Typically, transplantation recipients suffered with immune response problems resulting in only 50% survival. Transplantation of alveolar epithelial cells derived from embryonic stem cells (mESCs) may provide a novel strategy to regenerate endogenous lung cells damaged by lung disease. A novel injectable hydrogel system with tuneable stiffness and fast gelation time has been synthesized as a tissue scaffold to support the proliferation and differentiation of pluripotent embryonic stem cells into lung epithelial cells. The stiffness of the hydrogels was readily tuned by changing the biocompatible cross-linked and polymer precursor

concentrations. In this study we have compared differentiated embryonic stem cells in 3D scaffolds in presence and absence growth factors into definitive endoderm by measuring Ecadherin and Foxa2 as markers for definitive endoderm. The expression of Pept2, Sema4F and Unc5b in hydrogel embedded cells were assessed using quantitative polymerase reaction (qPCR), the expression of these markers were up-regulated expression both in presence and absence growth factors. Moreover, the expression of E-Cadherin and Pept2 confirmed that the hydrogel support the differentiation of mESCs to lung distal airway epithelial cells. Background Lung diseases are a significant disorder causing morbidity and mortality in the western world. There is no therapy for most of lung diseases including chronic obstructive pulmonary diseases (COPD), idiopathic pulmonary fibrosis (IPF) and cystic fibrosis. Cell therapy displays a promising approach to treat multiple diseases in tissue engineering and regenerative medicine areas as well as it plays a critical role in repair of damaged tissues and organs in vivo and formation tissue constructs in vitro for transplantation. Materials and Methods The scaffold was synthesized using a general method and characterised using swelling ratio and rheology test. Cytotoxicity of scaffold was assessed using Calcien AM kit by measuring cell viability. Proliferation rate and gene expression for differentiated embryonic stem cells to lung epithelial cells were determined using micro- plate reader and q-PCR. Results we have compared differentiated embryonic stem cells in D scaffolds in presence growth factors into definitive endoderm by measuring E - cadherin and Foxa2 as markers for definitive endoderm. The expression of Pept2 Sema4F and Unc5b in hydrogel embedded cells were assessed using quantitative polymerase reaction ( q - PCR ), the expression of these markers were up - regulated expression both in presence and absence growth factors . Conclusion Scaffold hydrogel has many properties that mimic lung tissue and all nutrients and oxygen can reach through diffusion as well as scaffold is very strong to make tissue engineered lung that can be suitable for lung tissue repair. Paper reference: Paper 13.2 Role of Low Level Laser in Tissue regeneration *Dr. Mohamed Helmy El Batanouny (El-Batanouny M), Emeritus Prof. of General and Vascular Surgery Faculty of Medicine Former head of Department of laser medical and Biological National Institute of laser enhanced Sciences, Egypt ([email protected]) Abstract Introduction: Almost 20 years ago the field of laser Biostimulation was full of controversies. Some of our studies are presented. Materials and Methods: Application of low level laser with different doses was used in stimulating healing in different studies as in repaired rabbit tendons, crushed rabbit median nerve, chronic leg ulcers in human with different causes, bone healing in rabbit, protection of

liver and skin in mice exposed to cummulative and acute ionizing radiation as well as to enhance the healing and approximating of teeth in orthodontic studies in humans. Results: Comparing the results in the study treatment group to control group showed definite significant role in enhancing regeneration towards healing as proved by parameter implemented according to each study assessing functional, histopathological and ultrastructural findings. Several factors should be considered as the laser dose, the disease or pathology tackled Paper reference: Paper 13.3 Differential effect of Epigenetic Modifiers *Dr. Ahmed Taher El-Serafi, University of Sharjah, UAE ([email protected]) Abstract Please see above. Background DNA demethylating agents and histone deacetylase inhibitors are chemical compounds that are approved for the treatment for certain malignant diseases. Such agents are supposed to inhibit the DNA methyltransferase and histone deacetylases, which should be associated with enhanced gene transcription. Thus stem cells differentiation should be more effective and cancer cell lines should be more responsive to treatment; which we found not to be the case. Materials and Methods Bone marrow and fetal derived stem cells and cancer cell lines were used in a 3 days course treatment regimen for the modifiers followed by culturing the cells in osteogenic and chondrogenic media to drive the stem cell differentiation (in two and three dimensional culture models) or in classical media to study the effect on the cancer cells. Results were verified by real time PCR, western blotting, histology and immunohistochemistry. Results Enhanced osteogenic matrix formation and osteogenic related genes with adult derived stem cells and not with the fetal with the DNA demethylating agents. Enhanced chondrogenic condition with histone deacetylase inhibitor in adult stem cells. Enhanced expression of markers of deceased response to chemotherapy in cancer cells. Conclusion The effect of such modifiers would vary according to the cell type. The action of both classes would not overlap or it may counteract each others in some instances. Paper reference: Paper 13.4 Effect of long term proliferation of adipose derived stem cell (ADSC) on immunomodulation and stemness markers *Dr. Oula Nabil El ATAT, Saint Joseph University, Lebanon ([email protected]) Abstract

Mesenchymal stem cells are known to have an immunomodulatory, antiapoptotic, angiogenic, anti-fibrotic, and chemotactic effect. Thus they play an important role not only in maintaining tissue homeostasis but also as potential future therapeutic tools in many tissues injuries. Adipose Derived Stem cells (ADSC) are being used therapeutically in many human diseases but also extensively in plastic surgery and arthritis. In both cases a large number of cells is essential to generate efficient results, where the need for culturing and expanding the cells in vitro for several weeks. The immunophenotypic properties of freshly isolated human adipose tissue derived stromal vascular fraction (SVF) and serial passaged ADSC (P0-P4) were observed by flow cytometry. In parallel, we compared the telomerase activity and the aldehyde dehydrogenase activity of SVF relative to ADSC (P0-P4) using a quantitative telomerase detection kit (QTDKit) and Aldehyde Dehydrogenase Based Cell Detection Kit (ALDEFLUOR™) respectively. The cytokines secretion profile of ADSC during passages was also analyzed by ELISA kits. SVF and ADSC were positive for CD 29, CD 44, CD 73, CD 90, and CD 105, and they were negative for CD 31, CD34, CD45 and CD 106. The telomerase activity was low in SVF and non-increased during P0 to P4 .However it always decreases at day 21 during each passage. Aldehyde dehydrogenase was detected in SVF with no changes with serial passages . The level of the cytokine IL-6, and IL8 increased significantly during passages. However, the level of TIMP1 and TIMP2 was not affected. Therefore, it seems that the expansion of ADSC does not affect the stemness of the cells and tends towards a negative cancerous capacity. Background Mesenchymal stem cells are known to have an immunomodulatory antiapoptotic angiogenic anti - fibrotic and chemotactic effect Thus they play an important role not only in maintaining tissue homeostasis but also as potential future therapeutic tools in many tissues injuries Adipose Derived Stem cells ( ADSC ) are being used therapeutically in many human diseases but also extensively in plastic surgery and arthritis In both cases a large number of cells is essential to generate efficient results where the need for culturing and expanding the cells in vitro for several Materials and Methods SVF and ADSC were isolated purified and cultured in vitro from lipoaspirates using a well established protocol The immunophenotypic properties of freshly isolated human adipose tissue derived stromal vascular fraction ( SVF ) and serial passaged ADSC ( P0 - P4 ) were observed by flow cytometer In parallel we compared the telomerase activity and the aldehyde dehydrogenase activity of SVF relative to ADSC ( P0 - P4 ) using a quantitative telomerase detection kit ( QTDKit ) and Aldehyde Dehydrogenase Based Cell Detection Kit ( ALDEFLUOR ™) respectively Results SVF and ADSC were positive for CD 29, CD 44, CD 73, CD 90, and CD 105, and they were negative for CD 31, CD34, CD45 and CD 106. The telomerase activity was low in SVF and nonincreased during P0 to P4 .However it always decreases at day 21 during each passage. Aldehyde dehydrogenase was detected in SVF with no changes with serial passages .The level of the cytokine IL-6, and IL8 increased significantly during passages. However, the level of TIMP1 and TIMP2 was not affected. Conclusion

Therefore, it seems that the expansion of ADSC does not affect the stemness of the cells and tends towards a negative cancerous capacity. Paper reference: Paper 13.5 Utilizing The Interrupted Electromagnetic Field For Autologous Growth Factors Release. *Dr. Aziz Denian, Denian Regenerative Rehabilitation Clinic – Jordan, Jordan ([email protected]) Abstract The preparation of PRP - PRF requires a significant amount of delicate platelets exposing force. At our practice we use two methods, one is always the interrupted electromagnetic field force. This ensure an effective and delicate GF concentration flow out, therefore the preparation could have its maximum clinical benefit. Paper reference: Paper 14.1 TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function Dr. Sherif Faruk El-Khamisy (El-Khamisy, S.F.), University of Sheffield, UK ([email protected]) Abstract The human genome is under continuous threat from environmental genotoxins and endogenous sources generated during normal metabolic activities. Failure to repair these breaks results in human disease, such as cancer, neurodegeneration, and immunodeficiency. It also contributes to complications associated with diseases with high social impact such as diabetes, reproductive, and cardiovascular disorders (El-Khamisy, EMBO Mol Med, 2011). One type of these breaks features a protein covalently linked to DNA termini. This class of DNA damage arises during the enzymatic cycles of DNA topoisomerases to overcome torsional barriers ahead of many DNA transactions. Beside their implications in the aetiology of human disease, these protein-linked DNA breaks underlie the clinical utility of an important class of anticancer topoisomerase ‘poisons’ that is widely used the clinic. Removal of the covalently linked topoisomerase (Top) from DNA termini is mandatory prior to subsequent repair steps. This is achieved by Tyrosyl DNA phosphodiesterases that hydrolyses the 3’-phosphotyrosyl bond between Top1 and DNA (El-Khamisy et al., Nature, 2005). Recently we identified the corresponding activity that removes Top2 from the 5’-teminus of a DNA strand break and it was subsequently named TDP2 (Ledesma & El-Khamisy et al., Nature, 2009; Gómez-Herreros et al., 2014). I will discuss our recent understanding of these pathways and ways to exploit them in neuroscience and as novel biomarkers and drug targets to improve cancer therapy. Paper reference: Paper 14.2

From the bench to bed side: Deciphering the molecular basis of Hereditary Spastic Paraplegia S M Wakil1, , K. Ramzan1, S. Hagos1, H. Aldossari1, R abothuraya1, D. Monies1, B. Meyer1, Z. Alhassnan2 , S.A. Bohlega3 1Department of Genetics, 2Department of Department of Medical Genetics, 3Department of Neurosciences, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia ([email protected]). Abstract Hereditary Spastic Paraplegia (HSP), a motor neuron disorder belonging to the group of neurodegenerative disorders is genetically heterogeneous characterized by progressive spasticity and weakness of the lower limbs. HSP is classified as pure or uncomplicated form or as complex or complicated if manifested with additional neurological features. A large no. of HSP loci have been mapped so far and in these loci, disease-causing mutations in approx. 34 genes have been identified (Online Mendelian Inheritance in Man). These loci have been found to increasing among Arabian Peninsula due to high rate of consanguinity and endogamy in the population. The HSP proteins involved have diverse functions, of which the most prominent are regulation of intracellular trafficking, organelle biogenesis, and shaping of membranes. We utilized combined homozygosity mapping and exome sequencing approaches to establish the genetic causes associated with different types and subtypes of HSP. More than 34 native families were recruited and their clinical, imaging and metabolic data has been collected. We identified SPG11 as the main cause of HSP and accounting for 18% of the families. In four families we identified novel variations in Alsin, erlin and B4GALNT1 genes. In 20 patients, no known gene was identified and Exome sequencing is in a process to further identify the genetic cause. The clinical variability observed in these families is supported by the large underlying genetic heterogeneity. Discovering such new mutations/genes should help in understanding HSP pathophysiology and possible therapeutic options. This will also provide prenatal as well as postnatal genetic counseling for these HSP families.

Paper reference: Paper 14.3 Global epigenetic status of endometrial tissue shows significant alterations in endometriosis *Dr. Elham Hosseini, Anatomy Department, School of Medicine ,Iran University of Medical Sciences, Tehran, Iran, Iran ([email protected]) Dr. Maryam shahhoseini, Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran, Iran Dr. Raha Favaedi, Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran, Iran Dr. Mahnaz Ashrafi, 3Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran, Iran Dr. Fariba Ramezanali, 3Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran,

Iran, Iran Dr. Fereshteh Mehraein, Anatomy Department, School of Medicine ,Iran University of Medical Sciences, Tehran, Iran, Iran Dr. Reza Aflatoonian, 3Department of Endocrinology and Female Infertility at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran, Iran Abstract Endometriosis defined as presence of endometrium like tissue outside of uterine, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is poorly understood, some evidence suggests that epigenetic factors are being considered as one possible mechanism through which endometriosis can develop. The two main epigenetic mechanisms, DNA methylation and histone modifications, regulate expression of genes undergoing epigenetic abnormality in diseases. Therefore, the aim of this study was to investigate the alterations of DNA methylation and histone acetylation/methylation in eutopic endometrium of endometriosis patients. For this respect, Informed consents were gained from all patients then eutopic endometrium samples (n=5) were obtained from endometriosis patients undergoing endometrial biopsy, as well as endometrial tissues from healthy women (control group, n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from all samples were prepared following fixation with formaldehyde and then shearing into fragments by sonication. Nucleosome ELISA was performed on extract chromatins using antibodies against H3K9ac, H3K9me2 and MeCP2, respectively. Our data revealed that eutopic endometrial samples were globally DNA hypermethylated compared to endometrium tissue from controls. Furthermore, a significant hyperacetylation as well as hypermethylation at histone H3K9 was observed in eutopic samples compared to control group. These results clearly show that epigenetic abnormalities such as aberrant DNA methylation and histone acetylation/methylation status may play a dynamic role in endometriosis and support the opinion that endometriosis may be an epigenetic disease. Background Endometriosis defined as presence of endometrium like tissue outside of uterine, is a common gynecologic disorder. Although endometriosis is a multifactorial disease and the exact etiology is poorly understood, some evidence suggests that epigenetic factors are being considered as one possible mechanism through which endometriosis can develop. The two main epigenetic mechanisms, DNA methylation and histone modifications, regulate expression of genes undergoing epigenetic abnormality in diseases. Therefore, the aim of this study was to investigate the alterations of DNA methylation and histone acetylation/methylation in eutopic endometrium of endometriosis patients Materials and Methods For this respect, Informed consents were gained from all patients then eutopic endometrium samples (n=5) were obtained from endometriosis patients undergoing endometrial biopsy, as well as endometrial tissues from healthy women (control group, n=5) during the proliferative phase of menstrual cycle. Chromatin extracts from all samples were prepared following fixation with formaldehyde and then shearing into fragments by sonication. Nucleosome ELISA was

performed on extract chromatins using antibodies against H3K9ac, H3K9me2 and MeCP2, respectively. Results Our data revealed that eutopic endometrial samples were globally DNA hypermethylated compared to endometrium tissue from controls. Furthermore, a significant hyperacetylation as well as hypermethylation at histone H3K9 was observed in eutopic samples compared to control group. Conclusion These results clearly show that epigenetic abnormalities such as aberrant DNA methylation and histone acetylation/methylation status may play a dynamic role in endometriosis and support the opinion that endometriosis may be an epigenetic disease. Paper reference: Paper 14.4 Massively Parallel Sequencing Identification of Novel Genes and Mutations for Breast Cancer and Hereditary Hearing Loss in Palestine *Dr. Moien Nihad Kanaan (kanaan,M), Bethlehem university, Palestinian Territory ([email protected]) Abstract BACKGROUNG AND PURPOSE: Identification of inherited mutations has been an ongoing challenge in human medical genetics. Using advanced targeted DNA capture and massively parallel sequencing technologies, in conjunction with homozygosity mapping relevant for consanguineous families, we are meeting this challenge for hereditary hearing loss, Breast cancer and other genetic abnormalities. Our cohort consists of Palestinian Arab families of variable size, and onset of hearing loss and breast cancer. METHODS AND MATERIALS We constructed two custom design arrays of cRNA oligonucleotides containing 250 genes, responsible for both human and mouse deafness. And a custom array containing 38 breast and ovarian cancer (BROCA) associated genes. We prepared paired-end libraries, followed by cluster amplifications on v4 Illumina flow cells with our bar-coded multiplexed samples. A 2x72bp paired end recipe was used, resulting in a median base coverage of 300-572x and overall, 94.7% of our targeted bases covered by more than 10 reads, which was our cutoff for variant detection. RESULTS We generated SNP and indel calls for our samples and filtered the variants against those of dbSNP 131 and the 1000 Genomes project to identify private and rare variants. Novel genes and mutations were discovered. Most compelling, a number of mutations were found in genes previously known only to be involved in mouse deafness. Protein structure predictions were made to provide insight into how the mutations lead to the predicted phenotypes. CONCLUSION Discovery of additional deafness, breast cancer genes and mutations will allow for early clinical diagnosis, enabling predication of phenotypes and enhanced management. Characterization of

the proteins encoded by these genes will enable comprehensive understanding of the biological mechanisms involved in the pathophysiology of these disorders. Paper reference: Paper 14.4 Healthy Nation through Healthy Newborns: Role of Genetic Carrier Screening through an Expanded Screening Platform before Pregnancy *J. Horcajadas, PhD1, S. Rodriguez, Sc.M.1, A. Bisignano1, B. Chu2, S. Munne, PhD1,2, N. Kumar, Sc.M.1 1. Recombine Inc, New York, NY 2. Reprogenetics, Livingston, NJ

Abstract Objective Genetic carrier screening is a genetic test performed on prospective parents to determine their risk to pass on a genetic disorder to their children. Typically, genetic carrier screening is performed for a selected few recessive genetic diseases based on ancestral background. Technological advancements in genomics now allow carrier screening to be performed for over 200 pan-ethnic genetic diseases in a cost-effective and high-throughput manner. Our goal is to introduce an expanded carrier screening platform, Recombine. Materials and Methods An Illumina Infinium HD Custom Genotyping platform was designed to perform expanded carrier screening. Genetic diseases and mutations were selected for inclusion based on frequency data in populations around the world, phenotype, and pursuit of preimplantation genetic diagnosis (PGD). Results Our custom genotyping platform can identify 1,679 mutations associated with 213 autosomal recessive and X-linked genetic diseases with high sensitivity and specificity. The platform has been for over 10,000 clinical referrals from reproductive endocrinologists, obstetricians, and genetic counselors. Analyses of our current data show that most carrier frequencies observed are consistent with the literature. Some diseases have been found to be more common than expected based on the literature. For example, we found a carrier rate of 1 in 17 for GJB2related nonsyndromic hearing loss and deafness (NSHL) while the literature-reported rate in the general population is approximately 1 in 43. Our data further demonstrate that 2.4% of couples are identified as carriers of the same genetic disease. Conclusions Couples identified to carry the same genetic disease based on this expanded pan-ethnic carrier screening platform have a 25% risk of having an affected child. Genetic counseling to discuss the many reproductive options available for such couples is critical. One option available is PGD to screen embryos prior to implantation. With population-based carrier screening, we therefore have the opportunity to reduce the incidence of genetic disease. Paper reference: Paper 15.1 Solubility Enhancement of Ethyl Acetate Fraction of The Artocarpus altilis (Parkinson) Fosberg Leaves with Addition of β-Cyclodextrin-HPMC by Using Kneading Method

Dr. sabrina binti dahlizar bin Dahlan, Islamic State University, Jakarta Indonesia, Indonesia ([email protected]) Abstract Ethyl acetate fraction of the Artocarpus altilis (Parkinson) Fosberg extract have a potency to treat the cardiovascular diseases have poorly solubility in water. The purpose of this study was to improve the solubility of the extract. One of method to improve the solubility of the extract by mixing with cyclodextrin polymers and their derivatives. Hydroxyl propyl methylcellulose (HPMC) as a water-soluble polymer can enhance the β-cyclodextrin (β-CD) activity. Three comparisons extract and ß-cyclodextrin were : 1:2, 1:4, and 1:6 by mixing with the addition hydroxyl propyl methylcellulose 0.12% of the total weight of extract and β-CD for each formula. The sample was prepared by kneading method. The sample characterization was used Karl Fischer titration, Scanning Electron Microscopy and solubility study.. Content of total flavonoid from the extract was 32.7%. The Result showed that the addition polymer combination of β-CD + HPMC caused increasing the solubility of extract in water 7.04% (F1), 19.47% (F2) and 59.92% (F3) compared to extract control with significant differences at level of confidence 95% (p ≤ 0.05). Background Ethyl acetate fraction of the Artocarpus altilis (Parkinson) Fosberg extract have a potency to treat the cardiovascular diseases have poorly solubility in water. Materials and Methods One of method to improve the solubility of the extract by mixing with cyclodextrin polymers and their derivatives Hydroxyl propyl methylcellulose ( HPMC ) as a water - soluble polymer can enhance the β- cyclodextrin (β- CD ) activity Three comparisons extract and ß- cyclodextrin were and by mixing with the addition hydroxyl propyl methylcellulose % of the total weight of extract and β- CD for each formula The sample was prepared by kneading method The sample Results polymer combination of β-CD + HPMC can increasing the solubility of extract in water Conclusion Content of total flavonoid from the extract was 32.7%. The Result showed that the addition polymer combination of β-CD + HPMC caused increasing the solubility of extract in water 7.04% (F1), 19.47% (F2) and 59.92% (F3) compared to extract control with significant differences at level of confidence 95% (p ≤ 0.05).

Paper reference: Paper 15.2 Fast, Accurate and Sensitive Quantitation of Genomic DNA Purified from FFPE Samples

*Dr. Robert Brazas, Promega Corporation, United States ([email protected]) Dr. Mark Denhart, Promega Corporation, United States Dr. Michael Bjerke, Promega Corporation, United States Abstract Careful DNA (and RNA) quantitation prior to downstream assays such as next-gen sequencing, PCR, transfection and cloning dramatically improves the success of those assays. Formalin-fixed, paraffin-embedded (FFPE) tissues present significant challenges to the extraction of sufficient amounts of pure genomic DNA due to the harsh fixation conditions and often long-term storage associated with these samples. As a result, common absorbance-based quantitation methods lack adequate detection sensitivity necessary to accurately quantitate FFPE DNA samples prior to analysis. Here we describe the use of fluorescence-based DNA quantitation which offers many advantages over absorbance-based methods for quantitating DNA extracted from FFPE samples. These advantages include improved sensitivity, greater specificity and minimal influence from contaminants that often inflate absorbance-based measurements. Background DNA quantitation prior to downstream assays such as next-gen sequencing, PCR, transfection and cloning dramatically improves the success of those assays. Formalin-fixed paraffinembedded ( FFPE ) tissues present significant challenges to the extraction of sufficient amounts of pure genomic DNA due to the harsh fixation conditions and often long-term storage associated with these samples. As a result, common absorbance-based quantitation methods lack adequate detection sensitivity necessary to accurately quantitate FFPE DNA samples prior to analysis. Materials and Methods DNA was extracted from FFPE samples using the Maxwell 16 Instrument and Kit. Samples were quantitated by (i) qPCR, (ii) absorbance using a NanoDrop 2000 Instrument, and (iii) fluorescence using a Quantus Fluorometer and various fluorescent dyes. Results from the various methods and dye systems were compared. Results Fluorescence-based DNA quantitation using the QuantiFluor dsDNA Dye is 200X more sensitive than absorbance-based measurements on a NanoDrop and is able to measure samples ranging from 0.01ng/ul to 200ng/ul when using 1ul of sample per assay. The QuantiFluor ONE dsDNA Dye, a pre-mixed dye system, is 10X more sensitive than the NanoDrop and capable of measuring samples ranging from 0.2ng/ul to 400ng/ul using 1ul of sample per assay. Conclusion Fluorescence-based DNA quantitation is more sensitive than absorbance-based measurements on a NanoDrop and is better suited to samples with low amounts of DNA, such as FFPE extracted samples. In addition, fluorescence-based quantitation better paralleled results obtained using qPCR, but fluorescence-based quantitation is faster and easier to perform than qPCR. Paper reference: 15.3

Estimating an Age of Splice-site Mutation Causing Hypotrichosis with Juvenile Macular Dystrophy Disease in Pakistani Population Dr. Adeel Ahmed, Quaid-i-Azam University, Islamabad, Pakistan,( [email protected]) Abstract The splice-site mutation at the locus on human chromosome 16q22.1 causes hypotrichosis with juvenile macular dystrophy (HJMD) disease in Pakistani population [1]. Different statistical methods have been used to estimate the age of mutations. We have analyzed a CDH3 gene dataset which consists of genotypes of the Pakistani family in order to compare two methods for estimating an age of IVS10-1 G → T mutation that occurrs in some affected members of a family. The first method is DMLE+ [2] that is a genealogy based method and estimate the mutation age with population growth rate and genotype data. A second method [3] that we used is based on allele frequency. We have found that the mutation age vary with population size, growth rate and mutant allele frequency. Estimates of IVS10-1 G→T mutation based on DMLE+ [2] are 232, 238 and 226 generations with three simulated runs and 95% credible interval, respectively. A method [3] gives an estimated time of 138 generations in units of 2N generations Background We have estimated a time of splice site ( IVS10 - G → T ) mutation occurs in a Pakistani family in CDH3 gene We have used two methods first method is based on allele frequency and is proposed by Kimura et al [ ] and second method is DMLE +, proposed by Rannala et al in [ ] for age prediction We compared two approaches for predicting the age of splice - site mutation with same parameters on the CDH3 gene dataset We have found a mutation age predicted Materials and Methods MCMC method, Simiulation Results TABLE I. ESTIMATES OF MUTATION AGE FOR IVS10-1 G → T Simulation Run (MCMC) Estimated mutation age (generations) 95% credible interval (DMLE) 1 232 [144, 364] 2 238 [152, 385] 3 226 [156, 374] Conclusion In this paper we have considered the problem of mutation age estimation We have analyzed a CDH3 gene dataset for a family of Pakistani population whose some members have splice - site ( IVS10 - G → T ) mutation that causes HJMD disease We have applied two approaches method [ ] and method [ ] over CDH3 gene dataset and successfully estimating a mutation age with % credibility We have observed that the mutation age vary with population growth rate and the allele frequency The estimated time of mutation

Paper reference: Paper 15.4 Isolation and Molecular Identification of cry gene in the Bacillus thuringiensis isolated from soils by Semi-conserve PCR *Dr. Ali Nazemi, Techer, Iran ([email protected]) Abstract Bacillus thuringiensis is a gram positive and spore forming bacterium. The most of the natural habitat this bacterium is soil and it is capable of producing the diversified varieties of crystalline proteins with insecticide property. The aim of this study has been isolation of strains of Bacillus thuringiensis carrying cry gene from soils of the west Mazandaran provinces. After screening the soil of 35 regions, 12 strains of Bacillus thuringiensis were isolated by the selective minimal medium contain sodium acetate and L-serine. After DNA extraction, Bacillus thuringiensis strains were confirmed using 16srDNA PCR sequencing. Also, relative molecular weight of crystalline protein of the strain containing the cry gene was evaluated through SDS-PAGE. Out of 12 isolated strains, cry gene from 1Aa class was identified only in one strain by Semiconserve PCR. Study of rDNA sequence of the isolated strain showed a 99% homology with IBL200 strain of the Bacillus thuringiensis. Also approximate weight of crystalline protein was measured in the range of 90 to 100 KDa. With regard to very much diversity of crystalline gene and lack of existence of accurate phenotypic methods to identify the presence of the crystalline protein using of this semi- conserve PCR method can identify the bacilli containing the crystal gene with low cost and fast method..

Paper reference: Paper 16.A The Role of Biotechnology in Development of Personalized Medicine *Professor Karim Nayernia Institute for Molecular Medicine and Cell Therapy, Düsseldorf, Germany Institute for Personalized Medicine, Cambridge, UK([email protected]( Abstract New Approaches such as pharmacogenetics and proteomics use biotechnology-based technologies and contributed to better diagnosis of a disease by using patients’ genetic information, and also to match the right drug, at the right dose, at the right time to the patient. The progressive elucidation of the molecular pathogenesis of cancer has been hugely enabled by genome sequencing and other large-scale omics approaches, leading to the discovery and development of molecularly targeted drugs and companion diagnostics for personalized treatment. New approaches to personalized medicine for breast cancer that involve molecular screening for clinically relevant genomic alterations and genotype-targeted treatments are emerging. Many of the first breakthroughs in personalized medicine have occurred in breast

cancer research. The identification of genes linked to breast cancer (BRCA1, BRCA2, CDH1, CHEK2, PTEN, p53, ATM) allows us to calculate increased risk for cancer decades before it might develop. Individuals can then make decisions to help prevent or detect cancer early. For women diagnosed with breast cancer, genetic testing on the cancer itself can help determine the need for chemotherapy and immunotherapy and whether they will work. In the talk, we will discuss applying personalized medicine for diagnostics of breast cancer and planning of a personalized treatment strategy with focusing on our recently developed personalized assay for breast cancer. Paper reference: Paper 16.1 Expression and characterization of recombinant human interferon beta using Escherichia coli Origami B and BL21 *Dr. Rehab I Alsahfy, Arabian Gulf University-College of Graduate Studies, Bahrain ([email protected]) Dr. Sonia Bourguiba-Hachemi, Arabian Gulf University-College of Graduate Studies, Bahrain Dr. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain Abstract Human Interferon β ( hIFN β) is a cytokine produced by macrophages and epithelial fibroblast cells in response to viral infections. Recombinant human IFN β is successfully used in therapeutic treatment of multiple sclerosis disease The high incidence of multiple sclerosis in the Gulf countries is raising the issue of patient ’ s access to such drug - class at reasonable cost In this work we carried out a comparative study of two different E coli host strains BL21 and Origami B cultured in two different media Biosilta and LB broth We first optimized hIFN β cDNA sequence to be fully conforming to the E coli codon - usage bias using GASCO and JCat software ’ s and then constructed a recombinant expression vector hIFN β- pGEX4T - We ’ ve also cloned the native hIFN β to serve as a reference The hIFN β was expressed as a fusion protein coupled to Glutathione - S - Transferase ( GST ). GST-IFN β protein produced by the two hosts in two different media showed a significant difference in protein localization We found that BL21 strain expresses higher amount of soluble fusion protein than Origami B strain The amount of soluble GST - IFN β protein is higher in BL21 transformed with the expression vector containing JCat optimized IFN β cDNA ( 40 %), cultured at 25° C in a fed - batch - like Enbase medium and induced with mM IPTG overnight The amount of GST - IFN β protein was very low in BL21 strain grown in a conventional LB broth medium However using LB medium supplemented with glucose as a carbon source with optimization of culture conditions 40% approximately of soluble GST was produced by BL21 transformed with the expression vector containing JCat optimized IFNβ cDNA.

Paper reference: Paper 16.2 AN IN SILICO APPROACH TO DEVELOP A TOOL BOX OF IgG Fc ISOFORMS WITH ENHANCED “ADCC” IMMUNE FUNCTION

*Dr. Dana Ashoor, Arabian Gulf University-College of Graduate Studies, Bahrain ([email protected]) Dr. Sonia Bourguiba-Hachemi, Arabian Gulf University-College of Graduate Studies, Bahrain Dr. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain Dr. Noureddine Ben Khalaf, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain Abstract Polymorphism of the Fc g Rs has been shown to be related to the efficacy of therapeutic antibodies as it plays a major role in forming strong inflammatory signaling complex with IgG Abs Recent studies pointed to the upper hinge region of IgGs as a good target for engineering to improve Antibody - Dependant Cell mediated Cytotoxicity ( ADCC ). To further investigate this issue we have developed an in silico approach for the design of recombinant IgGFc isoforms endowed with higher affinity to the Fc g receptors CD16 and CD32 natural variants to enhance their ADCC function Our approach consisted in modeling the D structures of the complex between HR and HL alleles of Fc g RIIa and Fc g RIIIa and using these models generate different upper hinge - mutated variants of IgG1 Fc region Indeed the generated models allowed the identification of the residues involved in the Fc Fc g R interaction and the development of mutated forms of the IgG1Fc region We have designed such mutated forms and used modeling of the interaction with Fc g R variants to measure the relative free energy of the complex The data collected revealed upper hinge mutants exhibiting increased Fc g Rs binding To confirm these results experimentally the extracellular domains of the Fc g RIIa and Fc g RIIIa four variants plus the wild type Fc region of IgG1 and the mutated forms were engineered and produced as recombinant proteins in Pichia pastoris The purified recombinant proteins are being analyzed by Biacore to confirm the in silico results Therefore this approach provides a novel strategy to create a toolbox of engineered human monoclonal antibodies Fc region variants exhibiting enhanced affinities to Fc g Rs and hence more efficient ADCC activity Keywords: Fc g Rs IgG1 Polymorphisms ADCC In

Paper reference: Paper 16.3 Enhancement of lipid production using genetic engineering approaches in Dunaliella salina to improve biodiesel production *Dr. Ahmad Farhad Talebi, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran, Iran ([email protected]) Dr. Meisam Tabatabaei, Agricultural Biotechnology Research Institute of Iran (ABRII), Karaj, Iran, Iran Abstract The third generation of biofuel producers is basically non-agricultural crop. Among them, unique characteristics such as natural tolerance to waste and saline water, huge biomass production and high lipid content, make microalgae as an appropriate source in biodiesel production. In the present study, Plasmid pGH, bearing the ME and AccD genes under the

control of atpB promoter with the chloramphenicol marker gene companionship, was used in cells bombardment. Primary selection was done on a medium supplemented by 80 mg l-1 chloramphenicol. After several passages, the survived cells were PCR-tested to confirm the stability of transformation. Southern hybridization of AccD probe with genomic DNA revealed stable integration of the cassette in the specific positions in the chloroplast genome with no readthrough transcription from outside promoters. The transformed cell lines then were studied to determine the ME/AccD activity in comparison with the control cells. The results successfully showed that overexpression of these two genes led to 12% increase in total lipid accumulation and great improvement in biodiesel properties especially by increase in oil oxidation stability. The whole process was successfully implemented as a pre-step to transform the algal cells by genes involved in lipid production pathway which will be helpful in large scale biodiesel production from microalgae. Background Todays, research efforts have concentrated on applying metabolic and genetic engineering approaches to in order to develop microalgae optimized for high productivity and energy value. This strategy will lead to making algae a viable commercial option to produce higher yielding and hardier strains. Although economic challenges always should be considered by increase the yield to a level that ensures micro-algae based biodiesel competitive with other biodiesel feedstock. Materials and Methods Since other factors such as nutrient management (like nitrogen and phosphor starvation), environmental conditions (like salinity, acidity and photon flux) and precursor addition would slightly affect the final productivity of premium algal strains, we aimed to improve the morphological and molecular characterization of a strain of microalga Dunaliella salina by manipulation of FAs synthase pathway in the chloroplast. Hence, particle bombardment was used as a method to transfer the pGH-ME-AccD vector into chloroplast genome of D. salina. Results The effect of transgenes presence in lipid metabolism was surveyed by LC measurement in 35days transformed and control cell line. The mean value of LC for the transformed cell line showed a slight increase and was around 25 which was 12 % higher than that of the control. Slim improvements in the estimated CP values were also recorded. It would help to an easier start up of a standard diesel engine. Overall, it was shown that the overexpression of AccD and ME led to improved fuel properties. Conclusion the feasibility of biodiesel production from genetically modified micro - algae can be expedited if large - scale production facilities can be integrated with other processes such as wastewater treatment and utilization of carbon dioxide from power plants. Screening of genetic variability between algal isolates could lighten the different potential of variant strains and the result could be used in prone strain selection for gene transformation. Paper reference: Paper 16.4 Recombinant production of hyaluronic acid in different E. coli strains.

*Dr. Heba T`Allah Ahmed Nasser (H.Nasser), University Of Ulm, Microbiology and Biotechnology Institute, Egypt ([email protected]) Dr. Bernhard J. Eikmanns, University Of Ulm, Microbiology and Biotechnology Institute, Germany - Deutschland Dr. Mohamed Ahmed Elazizy, GUC(The German Univeristy in Cairo), Egypt Dr. Khaled Ahmed Abou-Aisha, GUC(The German Univeristy in Cairo), Egypt Abstract Hyaluronic acid (HA) is a linear mucopolysaccharide consisting of disaccharide repeats of Dglucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) joined alternatively by β-1, 3 and β1, 4 glycosidic bonds. HA has many biomedical applications and its market demand is expected to increase in the following years. In this study, the hasABC operon was PCR amplified from the genomic DNA of S. equi subspecies zooepidemicus (The orientation of the PCR fragment was confirmed by sequencing) and cloned into the expression plasmid pPT7 that has a T7 RNA polymerase promoter which is considered as a strong promoter before being transformed into three Escherichia coli host strains (BL21lysY/Iq, Rosetta DE3 (pLysS), and Rosetta-gami 2(DE3)pLysS). The three E. coli strains were compared for their capability of production of HA using LB medium with glucose 1%, SOC medium and TB medium. Rosetta-gami 2(DE3)pLysS E. coli cells showed the highest HA productivity. The HA titer reached 0.5 g/L in TB medium in shaking flasks as a batch mode and it is expected to be even higher on media optimization. Paper reference: Paper 17.1 New molecular method for rapid and simultaneous nano-bio-sensing of Salmonella Enteritidis and Salmonella Typhimurium *Dr. Hamidreza Mollasalehi (H Mollasalehi), Shahid Beheshti University, Iran ([email protected]) Dr. Razieh Yazdanparast

[email protected]

Iran

University of Tehran

Abstract Nano-bio-sensing applied to foodborne pathogens may provide ideal molecular detection methods for rapid and simple differentiation of major salmonellosis-causing agents. Besides, targeting 16s ribosomal RNA of cells would assist amplification-based detection techniques to improve the sensitivity and specificity aspects mainly as a result of present multiple copies per cell and hyper-variable regions in the gene. Therefore, we develop a novel gold nanoprobenucleic acid sequence-based amplification (NASBA) in an advanced non-crosslinking mode for nanodiagnosis of the most important serovars in the Salmonella genus: Salmonella Enteritidis and Salmonella Typhimurium. In that regard, a specific primer along with thiolated oligonucleotides were designed for fabrication of bio-sensing gold nanoprobes. Moreover, the assay was evaluated regarding speed, limit of detection and specificity and also improved to

lower the elapsed time. We could successfully distinguish both bacteria simultaneously with a single primer set in relatively short period of 80 min. In addition, the sensitivity of 5 CFUs/mL of the bacteria was specifically achieved among 17 Salmonella and closely related non- Salmonella species. Overall, the developed method could provide a cost-effective promising assay in early bio-sensing of foodborne pathogen-outbreaks and its proven strategy could be extended to further target genes. Background In the absence of an appropriate detection technique for simple and rapid differentiation of food-borne pathogens, ribonucleic acid-based amplification and nanodiagnostic methods could provide ideal approaches for solving some of the common problems related to the conventional and molecular detection methods. Nano-bio-sensing applied to foodborne pathogens may provide ideal molecular detection methods for rapid and simple differentiation of major salmonellosis-causing agents. Materials and Methods In this study, we used nucleic acid sequence-based amplification (NASBA) together with nanotechnological approaches of NASBA product detection for discriminative differentiation of major serotypes in Salmonella genus and gastroenteritis-causing agents in human, Salmonella Enteritidis and Salmonella Typhimurium. Results We could successfully distinguish both bacteria simultaneously with a single primer set in relatively short period of 80 min. In addition, the sensitivity of 5 CFUs/mL of the bacteria was specifically achieved among 17 Salmonella and closely related non- Salmonella species. Conclusion The simple, developed ribonucleic acid-based assay could provide an ideal and cost-effective method for rapid diagnostics of pathogens especially foodborne agents in various circumstances e.g. outbreaks and also prevent epidemiological surveillance disasters.

Paper reference: Paper 17.2 Genotypic assessment of Drug Resistant Tuberculosis in Iraq using low-cost and density (LCD) DNA microarrays *Dr. Dalal Saleh Qader, Baghdad University, Iraq ([email protected]) Dr. Till bachmann Bachmann, 2Division of Pathway Medicine, University of Edinburgh, Medical School, Chancellor’s Building, Little France Crescent, Edinburgh EH16 4SB, UK, UK Abstract This is the first molecular investigation throughout Iraq to ascertain the prevalence of drug resistant TB and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH). One hundred and ten clinical isolates from category II TB cases from Baghdad and several Iraqi provinces were analyzed using colorimetric, low-cost and density (LCD) microarrays (MYCODirect and MYCOResistance LCD-array kits, Chipron GmbH, Germany)

to identify M. tuberculosis complex (MTBC) and the point mutations responsible for resistance. All samples hybridised the IS6110 probe on the MYCO-Direct LCD- array, confirming the presence of MTBC. Seventy-six patients (69.1%) had resistant strains, of which 44 (57.9%) were MDRTB. Where mono-resistance was identified, it was found to be predominantly to RIF (78.1%). The most common mutations were rpoB S531L (45.5% frequency), inhA C15T and katG S315T (each at 20% frequency). The LCD-arrays were easy to use and provided rapid results without the need for special lab equipment. Excellent correlation was found between the MYCOResistance LCD-array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDRTB in 40% of category II TB cases in Iraq. This closely reflects World Health Organisation (WHO) findings based on. phenotypic studies and confirms Iraq to be amongst the highest prevalence counties in the WHO Eastern Mediterranean Region. Paper reference: Paper 17.3 Simultaneous detection and molecular characterization of drugs resistant of Mycobacterium tuberculosis using multiplex allele- specific PCR *Dr. Amr Babiker Awadelkarim (Babiker), university of west kurdofan, UAE ([email protected]) Dr. Imadeldin Altahir aradaib (aradaib), university of Khartoum, UAE Abstract Aims: Rapid and reliable methods for detection drug resistant in TB are urgently needed. In this study, multiplex allele-specific polymerase chain reaction (MAS-PCR) was evaluated to detect the most commonly observed isoniazid (INH), rifampin (RIF), and ethambutol resistant associated mutations in a single assay. Methods: 120 sputum specimens were collected from patients diagnosed with pulmonary tuberculosis in Alshaab and Aboanga hospitals, Khartoum, Sudan. MAS-PCR were used to detect the resistance in identified isolates of M. Tb targeting mutations conducted in six genes in a single assay, and the results were compared with proportion method in LJ media (standard method). Result: The sensitivity, specificity and accuracy of MAS- PCR were for INH 97%, 100% and 98% respectively, and for RIF were 96.5%, 100% and 98% respectively, and for ETH were 84.9%, 95% and 91.6 respectively, the sensitivity and specificity in detection MDR were 95.2% and100% respectively with significant difference (P.v=0.001). The KatG 315 mutation was most found in 92.6% INH-resistant isolates, whereas Inh A-15 mutation was found in 63.4% -resistant isolates, regarding RIF, the majority were found to be associated with genes of rpoB 531 and rpo B 326 (94.3% and 28.3%) respectively and the less frequency associated with gene of rpoB 516. Conclusion: MAS-PCR compared with many genetic methods is rapid, inexpensive, and it has high sensitivity and specificity, particularly for detection MDR, and it can simultaneously screen resistance to anti- tuberculosis drugs.

Paper reference: Paper 17.4

Mutation pattern in rifampicin resistance determining region of rpoB gene in multidrugresistant Mycobacterium tuberculosis isolates from Pakistan *Dr. Obaidullah Qazi, Institute of Public Health, Pakistan ([email protected]) Dr. Zarfishan Tahir, Institute of Public Health, Pakistan Abstract The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1,080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan. Background Multidrug resistant tuberculosis ( MDR - TB ) is an important global health problem due to limited treatment options Resistance to rifampicin develops due to mutations in the gene encoding the beta subunit of the RNA polymerase ( rpoB ) of Mycobacterium tuberculosis ( M tb ). Mutations in rpoB gene are considered as marker for MDR - TB The pattern and frequency of mutations in the rpoB gene has variable geographical distribution and limited data is available on specific rpoB mutational patterns in Pakistan. Materials and Methods A total suspected TB patients referred from TB centers of Punjab were included in the study Sputum samples from these patients were collected aseptically at Institute of Public Health Lahore Manuscript Click here to download Manuscript Obaid et al _WJMB doc Click here to view linked References2 Pakistan and were subjected to microscopic ( ZN staining ) and culture based detection of M tb All the suspected TB samples were screened for MDR using drug susceptibility testing Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing Results Among referred TB cases ( %) were resistant against at least one of the four first line TB drugs Out of these resistant isolates isolates ( %) were found to be resistant to isoniazid and rifampicin Sequence analysis of MDR - TB isolates detected single mutation in rpoB gene at codon ( n = %), ( n = %), ( n = %), ( n = %), and ( n = %). One isolate reveal a double mutation at codon and while one has novel silent mutation at codon Moreover five MDR - TB isolates lack any mutation. Conclusion

rpoB gene mutations in drug-resistant M. tb were analyzed. Further study about these mutations might be helpful in the development of diagnostic tools for rapid detection of M. tb in high TB endemic area like Pakistan Paper reference: Paper 17.5 Comparative Diagnostic Applications of Antigen Capture ELISA and Immunohistochemistry for Detection of Bovine Viral Diarrhea Persistent Infection *Dr. ARFAN AHMAD, University of Veterinary and Animal Sciences, Lahore Pakistan, Pakistan ([email protected]) Dr. Masood Rabbani, University of Veterinary and Animal Sciences, Lahore Pakistan, Pakistan Dr. Khusi Muhammad, University of Veterinary and Animal Sciences, Lahore Pakistan, Pakistan Dr. Rana Muhammad Younus, Colllege of Vety and Animal Sciences, Jhang, UVAS, Lahore, Pakistan Dr. Muhammad Zubair Shabbir, University of Veterinary and Animal Sciences, Lahore Pakistan, Pakistan Abstract The diagnostic ability of antigen-capture enzyme-linked immunosorbent assays (AC-ELISA) and immunohistochemistry using two enzymes labels, alkaline phosphatase (AP) and peroxidase (P), to detect bovine viral diarrhea (BVD) persistent infection (PI) was assessed using serum and ear notch biopsy pairs (n= 469) collected from 12 Holstein dairy herds located in Charlottetown, Canada. The sampled animals were divided into two age groups, A (≤ 6 month of age, n = 146) and B (≥ 6 months of age, n = 323). All the animals of group B were pre-screened by serum neutralization test (SNT), and those animals (n=52) which had SN titer ≤ 1:64, as well as all ear notch biopsies of group A (n=146) were processed to confirm the BVDV persistent infection. Two ear notch biopsies of each groups A (2/146, 1.37%) and B (2/52, 3.48%) were found positive on first and follow up testing by AC-ELISA and immunohistochemical technique using AP enzyme label. Peroxidase label could not be distinguished from skin melanin, and thus was found unsuitable to differentiate between positive and negative tissue sections. There was no significant difference (P>0.05) between AC-ELISA and IHC-AP. Real time RT-PCR validated the results of IHC and ELISA assays used in the study. All the four positive animals were harbouring genotype1 of BVDV. The study concluded that AC-ELISA and IHC-AP were equally suitable for detection of BVDV persistent infection. However, AC-ELISA was found to be quicker, cheaper, and easier to perform within one day. Background Bovine viral diarrhoea virus ( BVDV ) belongs to pestivirus genus of flaviviridae and it is a significant viral pathogen associated with reproductive respiratory and gastrointestinal diseases of cattle Several methods have been used to screen persistent infection However in neonatal calves presence of maternal antibodies in blood can make the virus unavailable to be detected. The present study was undertaken to compare AC-ELISA and two IHC methods systems, AP and P, for the detection of BVDV persistently infected animals in ear notch sample

Materials and Methods The diagnostic ability of antigen - capture enzyme - linked immunosorbent assays ( AC - ELISA ) and immunohistochemistry using two enzymes labels alkaline phosphatase ( AP ) and peroxidase ( P ), to detect bovine viral diarrhea ( BVD ) persistent infection ( PI ) was assessed using serum and ear notch biopsy pairs ( n = ) collected from Holstein dairy herds located in Charlottetown Canada. Results Two ear notch biopsies of each groups A (( %) and B ( %) were found positive on first and follow up testing by AC - ELISA and immunohistochemical technique using AP enzyme label Peroxidase label could not be distinguished from skin melanin and thus was found unsuitable to differentiate between positive and negative tissue sections There was no significant difference ( P > 0.05 ) ) between AC - ELISA and IHC - AP. Real time RT - PCR validated the results of IHC and ELISA assays. Conclusion The study concluded that AC-ELISA and IHC-AP were equally suitable for detection of BVDV persistent infection. However, AC-ELISA was found to be quicker, cheaper, and easier to perform within one day Paper reference: Paper 18.1 Princess Haya Biotechnology Center Moving towards Globalization *Dr. Laith Naser Al-Eitan, Princess Haya Biotechnology Ceter, Jordan ([email protected]) Abstract Princess Haya Biotechnology Center (PHBC) lies strategically in a hyperactive political region in the Middle East. Two new training divisions are in final renovation phase at PHBC. They have been conducting standard courses in Biorisk Management and Genomics for three years in the MENA region. Our vision to build a regional center of excellence enhances the capabilities of laboratories in the developing world, and at the same time promotes a culture of laboratory biosafety and biosecurity to combat infectious diseases. Technologically, PHBC will soon realize the establishment of a new Next Generation Sequencing (NGS) platform and database. Thus, the training divisions will play role to enable the education of the next generation of MENA life scientists both in culture and technology. These roles require new measures of scientific diplomacy. The new training divisions are funded by a network of collaborators from the US, UK and Canada, while conducting training for a network of MENA countries. In other words, PHBC is trying to bridge new ties between the West and the Islamic World. This move towards globalization is the natural sustainable way for development because global problems require global measures of action. In addition, biosafety and biosecurity are considered situations of global sensitive matters. Therefore, the collaboration between developed and developing countries plays significant role in providing the scientific integrity, reputation and development that is necessary for our countries

Paper reference: SPS1164-14 Importance of genetic testing and genetic counseling in the Middle Eastern population Dr. Sonika Sachanandani, Eastern Biotech & Life Sciences, UAE, [email protected] *Dr. Sanjida Ahmed, Eastern Biotech & Life Sciences, UAE Abstract There are approximately over 400 genetic diseases in the UAE, 60% of which follow the autosomal recessive pattern of inheritance. We collected a total of 123 samples in the past 2 years for genetic testing. The study revealed that 25 out of 123 samples were positive. 68% were found to be among the Middle East population, 24% South Asian, 4% in Europeans and the remaining from other populations. This signifies that incidence of inherited genetic conditions are higher among the Middle East population which might be due to higher rate of consanguinity. Majority of samples (58%) that we received were for the non-invasive pre-natal diagnosis test (NIPD) for trisomies. Of these 71 samples; 1 was positive for trisomy 18 and 2 for trisomy 21. 11% of the samples were collected by invasive methods. 2 out of total 14 samples tested positive (compound heterogygotes) for a recessive condition. Half the samples (7 out of 14) were carriers of the known family mutation. 13% of the samples were collected to diagnose genetic conditions in adulthood. Half these samples tested negative and half were carriers of a condition, such as spinal muscular atrophy, etc. 11% of samples were collected to test for breast and ovarian cancer gene mutations. 11 out of the 14 samples tested negative and a variant of unknown significance (VUS) was found in the remaining 3. The remaining tests were for diagnosis of genetic disorders that present in childhood. Half of them tested negative, 1/ 8 was positive for sickle-cell anemia and 1/8 for biotine deficiency. 1 /8 was found to be a carrier of the R211H mutation. Genetic testing and genetic counseling are vital steps to take in the process of understanding and devising treatment and management of a condition to make an informed decision regarding the medical future of the family. Paper reference: Paper 18.3 Legal Implications of Personal Medicine *Dr. A. James Cuticchia (Anthony Cuticchia), AJC Legal Services, United States ([email protected]) Abstract Personal Medicine has been heralded for years as the culmination of the success of the human genome program, HAPMAP, translational research, biomarker discovery, and medical informatics. Researchers and physicians have brought forward a new and expanded scope of medicine; and as such; have created a legal vacuum which the courts will fill over the next several years. There are several areas where Personal Medicine opens its non-patient participants to legal action. First, quality control needs to be in place in order to protect practitioners? This is not only the diagnostic laboratories (is CLIA enough?) but also the basic researchers. How will simple mistakes be treated in the legal community versus outright fraud?

Second, since the prescription of a particular drug will most likely be one chosen from a set of drugs in the standard of care; how will the courts treat the choice of Drug A with a statistically predicted 5% better chance of treatment than Drug B? What if the physician fails to perform a test and is practicing medicine where the "physician in the same community" doctrine would show that it more likely than not that such a test have been provided? A discussion of the issues, the existing laws, and extrapolations to what the courts are likely to hold will be discussed in their importance of the adoption of Personal Medicine. Background Personal Medicine has been heralded as the next great step for medicine. However, as any new technology matures, a body of law will most likely be created and mature with the technology. Medical Malpractice is a serious cost component to health care. Personal Medicine opens up new facets of legal exposure for health care providers, testing laboratories, and even basic researchers. Here we will present several scenarios where legal exposure can occur and discuss existing litigation. Materials and Methods There are several scenarios where legal exposure can occur in Personal Medicine. First, is the research sound? Has there been proper controls put in place to prevent everything for honest laboratory mistakes to outright fraud? Second, what criteria in the diagnostic laboratory will be sufficient for legal protection? Is CLIA enough? Finally, when can a health care provider be sued for malpractice for making a decision based on personal medicine or failing to? What standards will physicians be held to? Results Here the present laws on medical malpractice and the "physician in the same community" doctrine will be applied to this topic. Second, regulations regarding testing will be examined. Third, where Personal Medicine is only a choice between two acceptable standards of care, this legal issue will be discussed in depth. Finally actual court findings, existing lawsuits, and public pleadings will be analyzed. Conclusion While there is a general distaste for lawyers in the medical profession, they act as a check-andbalance against bad medicine in such a way that any government agency (e.g., FDA) does not have the resources to do so. While the landscape will certainly change over time, the courts are generally much slower to react to change than science; thus, one must no only look at the present laws, but make sound guesses as to what the courts shall due in the future Paper reference: Paper 18.4 Characterization of cytokine profiles and signaling pathways up on activation by the Immune System- Released Activating Agent (ISRAA) *Dr. Sahar Elsaid Ahmed EA Elhannan, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain ([email protected]) Dr. Mohamed-Dahmani Fathallah, Arabian Gulf University-College of Graduate Studies, Bahrain Dr. Moiz Bakhit, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain Dr. Safa Taha, Arabian Gulf University-College of Medicine and Medical Sciences, Bahrain

Abstract ISRAA is an immune mediator produced as a result of a nerve stimulus initiated by immune challenge In this work a broad spectrum of pro - and anti - inflammatory cytokines was measured in response to rISRAA stimulation of human PBMCs The signaling pathways used by rISRAA to induce cytokine production were studied by examining phosphorylation of signaling proteins and nuclear translocation of transcription factors To study the production of cytokines by hPBMCs triggered by rISRAA and to characterize the signaling pathways we examined phosphorylation of signaling proteins and nuclear translocation of transcription factors hPBMCs were treated with different concentration of rISRAA at different intervals MTT and flow cytometer ( lymphocytes count ) and the quantitative analysis of pro and anti - inflammatory cytokines immunoblot and western blot analysis were used to detect phosphorylation of signaling proteins Reverse Transcriptase polymerase chain reaction ( RT - PCR ) and quantitative ELISA assay were utilized to detect the cytokines High levels of IL - IL - IL - and TNF -α, IL - β and IFN -γ production was observed but no production of IL - IL - IL - or TGF -β was noted IL showed the highest measurable levels Studies on signaling pathways revealed that Erk1 signals were activated While the MAPKs has no effect on cell proliferation but was fully involved on the apoptotic effect using the FAS FADD pathway by increasing the production of the TNF α, also it showed that ISRAA as other PIAS like protein works as a negative regulator of the STAT3 regulating the JAK STAT pathway Mechanisms used by ISRAA to induce cellular activity on PBMCs cells demonstrated preferential expression of the cytokine IL - and that the MAP kinase pathway and its downstream Erk1 signals were critically involved during this process Understanding

Paper reference: Paper 19.1 Role of Vitamin D and its genetics in pulmonary Tuberculosis in Pakistan *Dr. kashaf junaid, Department of Microbiology and Molecular Genetics , University of the punjab, Lahore, Pakistan ([email protected]) Dr. Abdul Rehman, Department of Microbiology and Molecular Genetics , University of the punjab, Lahore, Pakistan Dr. Adrian Martineau, Queen Mary University of London, UK Dr. Tahir Saeed, Gulab Devi Chest Hospital, Lahore, Pakistan Abstract From the last few years vitamin D deficiency is a hot topic understudy and its deficiency is related not only with bone disorders but also with many infectious diseases. Genetics of vitamin D receptor gene showed susceptibility with some infectious diseases as well. Vitamin D deficiency and Tuberculosis has an association as reported by many studies yet about the genetics of VDR and TB it is different in different ethnic groups. In this study genetics of vitamin D and tuberculosis susceptibility and response of treatment was observed. For this 300 patients were recruited from Gulab Devi hospital, Lahore from 2012 to 2013. Patients were followed up

till eight weeks to see the sputum conversion time. Vitamin D status was observed by IDS immunoassay method and DNA extraction was done. TaqMan allelic assay was used to study different SNPs in TB patients and controls. Three SNPs of VDR, three SNPs of CYP2R1 and two SNPs of Vitamin D binding protein were analyzed. All statistical analysis was done on SPSS version 20 , Stata 13 and Prism. Results indicate high prevalence of Vitamin D deficiency in TB patients however no significant association was observed in multivariate analysis with vitamin D deficiency and patient’s response to treatment. It was also observed that genotype of Bsm in VDR gene has association of vitamin D status in TB patients. Further analysis of vitamin D binding protein in patients and controls is under study. Paper reference: Paper 19.2 DETECTION OF P53 GENE MUTATIONS IN HELICOBACTER PYLORI INFECTED GASTRIC CANCER PATIENTS IN JORDAN *Dr. Suhaila Al-Sheboul, Jordan University of Science and Technology, Jordan ([email protected]) Dr. Taghreed Muhareb, Jordan University of Science and Technology, Jordan Abstract Background: Helicobacter pylori is the most common infection in the world. It has been classified as a major cause of peptic ulcer disease (PUD) and a risk factor for gastric adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. Inconclusive data are available on the role of p53 gene mutational patterns in gastric cancers in Jordan. It is unclear whether mutations in the tumor suppressor p53 gene, is associated with H. pylori infection and cancer. The present study was conducted among Jordanian individuals to investigate the possible association of mutations in p53 gene with gastric adenocarcinoma and peptic ulcer patients infected with H. pylori. This study aimed to detect mutations and their type in p53 gene at exons (5-8) in H. pylori-infected gastric cancer patients in Jordan. Method: Polymerase Chain Reaction (PCR) and Second Generation Automated Sequencer were both used to detect such mutations in p53. Results: Formalin fixed paraffin embedded gastrectomy tissue samples were obtained from 77 patients with gastric cancer, and 33 samples were obtained from patients with peptic ulcer disease patients. The samples were collected from three different hospitals in Jordan. Among the samples examined from gastric cancer patients, 54.5% (44/77) were H. pylori positive, while among the samples examined from peptic ulcer patients 100% (33/33) were H.pylori positive. We found that p53 mutations were higher in H. pylori infected-gastric cancer samples than in H. pylori infected-peptic ulcer disease. The difference was statistically significant: 37.7% (29 out of 77) of gastric cancer patients and 9% (3 out of 33) of peptic ulcer disease patients had mutations in p53 gene. These results show the dependence on H. pylori infection which in turn indicates the role of p53 gene mutations in gastric carcinogenesis. Paper reference: Paper 19.3

Detection of Human Papilloma Virus (type 16 and 18) in bronchial wash samples of patients with lung cancer: An evaluation by chromogenic in situ hybridization technique *Dr. Ban Abbas Abdul-Majeed, University, Iraq ([email protected]) Dr. Nada Hamza Al-Shabbani, MOH (Hospital), Iraq Dr. Raji Hssain Al-Hadithi, University, Iraq Abstract Background & objectives Lung cancer is a common neoplasm widely affecting both sexes and is the leading cause of cancer - related death worldwide Some studies suggested that Human Papilloma Virus ( HPV ) may play an etiologic role in bronchial carcinogenesis and the possibility of a latent HPV infection as a co - carcinogen cannot be excluded High risk types of HPV ( & ) may affect the cell cycle and inhibit apoptosis allowing uncontrolled cell division It was necessary to study the role of these viruses as causative agents in lung cancer The aim of this study was to determine the presence of DNA of high risk types ( & ) of HPV in bronchial wash samples of patients with lung cancer using in situ hybridization technique in correlation with histological types of lung cancer and clinical data ( age & sex ) Patients materials and methods This is a prospective case control study involving bronchial wash samples of patients who underwent diagnostic bronchoscopy for lung cancer The diagnosis was further confirmed by cytopathology examination in samples which served as the study group The remaining negative samples were studied as a control group For the purpose of conducting in situ hybridization procedure to detect the DNA of HPV & smears were made on positively charged slides and submitted for chromogenic in situ hybridization technique The results were statistically analyzed Results The main histological type of lung cancer was squamous cell carcinoma representing % of all cases Forty percent of malignant bronchial samples showed positive in situ hybridization signal for human papilloma virus and % of them showed positive in situ hybridization signal for human papilloma virus Positive signals of HPV were seen in % of female samples and those for type were detected in % of female cases Paper reference: 19.4 A novel multiplex PCR for detection of Pseudomonas aeruginosa: A major cause of wound infections *Dr. Muhammad Salman (Salman), National Institute for Biotechnology and Genetic Engineering (NIBGE), Pakistan, Pakistan ([email protected]) Abstract Our objective was to establish a comprehensive and reliable multiplex PCR to confirm diagnosis of P. aeruginosa. Background Wound infections are often difficult to treat due to various bacterial pathogens. Pseudomonas aeruginosa is one of the common invaders of open wounds. Precise diagnosis of this etiological agent in wound infections is of critical importance particularly in treatment of problematic

cases. The existing diagnostic methods have certain limitations particularly related to specificity. Materials and Methods A multiplex PCR test was developed for rapid and comprehensive identification of P. aeruginosa. Four highly specific genes were targeted simultaneously for detection of genus, species and exotoxin production (16S rDNA, gyrB, oprL and ETA) in P. aeruginosa; additionally one internal control gene invA of Salmonella was used. The specificity of the multiplex PCR was confirmed using internal and negative controls. Amplified fragments were confirmed by restriction analysis and DNA sequencing. Results The developed method was applied on 40 morphologically suspected P. aeruginosa isolates (from 200 pus samples) and 18 isolates were confirmed as P. aeruginosa. In comparison, only 12 could be identified biochemically. Conclusion Combination of the four reported genes in multiplex PCR provided more confident and comprehensive detection of P. aeruginosa which is applicable for screening of wound infections and assisting treatment strategy.

OSTER

Poster Abstracts

If the presenting author is not the corresponding author, the email of the corresponding author will appear.

Paper reference: SPS1263-14 Antimicrobial activity and molecular typing of Bacillus licheniformis Isolated from Korean traditional food sources against multi-drug resistant Escherichia coli Dr. Ho-Seong CHO

[email protected]

South Korea 한국

Bio-Safety Research Institute and College of Veterinary Medicine,

Abstract The purpose of this study was to analyze and evaluate the antimicrobial activity and molecular typing of 63 isolated from Korean traditional food sources against multi-drug Escherichia coli in pigs. Among the 50 strains were defined as STs whereas 13 strains were defined as new STs. These results suggest that pre-conditioning with probiotic B. licheniformis isolated from Korean traditional food sources may protect against porcine enteric bacteria. Background Probiotics are living microorganisms that benefit their host by providing intestinal flora balance through higher levels of favorable activities. In particular, fermented soybean products with Bacillus spp indigenous to Asian and African countries have long been considered traditional and nutritious foods has for many years been used in the industrial production of enzymes antibiotics and detergents. Therefore the aim of this study was to analyze and evaluate the antimicrobial activity and molecular typing of 63 isolated from Korean traditional food sources against multi-drug Escherichia coli in pigs. Materials and Methods A multi-locus sequence typing (MLST) analysis, based on the sequence of six house-keeping genes of 63 strains was performed. The result of the MLST analysis supported previous findings of two different sub groups (lineages) within this species, named “A” and “B”. Statistical analysis of the MLST data indicated a higher rate of recombination within group “A”. Results Among the tested strains, 50 strains were defined as STs [ST26 (19 strains), ST3 (12 strains), ST2 (6 strains), ST14 (5 strains), ST9 (4 strains ), ST24 (2 strains), ST 12 (1 strain) and ST 13 (1 strain)] and 13 strains were defined as new STs. The results of antibacterial activity, one strain of named SRCRM100160 demonstrated a high antimicrobial potency. Especially, B. licheniformis (ST14) were showed high antibacterial activity against porcine enteropathogenic bacteria. Conclusion Our findings suggest that pre-conditioning with probiotic B. licheniformis isolated from Korean traditional food sources may protect against porcine enteric bacteria. Paper reference: SPS1344-14 DNASE I GENE AND DIABETES: A FAMILY BASED STUDY Dr. nageen HUSSAIN

[email protected] Pakistan

university of the punjab

Abstract DNASE I SHOWED A SIGNIFICANT ASSOCIATION WITH SLE BUT OUR INTEREST IS TO DO MUTATIONAL ANALYSIS OF DNASE I GENE IN DIABETIC PATIENTS.DNA ISOLATION, PCR, SEQUENCING TECHNIQUES WERE USED NUT NO MUTATION WAS FOUND IN DNASE I GENE EXON 8 IN DIABETIC PATIENTS. Background DNASE I SHOWED A SIGNIFICANT ASSOCIATION WITH SLE BUT OUR INTEREST IS TO DO MUTATIONAL ANALYSIS OF DNASE I GENE IN DIABETIC PATIENTS. Materials and Methods DNA ISOLATION, PCR, SEQUENCING Results NO MUTATION WAS FOUND IN DNASE I GENE EXON 8 IN DIABETIC PATIENTS. Conclusion NO MUTATION FOUND Paper reference: SPS1340-14 MOLECULAR CHARACTERIZATION OF ANTIMICROBIAL RESISTANCE GENES IN SALMONELLA ISOLATES FROM POULTRY DRINKING WATER

*Dr. Zarfashan Tahir

[email protected]

Pakistan

Institute of Public Health

Dr. Saba Zeb Khan Dr. Tahir Yaqub Dr. Sehrish Firyal Dr. Nadia Mukhtar Dr. Muhammad Tayyab

University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan

Abstract The purpose of this study was to investigate the presence of Salmonella in the poultry and to find out the genes involve in the development of tetracycline resistance in animals. Water samples were collected from water containers of different poultry meat shops and poultry farms in Lahore. Total 50 water samples were collected and were utilized for the isolation of Salmonella. Various biochemical tests were performed to confirm the bacterial strains as Salmonella. The tetracycline resistance of the isolates was examined by disk diffusion method. Out of 50, 25 samples were found to be resistant against tetracycline. Plasmid DNA was isolated from these tetracycline resistant strains and was utilized for the detection of various tet genes (tetA, tetB, tetC, tetD, tetG). PCR confirmed the presence of tetA in all the 25 samples while tetB was present in combination with tetA gene only in 16 samples. No amplification of tetC, tetD and tetG was examined. We reported that our local population of Salmonella contains the tetA alone or in combination of tetB gene only. Background The purpose of this study was to investigate the presence of Salmonella in the poultry and to find out the genes involve in the development of tetracycline resistance in animals.

Materials and Methods Water samples were collected from water containers of different poultry meat shops and poultry farms in Lahore. Total 50 water samples were collected and were utilized for the isolation of Salmonella. Various biochemical tests were performed to confirm the bacterial strains as Salmonella. The tetracycline resistance of the isolates was examined by disk diffusion method. Results Out of 50, 25 samples were found to be resistant against tetracycline. Plasmid DNA was isolated from these tetracycline resistant strains and was utilized for the detection of various tet genes (tetA, tetB, tetC, tetD, tetG). PCR confirmed the presence of tetA in all the 25 samples while tetB was present in combination with tetA gene only in 16 samples. No amplification of tetC, tetD and tetG was examined. Conclusion We reported that our local population of Salmonella contains the tetA alone or in combination of tetB gene only Paper reference: SPS1335-14 Resolving the taxonomic status of Genus Urocleidus(Mueller,1936) with Indian monogenoids on the basis of 28S ribosomal RNA based study. *Dr. Amrin Ali

[email protected]

India

Dr. Nirupama Agrawal

[email protected] India

University of Lucknow University of Lucknow

Abstract Sequences of ribosomal subunits among monogenoideans dwelling on piscine hosts are used to establish phylogenetic relationships at the generic and species level.It also facilitates to study evolutionary associations between parasites and their hosts(Desdevises et al,2002; Simkova et al, 2004).The present work shows a degree of variation in the sequence length of 28S rRNA genes of the monogenoidean species which have been taken up for study on account of being host specific. All these monogenoids are of Indian origin and have been compared with a species of Urocleidus which is of North American origin. The doubted inclusion of all these monogenoids under a North American genus Urocleidus has been restudied and tried to resolve on the basis of molecular characters.This study confirms the validity and distinctness of Mastacembelocleidus bam (Tripathi,1959) Kritsky et al.,2004, Mastacembelocleidus heteranchorus (Kulkarni, 1969) Kritsky et al., 2004, Chandacleidus lucknowensis Agrawal et al.,2005, Chandacleidus recurvatus (Jain, 1961) Agrawal et al., 2005, Xenentocleidus xenentodoni (Jain,1961), Tripathi, Agrawal and Pandey.,2006 and Spicocleidus namae Agrawal et al.,2005 from India by the use of molecular characters and secondary RNA structure predictions. Background

Molecular Taxonomy of Monogenoids found on Piscine hosts play an important role in the ecology of aquatic environment because these ectoparasites if not checked can lead to high rates of fish mortality.Their correct identification is very essential to study their biodiversity and ecology in relation with the edible and economically important fish hosts.Hence an attempt has been made here to identify and validate these unique parasites with the help of molecular methods and computational biology. Materials and Methods Fishes for the purpose of parasite collection were collected from various water bodies and their gills were examined carefully for the ectoparasites found on them .Various freshwater fishes were caught alive to collect these monogenoids. Results The results yielded from the present study are that all these monogenoids have been identified at the species level with the help of molecular methods employed and their distinctness is seen in relation with their respective hosts. The bioinformatic tools have been important and essential method towards validation of these parasites along with conventional morphological methods. Conclusion This study confirms the validity and distinctness of Mastacembelocleidus bam (Tripathi,1959) Kritsky et al.,2004, Mastacembelocleidus heteranchorus (Kulkarni, 1969) Kritsky et al., 2004, Chandacleidus lucknowensis Agrawal et al.,2005, Chandacleidus recurvatus (Jain, 1961) Agrawal et al., 2005, Xenentocleidus xenentodoni (Jain,1961), Tripathi, Agrawal and Pandey.,2006 and Spicocleidus namae Agrawal et al.,2005 from India.Hence the doubted inclusion of these parasites under the Genus Urocleidus is resolved her Paper reference: SPS1334-14 The incidence of appendicitis among the appendectomies performed at Al Qassimi Hospital in the years 2013 – 2014 *Dr. Nada Obaid Al Dabbah Dr. Nishi Singh Dr. Muhammad Zaman

UAE UAE

[email protected] UAE Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology

Abstract Appendectomies common are surgical procedures, but there is still miss-diagnosis with confirmed reports especially in young females most often because of misdiagnosis of the ovarian cyst. In addition, a retrospective study was done to see if there is a correlation between appendectomies and misdiagnosing of appendicitis in that specific period and population. The results showed there is no correlation in any type of groups and either between the genders vs. diagnosis. Background The appendix accumulates bacteria and viruses from feces, resulting in possible infection or appendicitis. Appendix obstruction from rapidly multiplying bacteria causes swelling and pus

accumulation. Treatment is appendix removal, and if untreated the appendix may rupture causing more and sever infection. More rarely appendicitis in is due a traumatic injury or genetic factors. Despite common occurrence of appendicitis, misdiagnosis is not uncommon. The aim of our study is to determine any correlation between appendicitis misdiagnosis and age or gender to highlight increased chance of misdiagnosis in some patient categories. Materials and Methods A retrospective cohort study was undertaken with population of 666 patients comprising of 117 Females and 548 Males, by looking at patients records in the years 2013-2014. Results 666 patients were sorted by gender and age groups 0-18, 19-35, 35- 55 and 56+. There was weak to no correlation between appendicitis misdiagnosis and age or gender. Conclusion No age or gender correlation with appendicitis was observed in the sample. Moreover, the study population does not contain any predominant nationality, making it harder to detect specific biases in specific groups of people correlation and this makes the result are subject to regional variation. However, if the samples were only from specific nationalities it would be easier to detect any correlation. Paper reference: SPS1332-14 Comparative Diagnostic Applications of Antigen Capture ELISA and Immunohistochemistry for Detection of Bovine Viral Diarrhea Persistent Infection *Dr. ARFAN AHMAD

[email protected]

Pakistan

University of Veterinary and Animal Sciences, Lahore Pakistan

Dr. Masood Rabbani Dr. Khusi Muhammad Dr. Rana Muhammad Younus Dr. Muhammad Zubair Shabbir

University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan Colllege of Vety and Animal Sciences, Jhang, UVAS, Lahore University of Veterinary and Animal Sciences, Lahore Pakistan

Abstract The diagnostic ability of antigen-capture enzyme-linked immunosorbent assays (AC-ELISA) and immunohistochemistry using two enzymes labels, alkaline phosphatase (AP) and peroxidase (P), to detect bovine viral diarrhea (BVD) persistent infection (PI) was assessed using serum and ear notch biopsy pairs (n= 469) collected from 12 Holstein dairy herds located in Charlottetown, Canada. The sampled animals were divided into two age groups, A (≤ 6 month of age, n = 146) and B (≥ 6 months of age, n = 323). All the animals of group B were pre-screened by serum neutralization test (SNT), and those animals (n=52) which had SN titer ≤ 1:64, as well as all ear notch biopsies of group A (n=146) were processed to confirm the BVDV persistent infection. Two ear notch biopsies of each groups A (2/146, 1.37%) and B (2/52, 3.48%) were found positive on first and follow up testing by AC-ELISA and immunohistochemical technique using

AP enzyme label. Peroxidase label could not be distinguished from skin melanin, and thus was found unsuitable to differentiate between positive and negative tissue sections. There was no significant difference (P>0.05) between AC-ELISA and IHC-AP. Real time RT-PCR validated the results of IHC and ELISA assays used in the study. All the four positive animals were harbouring genotype1 of BVDV. The study concluded that AC-ELISA and IHC-AP were equally suitable for detection of BVDV persistent infection. However, AC-ELISA was found to be quicker, cheaper, and easier to perform within one day. Background Bovine viral diarrhoea virus ( BVDV ) belongs to pestivirus genus of flaviviridae and it is a significant viral pathogen associated with reproductive respiratory and gastrointestinal diseases of cattle Several methods have been used to screen persistent infection However in neonatal calves presence of maternal antibodies in blood can make the virus unavailable to be detected. The present study was undertaken to compare AC-ELISA and two IHC methods systems, AP and P, for the detection of BVDV persistently infected animals in ear notch sample Materials and Methods The diagnostic ability of antigen - capture enzyme - linked immunosorbent assays ( AC - ELISA ) and immunohistochemistry using two enzymes labels alkaline phosphatase ( AP ) and peroxidase ( P ), to detect bovine viral diarrhea ( BVD ) persistent infection ( PI ) was assessed using serum and ear notch biopsy pairs ( n = ) collected from Holstein dairy herds located in Charlottetown Canada. Results Two ear notch biopsies of each groups A (( %) and B ( %) were found positive on first and follow up testing by AC - ELISA and immunohistochemical technique using AP enzyme label Peroxidase label could not be distinguished from skin melanin and thus was found unsuitable to differentiate between positive and negative tissue sections There was no significant difference ( P > 0.05 ) ) between AC - ELISA and IHC - AP. Real time RT - PCR validated the results of IHC and ELISA assays. Conclusion The study concluded that AC-ELISA and IHC-AP were equally suitable for detection of BVDV persistent infection. However, AC-ELISA was found to be quicker, cheaper, and easier to perform within one day

Paper reference: SPS1331-14 Abuse of Serum Vitamin B12 and Folate Testing in Anemia Investigation in MOH Health Care Centers *Dr. Maythaa Abdulla Juma Meftah AlHamidh

[email protected]

Dr. Nishi Singh Dr. Muhammad Zaman

UAE UAE

Abstract

UAE

Sharjah Higher Colleges of Technology

Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology

Vitamin B12 and folate are B complex vitamins not produced in the human body, but obtained from external sources. Vitamin B12 and folate deficiencies can cause megablastic anemia, resulting from the disruption of RNA, DNA and protein synthesis but not cell growth prior to division.There has been increased testing of vitamin B12, resulting in an 72% increase in vitamin B12 testing from 2003 - 2008. This increase has resulted in the introduction of vitamin B12 testing guidelines, resulting in a reduction of vitamin B12 processed samples by 70% in the first year of application. Taken together these observations suggest widespread unnecessary B12 testing occuring at many laboratories. The objective of this studyis to find the importance of serum vitamin B12 and folate testing in anemia investigation. Background Folate, a B complex vitamin is obtained from vegetable and animal sources It has roles in DNA RNA and protein synthesis. Folate deficiency will have an effect on cell division including megaloblastic anaemia. Vitamin B12 is a water soluble vitamin similarto folate, but is only found in animal products. The anemia caused by this deficiency is similar to folate deficiency. In this report we analysed if anemia diagnosis requires both B12 and folate markers to be tested together or if one marker could replace the other. Materials and Methods A cross sectional quantitative and correlation study was done on 364 patients tested for serum vitamin B12 and serum folate from the period of July 2013 until March 2014. Serum ferritin, serum iron and red cell indices including MCV, MCH and MCHC results were collected. Samples are from Ministry of Health (MOH) hospitals and clinics in Dubai. Results We found no correlation between vitamin B12 and anemia in any subset of the population. Our study show while there is a low prevalence of folate deficiency which has a correlation with anemia. Conclusion From data analysis we conclude that vitamin B12 should not be tested in anemia investigation while folate should be tested in anemia investigation. Paper reference: SPS1330-14 ISOLATION, IDENTIFICATION AND MOLECULAR BASED INVESTIGATION OF BOVINE ROTAVIRUS *Dr. Tahir Yaqub

[email protected]

Dr. Ambreen Masood Dr. Jawad Nazir Dr. Sehrish Firyal Dr. Urwah Zia Dr. Nadia Mukhtar

Pakistan Pakistan Pakistan Pakistan Pakistan

Abstract

Pakistan

University of Veterinary and Animal Sciences University of Veterinary and Animal Sciences University of Veterinary and Animal Sciences University of Veterinary and Animal Sciences University of Veterinary and Animal Sciences University of Veterinary and Animal Sciences

A total of 100 diarrheic faecal samples of cattle and buffalo (n=50 each) calves less than three months of age were collected from Lahore district. The Samples were analyzed for the presence of bovine rotavirus by Ag-capture ELISA and molecular based techniques. Prevalence of bovine rotavirus was found 12% among total diarrheic samples. RT-PCR of ELISA positive samples for BRV VP4 gene showed only 5 samples (3 buffalo calf samples and 2 cattle calf samples) with desired product of 880 bp. Sequence and bioinformatics analysis of these BRV-VP4 gene showed that Pakistani bovine rotavirus VP4 gene (BRV/QOL/13) has maximum identity of 98% with Indian bovine rotaviruses VP4 gene (Accession no. AB625614, AB625613, AB625615, AB625616). Background Neonatal calf diarrhea caused by rotavirus has negative effect on the growth and survival of bovine calves Materials and Methods A total of 100 diarrheic faecal samples of cattle and buffalo (n=50 each) calves less than three months of age were collected from Lahore district. The Samples were analyzed for the presence of bovine rotavirus by Ag-capture ELISA and molecular based techniques. Results RT-PCR of ELISA positive samples for BRV VP4 gene showed only 5 samples (3 buffalo calf samples and 2 cattle calf samples) with desired product of 880 bp. Sequence and bioinformatics analysis of these BRV-VP4 gene showed that Pakistani bovine rotavirus VP4 gene (BRV/QOL/13) has maximum identity of 98% with Indian bovine rotaviruses VP4 gene (Accession no. AB625614, AB625613, AB625615, AB625616). Conclusion The prevalent strains of bovine rotavirus is quite similar to those reported from other neighboring countries to Pakistan Paper reference: SPS1382-14 Isolation, Molecular Diagnosis and Antibiotic Resistance Profile Study Of Acinetobacter From Human Origin *Dr. Nadia Mukhtar

[email protected]

Dr. Muhammad Shabbir Dr. Tahir Yaqub

University of Veterinary and Animal Sciences, Lahore Pakistan

University of Veterinary and Animal Sciences, Lahore Pakistan University of Veterinary and Animal Sciences, Lahore Pakistan

Abstract Strains of Acinetobacter were isolated from human clinical samples and were studied at molecular level. 100 samples were collected. Samples were identified by using preliminary biochemical tests and an identification kit API20 NE. Moreover isolates were diagnosed at molecular level by 16S rRNA gene sequencing. Antibiotic resistance profile was also determined which showed that percentage resistance of isolates toward Lincomycin, Ciproflocacin, Kanamycin, Ofloxacin, Tetracyclin, Imipenem, Cefixime, Ampicillin, Chloremphinicol, Amoxicillin

was 90, 70, 90, 90, 60, 70, 60, 60, 80 and 80 percent respectively. PCR and sequencing were also done for the amplification of specific antibiotic resistant gene that was gyrA. Comparative analysis was done using NCBI-BLAST and CLUSTAL W. On the basis of gyrA gene, phylogenetic tree of Acinetobacter was also generated by neighbor-joining method using MEGA 5 software Background Considering the emergence of novel pathogens and its subsequent resistance to antibiotics, studying Acinetobacter is of immense importance. Materials and Methods Strains of Acinetobacter were isolated from human clinical samples and were studied at molecular level. 100 samples were collected. Samples were identified by using preliminary biochemical tests and an identification kit API20 NE. Moreover isolates were diagnosed at molecular level by 16S rRNA gene sequencing Results Antibiotic resistance profile was also determined which showed that percentage resistance of isolates toward Lincomycin, Ciproflocacin, Kanamycin, Ofloxacin, Tetracyclin, Imipenem, Cefixime, Ampicillin, Chloremphinicol, Amoxicillin was 90, 70, 90, 90, 60, 70, 60, 60, 80 and 80 percent respectively. PCR and sequencing were also done for the amplification of specific antibiotic resistant gene that was gyrA. Comparative analysis was done using NCBI-BLAST and CLUSTAL W. On the basis of gyrA gene, phylogenetic tree of Acinetobacter was also generated by neighbor-joining method using MEGA 5 software. Conclusion The prevalent strain of Acinetobacter are resistant to antibiotics and are relatively novel than those reported before Paper reference: SPS1327-14 The Prevalence of Thyroid Diseases among National and Non-National in Both Genders in Umm Al Quwain *Dr. Noorah Taresh Obaid Al Kharsi Dr. Nishi Singh Dr. Muhammad Zaman

UAE UAE

[email protected] UAE

Sharjah Higher Colleges of Technology

Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology

Abstract Thyroid disorders are hard to diagnose due to their symptoms associated with other health problems. Furthermore, each category of thyroid disease has many symptoms associated with the condition. Hyperthyroidism patients can suffer from sudden weight loss, heat intolerance, tachycardia, increased appetite, anxiety, tremor, sweating, changes in menstrual patterns, enlarged thyroid gland (goiter), fatigue, muscle weakness, difficulty sleeping and brittle hair. By contrast, hypothyroidism can result in obesity, joint pain, infertility and heart disease, fatigue, constipation, weight gain, muscle weakness, elevated blood cholesterol level, irregular menstrual periods, cold intolerance, slowed heart rate, depression and impaired memory. As there are various types of thyroid disorders, there cannot be a single laboratory test able to

detect every type of thyroid disease, consequently abnormalities in thyroid functions are detected by minimum of two or more tests. Population studies to determine any correlations between various thyroid diseases and nationality and between thyroid diseases may help to streamline the diagnosis process. The aim of this study is to identify number of people in Umm Al Quwain in the UAE suffers from thyroid diseases and to determine if there are nationality and gender bias to the disease. Background The thyroid gland secretes hormones known as tetraiodothyronine (T4) and triiodothyronine (T3), and is regulated by Thyroid Stimulating Hormone (TSH) from the pituitary gland. Additionally, Thyroid Stimulating Hormone Releasing Factor (TRF) secreted by the hypothalamus regulates pituitary TSH. The complex interplay of T3, T4, TSH and TRF make diagnosis of thyroid diseases difficult. Population studies to determine any correlation between different thyroid diseases and nationality, or between different thyroid diseases and gender may help streamline the diagnosis process. Materials and Methods Blood samples were collected into EDTA tubes in Umm Al Quwain Hospital in Umm Al Quwain from national and non-national people from both males and females starting of age 18 and above.The blood samples were then analysed for T3, T4 and TSH levels by the Roche Cobas system and these values were compared to physician's diagnosis of thyroid disorders. Results The profiles of both non-national males and national males are similar except for TSH. They have higher levels of TSH than non-national males, and this applies to hyper, hypo and normal males. The profiles of men and women are different, and those of non-national females are most different from the other three. They have the lowest levels of T3, T4 and TSH, making thyroid diagnosis difficult. Conclusion There is wide variation in hyper, hypo and normal levels between genders and between nationals and non-nationals, making the application of universal ranges for diagnosis very challenging. This high variability in the UAE is likely due to the presence of many national subgroups which are represented more or less equally in the whole population. Paper reference: SPS1325-14 Anti Fibronectin expression in lung, liver, spleen, Kidney and Thymus during aspergillosis infection using Immunohistochemical technique. *Dr. Batol Imran Dheeb

[email protected]

Dr. Basim mohammed khashman

Iraq

Iraq

Iraqia University

College of dentistry, Baghdad university, department of oral diagnosis

Abstract The major function of fibronectin is probably related to its ability to mediate substrate adhesion to mammalian cells a process that involves the binding of specific cell surface receptors to discrete domains in the fibronectin molecule Anti fibronectin were investigated in BALB c mice infected intravenously with × virulent Aspergillus fumigatus conidia by using immunohistochemistry technique using detection kit ( ab80436 ) and anti fibronectin marker (

ab2413 ) Five groups of animals were studied including control group ( mice not infected with A fumigatus ) these groups were used to study the expression in Two separated periods and day post infection Each section of socket tissue from organs sample was evaluated for the presence of intracellular brown DAB precipitate indicative of antibody binding The staining intensity was assessed using a designed scoring system Expression of Anti fibronectin in the studied organs were shown in the cytoplasm of the tissue cells and detected by IHC technique Depending on the scoring system used for the Anti fibronectin were the intensity of the staining of the cytoplasim used as parameters dependent The intensity of the cytoplasim of the stained cells was negative if there is no expression Expression was found positive in all the studied organs but different in intensity in lung over expression represented by strong staining ( score +) in and days post infection with moderate staining at day and intense staining at day respectively in liver expression was ( score + and +) with moderate staining at and day post infection The expression in the spleen kidney and thymus was ( score1 +) with light staining at the period between to day Anti fibronectin expression in all studied sample compared with the positive control Results show significant difference ( P value > ) in the expression between the studied organs Background The proteins laminin and fibrinogen are candidates for mediating adherence of conidia to the extracellular matrix to basement membranes and to the fibrin and fibrinogen deposits formed in response to the inflammatory reactions at the surfaces of wounded epithelia conidia of Aspergillus fumigates are able to adhere to fibrinogen laminin and complement via proteins of the outer cell wall Fibronectin is a disulfide - linked dimeric glycoprotein present in a soluble form in blood plasma and other body fluids and in a fibrillar form in extracellular matrices . Materials and Methods Anti fibronectin were investigated in BALB c mice infected intravenously with × virulent Aspergillus fumigatus conidia by using immunohistochemistry technique using detection kit ( ab80436 ) and anti fibronectin marker ( ab2413 ) Five groups of animals were studied including control group ( mice not infected with A fumigatus ) these groups were used to study the expression in Two separated periods and day post infection Each section of socket tissue from organs sample was evaluated for the presence of intracellular brown DAB precipitate indicative of antibody binding . Results in lung over expression represented by strong staining ( score +) in and days post infection with moderate staining at day and intense staining at day respectively in liver expression was ( score + and +) with moderate staining at and day post infection The expression in the spleen kidney and thymus was ( score1 +) with light staining at the period between to day Anti fibronectin expression in all studied sample compared with the positive control Results show significant difference ( P value > ) in the expression Conclusion In conclusion, Anti fibronectin expression levels was time-dependent increased and related with the progress of infection that can significantly enhance resistance to A. fumigatus in BALB/c mice.

Paper reference: SPS1323-14 Metagenomic profile of bacterial communities present in the respiratory system of clinically healthy birds raised in different management systems *Dr. Muhammad Zubair Shabbir

Dr. JiHye Park Dr. Yury Ivanove Dr. Muhammad AbuBakr Shabbir Dr. Tahir Yaqub Dr. Masood Rabbani Dr. Eric Thomas Harvill

[email protected]

United States United States Pakistan Pakistan Pakistan United States

University of Veterinary and Animal Sciences, Lahore Pakistan

PennState University, USA Pennstate University K and N poultry University of Veterinary & Animal SC. University of Veterinary & Animal SC. PennState University

Abstract Unraveling microbiota residing in the respiratory system of clinically healthy birds is important quality control in monitoring endemic infections and surveillance of pathogens in a particular geographical region. Information obtained from identified resident bacterial communities improves current molecular diagnostics and disease control strategies but most importantly helps in isolation and characterization of novel microorganisms. To our knowledge, culture independent analysis of respiratory microbiome of birds has not been explored to date. Using 454 pyrosequencing of 16S rRNA gene from tracheo - bronchoalveolar lavage of birds in free range, open house and controlled house system produced substantial numbers of reads passing rigorous quality control. The sequences were taxonomically profiled using homology - based computational method allowing identification of various taxonomic nodes and their placement in a phylogenetic tree to its closest known ancestor. The analysis revealed a far more diverse consortium of bacteria in birds than previously known including several bacteria of significance to public health. Overall 10 phyla, 65 families, 85 genera and 30 bacterial species were identified, the majority of them not identified as specific species indicating there may be many novel species in these birds. The sequence reads corresponding to the phyla Proteobacteria Firmicutes Bacteroidetes Actinobacteria and Tenericutes were common to birds in each management system. Fusobacteria and Cyanobacteria were identified in free range and controlled house whereas Deinococcus - Thermus Chloroflexi and Verrucomicrobia were identified exclusively in controlled house system. A high taxonomic diversity was observed between the management systems. Within the management system the taxonomic diversity was greater in open house system followed by controlled house and free range system. Culturing of organisms identified as genera or other classification with subsequent 16S rRNA gene sequencing and phylogenetic analysis will provide better understanding of the healthy microbiota and potential opportunistic pathogens in birds. Background

Unraveling microbiota residing in the respiratory system of clinically healthy birds is important quality control in monitoring endemic infections and surveillance of pathogens in a particular geographical region. Information obtained from identified resident bacterial communities improves current molecular diagnostics and disease control strategies, but most importantly helps in isolation and characterization of novel microorganisms. To our knowledge, cultureindependent analysis of respiratory microbiome of birds has not been explored to date. Materials and Methods Using 454-pyrosequencing of 16S rRNA gene from tracheo-bronchoalveolar lavage of birds in free range, open house and controlled house system produced substantial numbers of reads passing rigorous quality control. The sequences were taxonomically profiled using homologybased computational method, allowing identification of various taxonomic nodes and their placement in a phylogenetic tree to its closest known ancestor. Results The analysis revealed a far more diverse consortium of bacteria in birds than previously known including several bacteria of significance to public health Overall phyla families genera and bacterial species were identified The sequence reads corresponding to the phyla Proteobacteria Firmicutes Bacteroidetes Actinobacteria and Tenericutes were common to birds in each management system A high taxonomic diversity was observed between the management systems Within the management system the taxonomic diversity was greater in open house system followed by controlled house and free range system. Conclusion Culturing of organisms identified as genera or other classification with subsequent 16S rRNAgene sequencing and phylogenetic analysis will provide better understanding of the healthy microbiota and potential opportunistic pathogens in birds. Paper reference: SPS1322-14 A Survey of cross-match to transfusion ratio to optimize blood use at Al Baraha Hospital *Dr. Maitha Rida Al Maazmi Dr. Nishi Singh Dr. Muhammad Zaman

[email protected] UAE

UAE UAE

Sharjah Higher Colleges of Technology

Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology

Abstract Blood cross-matching is performed to assess compatibility between donor’s cells and patient’s plasma at the immunology level. Ordering cross-match but not transfusion is common which leads to blood wastage and is manifested as a high of cross-match to transfusion ratio. Such wastage of blood units has a negative impact on blood supply service. This study aims to correlate the cross-match to transfusion ratio with individual blood groups and to predict number of blood units needed for future. Background Prior to transfusion blood should be tested for compatibility by cross - match ( CM ) by mixing donor red blood cells and recipient serum in vitro. During CM an incompatible recipient will have antibodies against donor red blood cells resulting in clotting or agglutination leading to

haemolysis. CM is also a indicator of blood supply wastage with higher CM to transfusion ratios (CM:T) suggesting greater wastage. This research paper aimed to prove if there was correlation between Cross - match wastage and blood type Materials and Methods Cross-matched to transfusion ratio (CM:T) data were collected from 2010 to 2014 along with the numbers of blood units and blood types used over same period. The data were analysed using SPSS and Excel. Results Negative correlations were observed between blood types (O negative, O positive, AB negative, AB positive, B negative and B positive) and cross-match to transfusion ratio. Positive correlations were observed for blood types (A positive and A negative). The demand of blood will be increase in the future to almost the double after 10 years. Conclusion Negative Rh groups units are used less relative to positive Rh groups in the lab. O negative AB negative and B negative are less common, have less usage and have weakly negative correlation with CM:T. O positive AB positive and B positive are more available and more used with strongly negative correlation with CM:T. In contrast A negative with low supply and A positive with high amount both are positive correlation between CM:T. Our results suggest wastage is dependent on blood supply availability. Paper reference: SPS1321-14 Determination of the optimal analysis parameters for the short tandem repeat DNA Polymerase Chain Reaction Assay *Dr. Fatema Al-Hammadi

Dr. Nishi Singh Dr. Muhammad Zaman Dr. Ban Altoumah Dr. Burae Abdulrahman

[email protected]

UAE UAE UAE UAE

UAE

Sharjah Higher Colleges of Technology

Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology Sharjah Higher Colleges of Technology DNA department, Forensic Laboratory, Sharjah Police

Abstract Experiments were conducted on DNA from two sources, a buccal swab from a volunteer donor and from the designated positive control DNA sample of the AmpFLSTR Identifiler Plus PCR Amplification Kit from Life technologies. This assay is used in the forensic DNA typing of biological evidence and representative identifying reference biological samples of victims and potential suspects. The assay amplifies STR from 16 loci from 13 autosomal human chromosomes and from both the sex chromosomes. The aim of the experiments was to determine the sensitivity of the assay and to define the minimum concentration of DNA required to obtain a reproducible full representation of the profile of 16 STR loci. In addition, experiments on the amplification of simulated DNA mixtures

made from 2 sources at set content ratios were performed to measure the limit concentration of input DNA at which full representation of the profile of 16 STR loci from a single contributor of the mixture fails to occur. Moreover, each sample has been done in triplicate and diluted to 8 different concentrations (50pg, 150pg, 200pg, 400pg, 600pg, 800pg and 1ng). Several stages have been done to reach to the last result including: collecting the DNA from buccal, extraction the DNA by Qiagen method, DNA quantification by real time PCR, DNA replication by PCR and finally STR. Conclusion, the limit of detection (LOD) has been varied between the two samples because the DNA extraction method was different. For the mixture analysis it is better to start with 400pg concentration of DNA and above. Background The human genome is diploid so consisting of two alleles per gene The alleles can be identical ( homozygous ) or different ( heterozygous ) The aim of doing this experiment is to determine the sensitivity of the assay and to define the minimum concentration of DNA required to obtain a reproducible full representation of the profile of STR loci As well to look at various human markers in different mixtures of combination to determine the optimal reaction conditions for resolving mixed samples Materials and Methods DNA was extracted from a buccal swab from a volunteer donor using the QIAamp DNA Investigator Kit from QIAGEN, Germany. The experiments that followed, subjected DNA from this sample as well as the designated positive control sample, Control DNA 9947A of the AmpFLSTR Identifiler Plus PCR Amplification Kit from Life technologies, USA to a series of assays namely DNA quantitation, DNA Amplification and Capillary Electrophoresis of the amplified DNA products. Results see below Conclusion Conclusion, the limit of detection (LOD) has been varied between the two samples because the DNA extraction method was different. For the mixture analysis it is better to start with 400pg concentration of DNA and above. Paper reference: SPS1317-14 Cancer stem cells: A paradigm shift in cancer treatment? *Dr. Asma Sultana Shaik Dr. Abjal Pasha Shaik

[email protected]

King Saud University, KSA

Saudi Arabia King Saud University, KSA

Abstract Cancer is the one of the leading causes of mortality and morbidity across the world. New and efficient therapies are continuously being investigated to prevent tumor growth and metastatis. Research over the past few years has indicated that cancer could be a stem cell disease. Cancer stem cells (CSCs) are typically found within tumors/cancers and are thought to possess the

ability to produce the cell types specific for a cancer. The tumorigenic ability of CSCs can thus produce new tumours through self-renewal and cell differentiation into other cell types. This feature can cause relapse and metastasis posing a challenge for therapy. CSCs constitute only a small percentage of the entire tumor but research indicates that the molecular pathways characteristic of cell cycle regulation are constitutively expressed in these leading to new and uncontrolled growth. The development of specific and targeted treatment options that can inhibit the survival of CSCs is the current focus of cancer research. This presentation will focus on the various cell signalling pathways that can operate in tumorigenesis induced by CSCs with specific focus on how nanomedicine can be used to not only track the CSCs but also to deliver drugs to the site of CSCs. Paper reference: SPS1310-14 IDENTIFICATION OF 12 STD PATHOGENS IN SEMEN USING POLYMERASE CHAIN REACTION (PCR) AND “FLOW-THROUGH” HYBRIDIZATION TECHNOLOGY *Dr. Rubina Ghani

[email protected]

Pakistan Pathological & Molecular Laboratories/Baqai Medical University

Abstract Background The prevalence of sexually transmitted Disease ( STDs ) among hotel - based sex workers ( HBSWs ) in Karachi Pakistan was studied These hotel workers are considered as high risk group because of their age economic independence low education and residence in a place away from their family Objective In poor countries data on STDs and related complications are limited which causes a substantial under estimation of the burden of these diseases Aim The aim of this study was to access in health care facilities for diagnosis and common pathogens of STDs those causing infertility and Chlamydia trachomatis Neisseria gonorrhoeae and Mycoplasma hominis Genital wart is a highly contagious sexually transmitted disease caused by some sub - types of human papillomavirus ( HPV ). Material and Methods Semen samples were obtained by masturbation into sterile containers after sexual abstinence of to hours Samples were subjected to semen analysis within one hour of collection and processed for freezing within two hours of collection The concentrations of sperm as well as sperm motility were also determined DNA extraction was extracted of all the samples and the PCR assay was performed. The amplified by The amplicons are subsequently hybridized to pathogen - specific capturing probes via “ Flow - through ” hybridization Result During our study we came across with the STI pathogens present in our population and the reason for infertility was the main cause When Chlamydia trachomatis and Neisseria gonorrhoeae were detected in their wife ’ s were also screened and these STI pathogens were identified The main route for the transfer of STI pathogens were the men special those who visited commercial sex workers or hotel - based sex workers as they were working in other cities.

Paper reference: SPS1309-14 Differential up regulation of the hypothetical transmembrane protein 66 (TMEM66) in Bahraini multiple sclerosis patients with potential inflammatory response *Dr. Safa Taha

[email protected]

Bahrain

Arabian Gulf University-College of Medicine and Medical Sciences

Abstract The prevalence of multiple sclerosis (MS) in the Gulf region has been reported to be markedly increased during the last decade, but the pathogenesis of the disease was not investigated. To understand the molecular mechanisms involved in the disease process, the present work utilized the microarray technology to study differentially expressed novel genes in MS Bahraini patients compared to healthy matched subjects. The results depicted that 493 transcripts were differentially expressed out of about 50,000 transcripts; among these, 230 transcripts were upregulated while 263 were downregulated, in MS patients compared to the healthy controls, as Fold Change (FC) was 1.5 Keywords: Cytokine, Chemokine, Proliferation, Gene expression, Microarray

Paper reference: SPS1308-14 Identification of polycyclic aromatic hydrocarbon degrading bacterial strain and its ability to degrade pyrene *Dr. Mervat Aly Mohamed Abo-State

[email protected] Egypt

National Center for Radiation Research and Technology (NCRRT), Egyptian Atomic Energy Authority (EAEA)

Abstract Four bacterial isolates were previously isolated from soil polluted with petroleum oil from Cairo Refining Company Al-Qalubia , Egypt. These isolates (MAM-26 , 29 , 62 and 68) and standard strain Enterobacter cloacae MAM-4 were grown on five concentrations of pyrene (Pyr.) as a sole carbon and energy source. The abilities of these isolates to degrade Pyr.have been investigated . The growth (O.D) and extracellular protein secretion were determined after 1,2,3,4,5,6,7,14 and 21 days incubation for each strain. Degradation of Pyr.was quantified by High Performance Liquid Chromatography (HPLC). The results revealed that isolate MAM-29 degrade 95.0% , 90.5% and 90.3% of 100 , 200 and 300 ug/L of Pyr. respectively .The best Pyr.degrader bacterial isolate MAM-29 was identified by 16S-rRNA. This isolated strain showed 100% similarity with Achromobacterxylosoxidansstrain R8-558 with accession No. JQ 659958.1 So isolate MAM-29 was identified as Achromobacterxylosoxidans with accession No. JN 038055. Pyrene degradation by B.amyloliquifaciens MAM-62, the most potent strain in degrading PAHs in general, produced 4 intermediates compounds after 24 hrs. incubation as determined by GC/ MS . These intermediates were benzeneethanol;hexanoic acid 3,5,5'-trimethyl;2,4,6-

cycloheptatriene -1-one and tetradecanoic acid . Mutant ofB.amyloliquifaciens MAM-62 (4) resulted from exposure to gamma radiation produced five different intermediates.

Paper reference: SPS1307-14 DEGRADATION OF CHLORPYRIFOS USING AUTOCHTHONOUS BACTERIAL CONSORTIUM *Dr. Elizabeth Mary John

[email protected]

Dr. Jisha M.S Dr. Divya K

School of Biosciences, Mahatma Gandhi University, Kottyam, Kerala School of Biosciences, Mahatma Gandhi University, Kottyam, Kerala

India India

India School of Biosciences, Mahatma Gandhi University, Kottyam, Kerala

Abstract Widespread and indiscriminate use of chlorpyrifos (CP)- an organophosphate pesticide, leads to severe environmental problems. It poses a great threat to different trophic levels of the ecosystem from soil microorganisms to human beings. As the risk of their off-site migration pose a health risk to non-target organisms, it is essential to remove this pesticide from their point source of contamination. The current research paper attempts to develop a bacterial consortium capable of remediating chlorpyrifos effectively. The degradatory efficiency of thirteen morphologically different soil bacterial isolates obtained by selective enrichment on mineral medium containing chlorpyrifos (25 ppm) or Trichloro2pyridinol (TCP- antimicrobial byproduct of CP degradation) (25 ppm) as sole source of carbon and nitrogen was investigated for five days. The isolates were equally capable of degrading TCP compared to chlorpyrifos. Using these isolates different bacterial consortia were developed and the consortium C2 (S5, S6, S12) was efficient and showed 95.38±0.02 % of degradation of CP (25 ppm) in three days. The efficiency of the consortium to mineralize chlorpyrifos was investigated under different culture conditions. The study revealed that the consortium could transform chlorpyrifos to nontoxic products. Immobilisation of developed consortium was done and rate of chlorpyrifos degradation using immobilized system in presence of inorganic fertilizer (NPK) and coir pith was investigated. The obtained results demonstrated that the immobilized consortium could be recommended for usage in biobeds as a viable alternative to prevent chlorpyrifos dissipation in the environment. Background Widespread indiscriminate use of chlorpyrifos, an organophosphate pesticide leads to severe environmental problems and poses a great threat to beneficial soil microorganism. Repeated applications of pesticides may have an adverse effect on soil microbial functional diversity and subsequently influence soil fertility and plant growth, which pose serious threats to the sustainability of agricultural soils. As the risk of their off-site migration pose a health risk to nontargets including humans, it is essential to remove this pesticide from the point source of contamination in the environment. Materials and Methods

Isolation of chlorpyrifos degrading bacteria was done by selective enrichment on mineral medium containing chlorpyrifos as sole source of carbon and nitrogen. Morphologically different colonies were selected and screened to obtain efficient isolates by HPLC and GC. Efficient six strains were used to develop a consortium.The ability of the consortium to mineralize chlorpyrifos was investigated under different culture conditions. The consortium was immobilized and rate of degradation of chlorpyrifos using immobilized system biostimulated with inorganic fertilizer (NPK) and coir pith was investigated. Results Thirteen morphologically different soil bacterial isolates were obtained by selective enrichment on mineral medium containing chlorpyrifos as sole source of carbon and nitrogen for mineralization of chlorpyrifos ( ppm ) and TCP ( ppm ) and were screened to obtain six effiecient isolates. A consortium of three bacterial strains - S5, S9, S10 - which were more efficient and showed 96 % of degradarion of 25 ppm chlorpyrifos in three days was developed. Immobilised consortium was also able to degrade chloryrifos. Conclusion An efficient consortium of chlorpyrifos degrading bacterial strains- S5,S9,S10- was developed and the rate of degradation of chlorpyrifos using immobilized system biostimulated with inorganic fertilizer (NPK) and coir pith was investigated. The obtained results demonstrated that the immobilized consortia biostimulated with NPK nutrient can be recommended in biobeds as a viable alternative of chlorpyrifos dissipation in the environment. Paper reference: SPS1306-14 Endophytic diazotrophic bacteria: An ecologically effective method for sustainable biological nitrogen fixation * Dr. SHABANAMOL SUBAIDABEEVI Dr. DR JISHA M S

[email protected] India

India

School of Biosciences, Mahatma Gandhi University, Kottyam, Kerala

School of Biosciences, Mahatma Gandhi University, Kottyam, Kerala

Abstract Lysinibacillus sphearicusis a bacterium currently used in the biological control of mosquitoes. We have got this bacterium as a nitrogen fixing endophyte from cultivated rice plants on Dobereiner’s N2 free BTB agar. Due to higher seed germination capacity, this bacterium was studied for its colonization efficiency and plant growth promotion in rice using double resistant antibiotic marker. It was found that this strain could colonize the roots of paddy plants and observed using light microscopic and SEM analysis. The bacterium is positive for production of plant growth promoters like IAA,ammonia but was negative for phosphate solubilization and ACC deaminase. The percentage of nitrogen in the growth solution was analyzed using micro kjeldahl method. Total NPK analysis, shoot length, root length and chlorophyll content of 15 days old inoculated plants were also higher than control and standard inoculated plants Background It has been a long goal of many biological nitrogen fixation researchers to transfer the ability of nitrogen fixation effectively to plants other than leguminous ones. Since endophytic

diazotrophs may have an advantage over root associated ones they can better exploit the substrates provided. Therefore a study was planned to isolate and characterize efficient endophytic nitrogen fixing bacteria with excellent plant growth promotory and biocontrol activities as nitrogen is the major limiting nutrient for the development and yield of rice plants. Materials and Methods Isolation of endophytic diazotrophic bacteria colonization and establishment of endophytic diazotrophic bacteria seed germination index Nitrogenase (ARA) activity o the isolates (in vitro- and in planta)Hardy et al,1968) nif gene analysis P.Gyaneswar et al., 2001 J.Hallmann et al.,1981 Elbeltagy et al., 2001 S M Tripathi et al., 2006 Results It was found that the isolate could colonize the roots of paddy plants and observed using light microscopic and SEM analysis. The bacterium is positive for production of plant growth promoters like IAA,ammonia. ARA analysis also confirmed the in planta nitrogenase activity. The percentage of nitrogen in the growth solution was analyzed using micro kjeldahl method. Total NPK analysis, shoot length, root length and chlorophyll content of 15 days old inoculated plants were also higher than control and standard inoculated plants Conclusion Through this study, it is clear that the isolated endophytic diazotrophic L sphearicus could colonize the rice plants and enhance plant growth promotion in a better way than do standard free living plant growth promoting bacteria. Paper reference: SPS1305-14 In vitro antimicrobial, antiprotozoal activities, phytochemical screening and heavy metals toxicity of different parts of Ballota nigra *Dr. Najeeb Ullah

[email protected] Pakistan Kohat University of Science & Technology, Kohat

Abstract The study was done to assess the phytochemicals ( flavonoids terpenoids saponins tannin alkaloids and phenol ) in different parts ( root stem and leaves ) of Ballota nigra and correlated it to inhibition of microbes ( bacteria and fungi ) and protozoan ( Leishmania ). In root and stem flavonoids terpenes and phenols were present in ethanol chloroform and ethyl acetate These fractions were the most active inhibiting fractions against bacteria and fungi Ethanol and chloroform fractions show maximum inhibition of 24 mm each against Enterococcus faecalis in root and chloroform against Staphylococcus aureus in stem 19 mm. Maximum inhibition of ethyl acetate fraction against Staphylococcus aureus is 24 mm in root. In leaves flavonoids terpenes and phenols were present in ethanol chloroform and butanol fractions which were the most active fractions against both types of microbes in in vitro study Ethanol and chloroform

fractions show maximum inhibition against Escherichia coli. Hexane fraction in leaves contain only terpenes which show excellent inhibition of mm against Klebsiella pneumoniae In antileishmanial results crude ethanolic extract chloroform and ethyl acetate fractions in roots of Ballota nigra have antileishmanial activity In stem the ethanol butanol and ethyl acetate fractions show inhibition of amastigote where as in leaves ethanol chloroform and butanol fractions show inhibition of leishmanial parasite The phytochemical and biological screening was correlated with the presence of heavy metals in selected plant Ballota nigra. Only few metals (Cr Ni Fe Cd Pb) were observed above WHO limits. Background The study was done to assess the phytochemicals ( flavonoids terpenoids saponins tannin alkaloids and phenol ) in different parts ( root stem and leaves ) of Ballota nigra and correlated it to inhibition of microbes ( bacteria and fungi ) and protozoan ( Leishmania ).The phytochemical and biological screening was correlated with the presence of heavy metals in selected plant Ballota nigra. Materials and Methods Collection and Drying of Plant Materials Extraction Procedure Antimicrobial Assay Preparation of the Test Compound Antibacterial Bioassay ( The six bacterial strains Escherichia coli Staphylococcus aureus Proteus mirabilis Klebsiella pneumoniae Enterococcus faecalis and Salmonella typhi were used ) Well Assay Method Antifungal Bioassay ( four fungal strains Aspergillus niger Aspergillus flavus Aspergillus fumigatus and Fusarium solani were tested ) Agar Tube Dilution Method Antileishmanial Bioassay Culturing of Parasite Leishmanicidal Procedure Phytochemical Analysis ( flavonoids terpenoids saponins tannin alkaloids and phenol were investigated ) Analysis of Plant Samples for Heavy Metals Results The results of antimicrobial ( antibacterial and antifungal ) study of different parts of Ballota nigra indicate that crude extract ethyl acetate and chloroform fractions were the most active fractions The crude extract ethyl acetate and chloroform fractions of Ballota nigra show good leishmanicidal properties But different parts have different inhibition properties So each part of plant should be checked for antimicrobial and antiprotozoal assay Ballota nigra accumulates different phytochemicals with different concentrations Only few metals (Cr Ni Fe Cd Pb) were observed above WHO limits. Conclusion The antimicrobial and antiprotozoal study of different parts of Ballota nigra indicate that Ballota nigra show good antimicrobial and leishmanicidal properties But different parts have different inhibition properties So each part of plant should be checked for antimicrobial and antiprotozoal assay Ballota nigra accumulates different phytochemicals in different parts with different concentrations the plants accumulate different metals in its different parts ( root stem and leave ) with diverse concentration.

Paper reference: SPS1304-14 PICRUSt metagenome functional content prediction using 16S rRNA pyrosequencing of gastrointestinal tract microbiome of broilers Dr. Muhammad Umar Sohail

[email protected] Pakistan

Abstract Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) is a recently developed computational approach for prediction of functional composition of a microbiome comparing marker gene data with a reference genome database. The procedure established significant link between the phylogeny and function in human subject studies and have been thoroughly reviewed. In the current study, we used PICRUSt for predicting metagenomics in broilers subjected to chronic heat stress (HS), and supplemented with prebiotics and probiotics. Ceca digesta were taken for DNA extraction. The sample DNA was sequenced using 16S rRNA high throughput pyrosequencing. The 16S rRNA genome sequences were analyzed using bioinformatics software PICRUSt to predict the functional capacities of the bacteria. Functional genes content inference was consigned according to Kyoto Encyclopedia of Genes and Genomics Orthology (KO) Hierarchy and compared using Linear Discriminant Analysis Effect Size (LEfSe) and the Mann-Whitney U test. The gene contents of gut microbiome were predominantly associated with metabolism (52%) and information processing (34.6%). Among metabolic processes, carbohydrates metabolism (18.5%) was highest (P < 0.05), followed by xenobiotics, amino acids and lipids metabolism. Membrane transportation (6.1%) activity was highest among different treatment groups, followed by carbohydrate metabolism (2.7%). Among different treatment groups metabolism and membrane transportation activity was not significantly different (P>0.05). In conclusion gut microbiome significantly contributes to host metabolism and membrane transportation. Background Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) is a recently developed computational approach for prediction of functional composition of a microbiome comparing marker gene data with a reference genome database. The procedure established significant link between the phylogeny and function in human subject studies and have been thoroughly reviewed. Materials and Methods In the current study we used PICRUSt for predicting metagenomics in broilers subjected to chronic heat stress ( HS ), and supplemented with prebiotics and probiotics Ceca digesta were taken for DNA extraction The sample DNA was sequenced using S rRNA high throughput pyrosequencing The S rRNA genome sequences were analyzed using bioinformatics software PICRUSt to predict the functional capacities of the bacteria. Functional genes content inference was consigned according to Kyoto Encyclopedia of Genes and Genomics Orthology (KO) Hierarchy. Results The gene contents of gut microbiome were predominantly associated with metabolism ( %) and information processing ( %). Among metabolic processes carbohydrates metabolism ( %) was highest ( P < ), followed by xenobiotics amino acids and lipids metabolism Membrane

transportation ( %) activity was highest among different treatment groups followed by carbohydrate metabolism (2.7%). Among different treatment groups metabolism and membrane transportation activity was not significantly different (P>0.05). Conclusion In conclusion gut microbiome significantly contributes to host metabolism and membrane transportation. Paper reference: SPS1303-14 Bioactive compounds from Spirastrella inconstans against wound infection Dr. TamilSelvi Arulmozhi Varman

[email protected]

UAE

Periyar University, India

Abstract Marine invertebrates, in particular sponge (Spirastrella inconstans), represent a source of a wide range of secondary metabolites, many of which have been attributed to various defensive capabilities against CA-MRSA (Community associated- Methicillin Resistant Staphylococcus aureus) obtained from wound infection among fisherman community. In this work spongederived low-molecular peptide-like compounds and associated analogs are investigated for bioactivity and pharmacological targets. 3,000 Da molecular weight of protein was isolated by SDS-PAGE. Amount of protein from Spirastrella inconstans was estimated by UV-Vis spectrometer were analysed by IR and UV spectrum. Absorption value obtained by UV spectrum using TECHCOMP 8500 spectrophotometer was 1.7141. Further confirmation of isolated compound was carried out by HPLC analysis as 254 nm wave length and 13C and 1H NMR. Scanning Electron Microscope (SEM) with constant voltage of 20 kV at X 850 illumination shows 20 μm between the compounds, at X 270 illumination 50 μm, at 2,300 X illumination 10 μm, at 6,000 X illumination 2 μm, at 2,700 illumination 5 μm confirms the compound by structural elucidation as glutamic acid. The novel compound in lyophilized form can favor fibroblast proliferation with collagen production, in the therapy for the healing of tendon lesions and of wounds by acting against MRSA. Paper reference: SPS1301-14 Development of a viral recombinant based vaccine for IPNV *Dr. Maryam Dadar Dr. Hamid Rajabi Memari

[email protected] Iran Iran

Shahid chamran University

Shahid chamran University, Nogen Company

Abstract Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in salmonids. Bacterial based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. VP2 is a structural viral protein of IPNV immunogenicity effects. In this study IPNV was isolated from diseased fry rainbow trout Oncorhynchusmykiss (Walbaum) using CHSE-214. Then an

expression vector was constructed for expression of viral protein VP2. The designed vector was constructed based upon pET-26b (+) with T7 promoter. A fragment containing the full length of the VP2 gene of Iranian Sp strain was amplified by PCR using genomic RNA of IPNV as template and cloned in pET-26b(+) plasmid. Recombinant structural viral protein VP2 was expressed as a soluble, N-terminal PelB fusion protein and secreted into the periplasmic space of Escherichia coli BL21(DE3) and Rosetta (DE3). The glucose, Isopropyl-β-D- thiogalactopyranoside (IPTG) was used as a chemical inducer for rVP2 production in 37º C. The rVP2 was extracted from the periplasm by osmotic shock treatment. The presence of gene in bacterial system of E.coli was confirmed by gel electrophoresis technique. The constructed vector could efficiently express the rVP2 into the periplasmic space of E.coli. The successful cloning and expression of the structural viral protein gene into E.coli can be used for developing a useful and safe vaccine to control IPNV infection in Iranian fish industry. Background Infectious Pancreatic Necrosis Virus (IPNV) is a member of the family Birnaviridae that has been linked to high mortalities in juvenile salmonids. Also structural viral protein VP2 is a protein of IPNV which different studies shown its immunomodulatory. Bacterial based systems as live vectors for the delivery of heterologous antigens offer a number of advantages as vaccination strategies. Materials and Methods Full length of the VP2 gene of Iranian Sp strain ( NCBI KC489465 ) was amplified by polymeras chain reaction ( PCR ) using genomic RNA of IPNV as template The amplified bp fragment was digested by NcoI and BamHI followed by ligation into the corresponding site on the PET26b (+) plasmid The ligated DNA was then transformed into E coli BL21 and Rosetta (DE3) by standard method. Verification of cloning was carried out using restriction enzyme digestion and DNA sequencing. Results The successful cloning and expression of the structural viral protein gene into E.coli can be developed as a useful and safe system to control IPNV infection in fish. one of the most promising and urgent applications of rVP2 is to investigate their use in introducing a recombinant vaccine. The recombinant VP2 can be used as part of a commercial injectable polyvalent and monovalent vaccines well as development of new diagnostic kits. Conclusion The presence of gene in bacterial system of E.coli was confirmed by gel electrophoresis technique, dot blot and western blotting methods. The constructed vector could efficiently express the rVP2 into the periplasmic space of E.coli. Paper reference: SPS1300-14 Biotechnological approaches to fish vaccine Dr. Hamid Rajabi Memari

[email protected] Iran

Shahid chamran University

Abstract Pathogens are the most important limiting factors in development of intensive aquaculture. Because of limitation in treatment, such as costs of treatment and delay in growth,

environmental pollutions, mortalities, and intensive labor, the prevention of disease is most preferable to disease treatment. The best approach for disease control is vaccines. A substantial amount of research has been done on protection of fish against pathogens affecting various species in aquaculture. This review will focus on the vaccination in aquaculture industry, as this is the most important part of development in fish farming. It will address the type of vaccines, some advantages and disadvantages of these vaccines, and discuss the future aspect in this field. Background Vaccination is the most effective method of combating disease and currently there are a number of vaccines commercially available for use in fish. Vaccines for some bacterial,viral and parasitic fish pathogens are proving more difficult to develop and researchers have begun to use molecular and biotechnological approaches to develop such vaccine. Materials and Methods Recombinant vaccines are consist of Recombinant protein vaccine Peptide vaccine Attenuate live vaccine via genetic engineer and DNA vaccine Recombinant vaccines is the best vaccine because of Stimulate humoral and cellular immunity but killed vaccine Stimulate mainly humoral immunity recombinant protein vaccine is used for viral and bacteriad disease in fish such as IPN IHN VHS A salmonicida A Hydrophila. Results vaccines of fish must be made at a low sales and application cost In additional of multivalent vaccine for salmonid we need additional antigens against ISAV pancreas disease virus ( PDV ), VHSV and IHNV for the North Hemisphere . In addition to development of recombinant technology in production of a new vaccine there is a need to develop new expression system which yields glycosylation of the proteins and restoration of the tertiary structure . Conclusion Advances in biotechnology have made an important contribution in reducing disease risks and their losses in aquaculture and allow vaccine development against pathogens These advances made it possible to identify protective antigens and provide safe and inexpensive manufacture of vaccine Vaccination in aquaculture is becoming an important part of the health management since it is considered a cost - effective approach of controlling important threatening pathogen. Paper reference: SPS1296-14 IL-21 Polymorphisms Linked with Recurrent Spontaneous Miscarriage Dr. Safia MESSAOUDI

Dr. Touhami Mahjoub

[email protected]

Tunisia

Laboratory of Human Genome and Multifactorial Diseases, College of Pharmacy, Monastir

Tunisia Laboratory of Human Genome and Multifactorial Diseases, College of Pharmacy, Monastir

Abstract Problem: Insofar as recurrent spontaneous miscarriage (RSM) is linked with dysregulated immunity and inflammatory changes, and given the role of interleukin-21 (IL-21) as a proinflammatory cytokine, we examined the association between IL-21 polymorphisms and RSM. Method of Study: IL-21 rs2055979, rs13143866, rs9992580, and rs4833837 were genotyped in 235 RSM cases and 235 control women. Results: Higher minor allele and genotype frequencies of rs2055979 and rs13143866, but not rs9992580 or rs4833837, were seen in patients than control women. IL-21 haplotype [rs9992580/rs4833837/rs2055979/rs13143866] analysis revealed lower frequency of TGCG haplotype, and higher frequency of GGCG and GAAA haplotypes in patients, thus conferring RSM protection and susceptibility, respectively. Regression analysis confirmed the association of TGCG [OR(95%CI) = 0.09(0.05-0.16)], and GGCG [OR(95%CI) = 2.52(1.34–4.54)] and GAAA [OR(95%CI) = 4.02(2.20–7.70)] haplotypes, after adjusting for age and BMI. Conclusion: Our findings indicate that IL21 is a novel susceptibility gene for RSM. Paper reference: SPS1291-14 A putative Novel Immune Signaling Pathway driven by the Nervous System revealed by computational analysis of the mouse ISRAA protein. Dr. Mohamed-Dahmani Fathallah

[email protected] Bahrain

Arabian Gulf University-College of Graduate Studies

Abstract We have previously described in mouse a novel immune system released activating agent ( Israa ) over - expressed by splenocytes following a nervous stimulation We showed that the Israa gene is embedded in intron of the Zmiz1 locus Israa encodes a AA protein that seems to be involved in T - cell signaling pathways No typical gene orthologs were found in other species and no significant homology with known proteins To investigate the precise biological function of ISRAA we carried out a computational approach to study its structure - activity relationship Functional motif scan showed the presence of an SFK specific SH2 binding domain surrounding Tyr ( YTEV ). We generated an abinitio model of ISRAA and carried out docking analysis with Fyn and Lck SH2 domains - ]. We used this model to design and produce recombinant wild type and mutant ( YTEV ) isoforms of ISRAA When stimulated by WT - recombinant ISRAA mouse splenocytes showed no significant proliferation by measuring radioactive - Thymidine incorporation However MTT assay revealed a raise in cellular activity which does not occur following stimulation by the YT - double mutant In vitro direct binding assay showed that WT - ISRAA interacts with active mouse rFyn while simultaneous mutation of Y102 and T103 abolished this binding The analysis of the phosphorylation status of SFKs ( Src Fyn Lck ) showed a decrease in phosphorylation of the activating Tyr - in the early stage of stimulation by WT ISRAA Meanwhile YT - Mutant showed a normal rate compared to untreated cells Considering that Src - Family Kinases ( SFKs ) Fyn and Lck play an essential role in the T - cell development homeostasis and activation our data strongly suggest that ISRAA

Paper reference: SPS1290-14 Genomic Structure of the israa locus and cross-species comparative analysis. *Dr. Noureddine Ben Khalaf

[email protected]

Dr. Mohamed-Dahmani Fathallah

Bahrain Arabian Gulf University-College of Medicine and Medical Sciences

Bahrain Arabian Gulf University-College of Graduate Studies

Abstract We have previously discovered, using a Differential-Display approach, the Immune system released activating agent: israa gene, an intron-embedded gene in the mouse Zmiz1 intron 8. Israa has been shown to be over-expressed in mouse splenocytes following a nervous stimulation. This gene encodes for a 125 AA protein that could play a role in immune signaling pathways and nervous-system induced immunity. Israa locus is located within intron 8 of the zmiz1 gene on mouse chromosome 14. In this study, we carried out a bioinformatic analysis to determine the structural organization of the zmiz1 gene and the israa locus. Our study showed interesting genomic features that could help identify the regulation of israa transcription and expression; in addition we noticed an uncommon splicing event occurring in the israa ORF. The analysis revealed also the presence of another nested gene upstream of the israa locus; this gene is annotated as a RIKEN gene, which indicates a transcriptional activity and gene expression in this part of the genome. Meanwhile across species (human, mouse, primate and rat) comparative analysis of homologous genomic region did not show a typical ortholog of the israa locus. Since zmiz1 is widely conserved among species, we compared the gene structure in the four species, and find numerous deletions and insertions in intronic regions of the gene despite the high conservation in exonic regions. Interestingly, we identified conserved genomic properties and features that might help identifying "functional-ortholog" of israa in these species. This study unveiled several structural features of intron-nested genes and gave new insights into novel functions that could be carried out by unexplored region of the genome. Keywords: Nested genes, Comparative genomics, Mouse, Gene

Paper reference: SPS1289-14 Seroprevalence of Echinococcosis in Healthy Blood Donors and Municipality Workers in UAE *Dr. Naglaa Oda Dr. Doaa Sultan Dr. Marwa Khalil Dr. Amin Al-Amiri Dr. Mahra Al - Marzouqi

[email protected] UAE UAE UAE UAE

UAE

Dubai Medical College Dubai Medical College Ministry of Health Ministry of Health

Dubai Medical College

Dr. Haya Al-Shawa UAE Dr. Hiba Al-Saman UAE Dr. Seyed Mahmoud Sadjjadi Iran

Dubai Medical College Dubai Medical College Shiraz University

Abstract Echinococcosis is an important public health problem in many parts of the world. High prevalence is found in the Middle East as well as Arabic North Africa. Among all available serological assays, the use of highly purified antigens improves the sensitivity of these assays, and the use of immunoblot is a powerful confirmatory technique. The present study was carried out to estimate the prevalence of Echinococcosis as measured by serum AntiEchinococcus antibodies (AB) among 1651 healthy individuals seen at “Sharjah blood transfusion and research center” and “Dubai municipality clinics” (UAE). Serum samples were tested for the presence of Anti-Echinococcus antibodies using enzyme linked immnunosorbent assay (ELISA) and western blot analysis as confirmatory test. The prevalence of Echinococcosis was 1.3 % among the studied population. The highest prevalence was found in males (89.5%) and most of them were of South Asian nationality (79%). Between all categories, the South Asian Nationality and male groups has a significant higher seropositivity level. Background Echinococcosis is an important public health problem in many parts of the world. High prevalence is found in the Middle East as well as Arabic North Africa. Among all available serological assays, the use of highly purified antigens improves the sensitivity of these assays, and the use of immunoblot is a powerful confirmatory technique. Materials and Methods The present study was carried out to estimate the prevalence of Echinococcosis as measured by serum Anti-Echinococcus antibodies (AB) among 1651 healthy individuals seen at “Sharjah blood transfusion and research center” and “Dubai municipality clinics” (UAE). Serum samples were tested for the presence of Anti-Echinococcus antibodies using enzyme linked immnunosorbent assay (ELISA) and western blot analysis as confirmatory test. Results The prevalence of Echinococcosis was 1.3 % among the studied population. The highest prevalence was found in males (89.5%) and most of them were of South Asian nationality (79%). Conclusion Between all categories, the South Asian Nationality and male groups has a significant higher seropositivity level. Paper reference: SPS1288-14 NUTRITIONAL GENOMICS AND PERSONALIZED NUTRITION *Dr. Dina Abdulrahman Muharib [email protected]

KSA

king saud medical city

Abstract Nutritional Genomics is the study of how foods affect gene expression(nutrigenomics) and how individual genetic variation affects the way an individual responds to nutrients in food(nutrigenetic) .

Genetic variation certainly has an important influence on human nutritional requirements, and the introduction of genomics has both highlighted the complexity of the interaction between genes and diet and offered opportunities to reevaluate the criteria used to determine RDAs and the contribution of genetic variation to optimal nutrition for individuals. As the interactions between genetic variation and nutritional requirements become more fully understood, it will allow dietary recommendations to be individualized according to genotype to ultimately reduce our risk of degenerative diseases and increase health and well-being in old age

Paper reference: SPS1287-14 Hypoxia may have a teratogenic effect on sex differentiation and determination of aquatic animals *Dr. Hamed Kolangi Miandare

[email protected]

Iran

Assistant Professor at Gorgan University of Agricultural Sciences and Natural resources

Abstract The sex of a fish is not only determined by its genotype, but may also be affected by environmental factors which can alter sex differentiation. While the gender of most organisms is determined at the time of fertilization, but in the most of the fish species sex can be effected by the environment after fertilization. One of the key environmental factors is oxygen; the hypoxia-inducible factors (HIFs) are the indicators of oxygen condition. HIFs are key transcriptional regulators of hypoxic response in embryonic organisms. HIFs are “master regulator” that either directly or indirectly regulate transcription of more than one hundred Genes those are important in divers function, HIFs also down regulate some of genes involved in steroid genesis that alter the ratio of testosterone (T) to estradiol (E2). In this study hypoxiainducible factors gene expression (HIF-1α, HIF-2α) were evaluated during development of an ancient fish species, Beluga sturgeon (Huso huso) in a normal oxygen condition. The hypoxia genes are affective on sex determination and differentiation, hypoxia genes regulating steroid genesis during embryonic development, thereby disrupting the hormonal balance and sex differentiation, subsequently may be leading change in sex ratio in sturgeon. The result of this study was suggested to study sox family gene and hypoxia genes expression in same time to find the relation between of them in future studies. Paper reference: SPS1284-14 Novel mutations in the gene HOXC13 underlying pure hair and nail ectodermal dysplasia in consanguineous families Dr. Raja Hussain Ali

[email protected] Pakistan Department of Biochemistry, Quaid i Azam University, Islamabad, Pakistan

Abstract Pure hair and nail ectodermal dysplasia ( PHNED MIM ) is a congenital disorder with hair growth disorder of scalp and boby and the appearance of dystrophic nails The autosomal recessive form has been mapped on chromosomes p12 – q21 and p11 – q21 encompassing type I and type II keratin gene clusters respectively Pathogenic mutations in a type II keratin gene KRT85 ( MIM ) causing the recessive form of PHNED have been reported previously Recently two research groups working on PHNED reported three mutations in the gene HOXC13 located in the HOXC cluster on chromosome12 p11 – q21 lIn the present study two unrelated consanguineous families ( A and B ), segregating the autosomal recessive form of PHNED were identified and sampled from remote regions of Pakistan for subsequent genetic analysis to identify the underlying defective gene Affected individuals in the two families A and B presented slight variations in clinical features including the hair and nails Genotyping data and haplotype analysis established linkage in both families to the type II keratin gene cluster on chromosome p11 – q21 Initially the gene KRT85 shown previously to cause PHNED was screened to search for potential sequence variants After failing to detect sequence variants in the gene KRT85 a member of the WNT family and three other keratin genes ( WNT10B KRT86 KRT81 KRT75 ) and HOXC13were screened Sequence analysis revealed a homozygous - bp duplication ( c – dupGCCA p His68Glnfs * ) and a nonsense mutation ( c C > A p Ser135 *) in HOXC13 in family A and B respectively These defective transcripts formed due to mutations are most likely degraded by nonsence mediated RNA decay The results shows the importance of a role played by HOXC13 in the development of hair and nails Background Pure hair and nail ectodermal dysplasia ( PHNED MIM ) is a congenital disorder with hair growth disorder of scalp and boby and the appearance of dystrophic nails. Earlier the autosomal recessive forms are known to be caused by Pathogenic mutations in a type II keratin gene KRT85. Recently three mutations including a nonsense mutation ( c.390 C > A; p. Tyr130 *), 27.6 kb deletion and a homozygous frameshift mutation ( c .delC; p. Leu119Trpfs *20 ) in three families of different ethnic backgrounds Materials and Methods Affected families were identified and their venous blood sampled after informed consent of affected and normal individuals. The study was approved by the Institutional Review Board (IRB) of Quaid-i-Azam University, Islamabad, Pakistan. Linkage in the two families was tested using microsatellite markers linked to type I and type II keratin gene clusters on chromosome 17p12–q21.2 and 12p11.1–q21.1, respectively. Sequencing of HOXC13 after convincing linkage on 12p11.1–q21.1 revealed two novel mutations in both families. Results Linkage in the two families was tested using microsatellite markers linked to type I and type II keratin gene clusters on chromosome 17p12–q21.2 and 12p11.1–q21.1, respectively. Haplotype anslysis revealed linkage on 12p11.1–q21.1, subsequent sequencing of HOXC13 in linkage interval revealed two novel mutations including a homozygous 4-bp duplication (c.200– 203dupGCCA; p.His68Glnfs*84) and a nonsense mutation (c. 390C>A; p.Ser135*) in family A and B respectively. Conclusion

HOXC13 is a member of the evolutionarily conserved homeobox ( HOX ) gene family encoding a set of transcription factors regulating downstream target genes involved in morphogenesis A cluster of amino acids form the DNA binding homeodomain regulates the transcriptional activity of various hair keratin genes and FOXN1 in hair follicles and nails The mutant transcripts formed as a result of these mutations and most likely degraded by nonsence mediated RNA decay This demonstrates the importance of role played by HOXC13 in the development of hair and nails in humans

Paper reference: SPS1283-14 Stability of Ester- Prodrugs with Alkaline Agent in the Simple Suspension Method Dr. nelly suryani djamain

[email protected] Indonesia

Islamic University Syarif Hidayatullah Jakarta

Abstract The simple suspension method involves allowing tablets or capsules to be suspended in warm water at 55°C without crushing. This method is an easy and rapid method for making suspensions of tablets and capsules to be administered via a feeding tube. This new method does not require crushing, thus, can avoid drug exposure to pharmacists and loss of drug due to adhesion to instruments In the present study, we evaluated the stability of two prodrugs suspended alone and in concomitant with an alkaline agent in the simple suspension method. We employed two ester-prodrugs, acemetacin (AMT) and cefpodoxime proxetil (CPP), which are known to be hydrolyzed at higher pH. Drug concentrations in the suspension were measured by high-performance liquid chromatography. AMT and CPP were not hydrolyzed when suspended alone, but decomposed in alkaline condition with the addition of magnesium oxide (MgO). Therefore, AMT concentration decreased to 55.5% and 39% at 30 min and 60 min, respectively, whereas its active metabolite, indometacin (IMT), increased from null to 60 µg/mL and 122.7 µg/mL at 30 and 60 min, respectively. CPP concentration decreased to 62%, 37.2%, 27.2% and 16.6% at 30, 60, 90, and 120 min, respectively. AMT and CPP may not be administered in the simple suspension method in concomitant with an alkaline drug which increases the suspension pH. Background Some medicines are administered to patients as suspensions in clinical practice, because it is sometimes difficult for patients to take medicines orally in the form of tablets and capsules. A traditional method to prepare such suspensions is to crush tablets and/or capsules and suspend them in water. Another method is to administer drugs as suspensions using warm water (55°C), which is called a “simple suspension method”(SSM)1) Materials and Methods Acemetacin (30 mg tablets, Rantudil®), was purchased from Kowa Co., Ltd. (Tokyo, Japan), cefpodoxime proxetil (100 mg tablets, Banan®) was purchased from Daiichi Sankyo Co., Ltd. (Tokyo, Japan), Results

Figure 7A shows that the concentration of AMT did not change when suspended alone in the simple suspension method. The pH of the suspension was 6.0. On the other hand, the concentration of AMT concomittan of MgO decreased to 832 and 584 µg/mL at 30 min and 60 min, respectively. The active metabolite of AMT, IMT, was found to increase in SSM, (Fig. 7B). The pH of the suspension was 9.5 when AMT and MgO were suspended together, and changed to 6.0 after the addition of methanol and phosphate buffer. Conclusion This study demonstrated that some ester prodrugs can be markedly hydrolyzed when applied to SSM in combination with MgO or Li2CO3 which can increase the pH of the suspensions. Paper reference: SPS1279-14 Evaluation of Her-2/Neu Gene Amplification in Saudi Female Breast Cancer Patients Using Quantitative Real-Time PCR and Fluorescence In situ Hybridisation *Dr. Mona Abdulqadir AlJuhani

[email protected]

KSA King Abdulaziz university

Abstract Nearly 25 % of breast tumours are caused by the overexpression of ( Her2 - neu ) gene or the overexpression of its protein product Although FISH has been widely used for the analysis of gene amplification we opted for a more comprehensive diagnostic strategy for Saudi female breast cancer patients Therefore we evaluated the quantitative real - time PCR ( qRT - PCR ) technique against the FISH technique in a limited group of patients Of the fifty paraffin embedded samples from Saudi female breast cancer patients that were analyzed using FISH ten cases were selected with +3 Her2 - neu amplification for quantitative measurement using qRT PCR Fresh - frozen sections were obtained from these patients followed by RNA extraction and subsequent cDNA synthesis The experiment was performed on a StepOne Plus - Real Time PCR platform from ABI in which the samples were analysed in duplicate along with standards and endogenous RNA controls This kinetic method exploits the use of fluorescent Taq ManTM probe technology with a reporter and quencher dye at the ’ and ’ ends respectively to quantify gene amplification in tumour DNA Of the ten samples analysed using qRT - PCR extra copies of the gene were observed in nine samples (90 %). These results demonstrate intra - experimental reproducibility and correlated well with the FISH data These results indicate that qRT - PCR is an effective technique for the rapid quantification of Her - neu The use of this technique made molecular analysis of Saudi female breast cancer samples simpler and more reliable by bypassing the human factor and labour - intensive process involved in FISH and other conventional techniques such as immunohistochemistry ( IHC ). Thus qRT - PCR for Her - neu should Background Almost % of breast tumors are caused by the amplification of the Her - neu gene or the overexpression of its protein product Recently fluorescence in situ hybridisation ( FISH ) has been widely adopted to determine Her - neu amplification which is widely used as a confirmatory test for conventional immunohistochemistry ( IHC ) results However recently newer and more stringent methods have been tested including quantitative - PCR ( qRT - PCR ), a relatively new and alternative technique for assessing Her - neu gene amplification

Materials and Methods fifty females suffering from breast cancer were included in this study. paraffin- embedded tissues were obtained from these patients. Her-2/neu gene amplification was evaluated using FISH and RT-PCR; then results were compared. Results The Her - neu to chromosome centromere 17 ratios varied from 0.87 to 9.66 with values greater than 2.2 demonstrating Her - neu gene amplification. Seventeen breast cancer samples showed amplification whereas 33 were negative (34 %). High concordance was found between FISH and Q - RT - PCR ( 90%), while the discordance was 10%, however statistical significance could not be observed due to small sample number Conclusion RT-PCR for Her-2/neu should find broad applications in comprehensive breast cancer diagnostic and prognostic settings

Paper reference: SPS1273-14 Pectic acid from apple induced cell cycle arrest in MDA-MB-231 human breast cancer cells *Dr. Ladan Delphi

[email protected] UAE

Dr. Houri Sepehri

Animal Biology Department, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran Endocrinology and Metabolic Research Institute, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

Dr. Mohammad Reza Khorramizadeh

Animal Biology Department, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran

Abstract Background Breast cancer an estrogen - dependent disease is the most common cancer in women that starts in the cells of the breast Chemotherapy is used to treat breast cancer in which cancer cells direct to commit apoptosis Apart from the chemical drugs natural origin substances have also been noted for cancer therapy These substances are usually free from chemical drug side effects Pectin a product of plant cell walls is one natural substances extracted from the citrus and apple wastes Pectins include potential for apoptosis induction and anticancer effects Objective Here apple pectin ( pectic acid ) was used as an anticancer agent to inquire on a cellular model of breast cancer Methods MDA - MB - human breast cancer cell line was cultured in RPMI medium supplemented with % fetal bovine serum The effect of pectic acid on cellular viability was determined by the MTT assay Morphological features of apoptotic cells were determined with AO EB followed by fluorescence microscopy The ratio of cells in the G0 G1 S and G2 M phases of cell cycle was determined by their DNA content using PI and flowcytometry analysis Results Cell viability of MDA - MB - in presence of pectic acid ( mg ml ) for h was concentration - dependent The high concentrations of pectic acid ( mg ml )

statistically reduced viable cell number compared to the untreated cells Also apoptotic features such as fragmented chromatin was determined with AO EB staining Cell - cycle analysis showed more than1mg ml pectic acid caused a large number of cells distributed in G0 region CONCLUSION Pectins such as pectic acid may contain great protective effects against cancer Our results indicated pectic acid induced a very strong sub - G1 arrest representing apoptosis in breast cancer cells Key words Pectic Background Breast cancer an estrogen-dependent disease is the most common cancer in women that starts in the cells of the breast. Chemotherapy is used to treat breast cancer in which cancer cells direct to commit apoptosis. Apart from the chemical drugs, natural origin substances have also been noted for cancer therapy. These substances are usually free from chemical drug side effects. Pectin a product of plant cell walls is one natural substances extracted from the citrus and apple wastes. Pectins include potential for apoptosis induction and anticancer effects. Materials and Methods MDA-MB-231 human breast cancer cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The effect of pectic acid on cellular viability was determined by the MTT assay. Morphological features of apoptotic cells were determined with AO/EB followed by fluorescence microscopy. The ratio of cells in the G0/G1, S and G2/M phases of cell cycle was determined by their DNA content using PI and flowcytometry analysis. Results Cell viability of MDA-MB-231 in presence of pectic acid (0, 0.01, 0.1, 0.5, 1, 2.5, 5 mg/ml) for 24h was concentration-dependent. The high concentrations of pectic acid (1, 2.5, 5 mg/ml) statistically reduced viable cell number compared to the untreated cells. Also apoptotic features such as fragmented chromatin was determined with AO/EB staining. Cell-cycle analysis showed more than1mg/ml pectic acid caused a large number of cells distributed in G0 region. Conclusion Pectins such as pectic acid may contain great protective effects against cancer. Our results indicated pectic acid induced a very strong sub-G1 arrest representing apoptosis in breast cancer cells. Paper reference: SPS1271-14 Simulation of DNA Polymerase beta phosphorylation-induced structural changes using molecular modeling *Dr. Haitham Idriss

[email protected] UK

Annals of Alquds Medicine

Dr. Shahir Shamsir Malaysia Universiti Teknologi Malaysia Abstract DNA polymerase beta is a 39 kDa enzyme that comprises two major domains, a 31 kDa domain responsible for the polymerase activity and an 8 kDa domain, which bind ssDNA and has a dRP Lyase activity. The atomic structure for the enzyme has recently been elucidated. DNA polymerase beta was shown to be phosphorylated in vitro with Protein Kinase C at serines 44 and 55, resulting in loss of its polymerase enzymic activity and ability to bind ss DNA. In this study, we set out to simulate potential phosphorylation-induced structural changes for DNA

polymerase beta using molecular modeling algorithm (CHARMM22) and published structural coordinates for the enzyme (pdb: 2FMS). Compared to the apo_enzyme (pdb: 2FMS) RMSF plot, Ser44P showed highest deviation. RMSF plot of Ser55P and Ser44/55P showed a similar fluctuation pattern except at aa 1-75, the major part for ssDNA binding and dRP lyase domain of the enzyme. The radius for gyration (Rg) for apo_enzyme and Ser55P were consistent throughout their respective simulation period. The Rg of Ser44P and Ser44/55P fluctuated throughout the simulation. Phosphorylation at Ser44, Ser55 and Ser44/55 induced major conformational fluctuations from apo_enzyme at the ssDNA binding domain and dNTP selection domain. This may explain the phosphorylation-induced loss of enzymic activity reported in vitro. Such studies may pave the way for simulating variations in the atomic structure for the phosphorylated and other post-translationally modified form(s) of proteins/enzymes. Paper reference: SPS1270-14 Extraction of Neural Crest Stem (NCS) Cells from Bone Marrow and Developing Gut by Magnetic Cell Sortingrting *Dr. Amir Ali Khan

[email protected]

Dr. Tee Jong Huat Dr. Soumya Pati Dr. Qaiser Iftikhar Sheikh

Malaysia Malaysia UAE

Dr. Jafri Malin Abdullah Dr. Hasnan Jafar

Malaysia Malaysia

UAE

Department of Applied Biology and Biotechnology, University of Sharjah

Universiti Sains Malaysia Universiti Sains Malaysia Department of Applied Biology and Biotechnology, University of Sharjah Universiti Sains Malaysia Universiti Sains Malaysia

Abstract Neural crest ( NC ) cells are group of a migratory cell population arising during neurulation stage After the epithelial - to - mesenchymal transition and their extensive migration they generate various tissues Neural crest stem ( NCS ) cells remain in the sites of the NC cells migration and demonstrate multipotency Recent studies suggest that the NCS may migrate to many developing tissues than previously thought including bone marrow We have isolated NCS cells from the developing gut of the E foetuses of Sprague Dawley ( SD ) rats and from the bone marrow of four weeks olds SD rat The pregnant females were euthanized after days of confirmed pregnancy The cells were removed from the developing gut using trypsin and collagenase The four weeks old rats were also euthanized and bone marrow harvested from the marrow compartments of the rat tibia and femoral The NCS cells were isolated from the developing gut and bone marrow by manual magnetic cell sorter The cells were labelled with primary antibody against the p75 protein followed by secondary antibody conjugated with micro - beads Then the cells suspension was placed onto MACS column in the magnetic field of a MACS separator The magnetic labelled neural crest stem cells were retained and eluted after separating from unlabelled cells The isolated cells were grown for more days before their

validation The NCS from bone marrow and the developing gut cells were then stained with monoclonal antibodies against p75 - FITC for quantitative validation using flow cytometer In this study we have shown that the extraction of the NCS cells from the developing gut and the bone marrow was successful. Background Stem cells extraction and characterization Materials and Methods Animal work, Stem cells extraction, Magnetic cell sorter, and Flow cytometery Results The result shown that the isolation of neural crest cells were P75 positive and were able to grow in vitro after their extraction from the developing gut and bone marrow using magnetic cell sorter. Conclusion In this study we have shown that the extraction of the NCS cells from the developing gut and the bone marrow was successful using magneitc cell sorter and antibody against P75.

Paper reference: SPS1269-14 Cytotoxic effects of Rubus idaeus polyphenols on HepG2 and L20B Cell line in vitro *Dr. Batol Imran Dheeb Dr. Raghad K. al-lihaibi Dr. Farooq Ibrahem Mohammad Dr. Baraa abdulhadi abdulhameed

[email protected] Iraq Iraq Iraq

Iraq

Iraqia University

biotechnology research center, Al- Nahrain university biotechnology research center, Al- Nahrain university biotechnology research center, Al- Nahrain university

Abstract The disease-prevention properties of fruits and vegetables are attributed to the biological activities of the dietary fiber, vitamins, minerals and phytochemicals in the plants, however many studies suggest the protective effects of fruits and vegetables against chronic diseases are due in large part to the phytochemical content of the plants in our study to investigate the cytotoxicity of polyphenols, the active compound isolated and purified from Rubus idaeus fruits, prepared at the same starting concentration, and tested on human hepatocellular carcinoma (HepG2) cell line and the mouse cell lineusin(L20B) using neutral red assay, cytotoxic effect showed a concentrations dependent inhibition rate increased with the increase of polyphenols concentration. The inhibition of cells increase was significantly higher for HepG2 (p< 0.05) ranged between 96.7- 33% while the inhibition of L20B raned between 76-12%. The cytotoxic effect of different concentrations for the Rubus idaeus Polyphenols after treating both cell lines HepG2 and L20B for 48 hours intervals, the concentrations showed there was a potent toxic effect on treated cell line especially at the higher concentration ( 125 ng/ ml then the cytotoxic effect decreased as the polyphenol concentrations decreased.

Background Rubus idaeus bioactives Blakberry have many roles in cancer prevention according to Stoner et al Laboratory studies show blakberry bioactives protect against oxidative DNA damage by direct scavenging of reactive oxygen species ( ROS ), often considered a first line of defense against the multistage process of carcinogenesis Berry bioactives are also effective in inhibiting the formation of carcinogen - induced DNA adducts enhancement of DNA repair and inhibition of carcinogen - induced tumorigenesis in animal models In addition berry bioactives modulate signaling pathways involved with cellular proliferation apoptosis inflammation Materials and Methods This work aimed to study the cytotoxic effects of polyphenols prepared from the fruit of Rubus idaeus in gradual concentration from 250ng/ml to 12.5 ng/ ml The cells Hep G2, and L20B were propagated and maintained in Minimum Essential Medium Eagle MEME medium, the cytotoxic assay was applied according to Fresheny method the cell incubated at 37 c for 48 hour with gradual concentrations of the poly phenol solution in three replicates Results The cytotoxic effect of different concentrations for the Rubus idaeus Polyphenols after treating both cell lines HepG2 and L20B for 48 hours intervals, the concentrations showed there was a potent toxic effect on treated cell line Conclusion Polyphenols exibit cytotoxic effects on both cancer cell lines need to further investigation to know mechanism by which the prepared polyphenls act in comparison to traditional anticancer drug that might give Rubus idaeus fruits an attention for being promise anticancer product

Paper reference: SPS1268-14 Genotyping of of Iraqi local Aspergillus flavus islates based on AFLAR1 gene sequencing. *Dr. Batol Imran Dheeb Dr. nemat al-judy Dr. zainab anas salman Dr. Majeed Arsheed Sabbah Dr. safa m Abdulateef

[email protected] Iraq Iraq Iraq Iraq

Iraq

Iraqia University

baghdad university- college of science- biology department baghdad university- college of science- biotechnology department Al nahrain university baghdad university- college of science- biology department

Abstract This study examined 11 isolates of Aspergillus flavus on its ability to produce aflatoxin B1 using molecular method, the regulatory gene AFLAR1 gene of 11 of these isolates was successfully amplified and sequenced. These PCR results suggested that the aflR gene is Presence in the studied isolates The sequenced aflR1 genes from the 11 positive strains had greater than 99.7% similarity,baswed on sequencing and Cluster analysis which was particularly conserved in the zinc-finger DNAbinding domain.

In this study, rapid assessment of 11 isolates of A. flavus was accomplished using an indigenously designed primer pair for the Aflatoxin B1 regulatory gene aflR1 in polymerase chain reaction (PCR). Background Aspergillus flavus mainly infect maize cotton peanuts tree nuts [ ], figs [ ] and spices [ – ]. The contamination of foods by aflatoxigenic fungi especially in tropical countries may occur during preharvesting processing transportation and storage [ ]. The toxic effects of aflatoxins have adverse consequences for human health and agricultural economics The aflR gene a regulatory gene for aflatoxin biosynthesis encodes a protein containing a zinc - finger DNA - binding motif [ ]. Materials and Methods Fungal strains and culture conditions Preparation of fungal genomic DNA PCR amplification of AFLR1 gene fragments: DNA sequencing and analysis The alignment of all sequences was visually assessed and optimized when necessary. Phylogenetic evaluation was accomplished by applying neighbour-joining program from the PHYLIP 3.63 package [15]. Phylogenetic trees were rooted with Aspergillus flavus (. Finally, a bootstrap analysis with 1000 replications was performed. Trees were viewed using TreeView [16]. Results Sequencing of coding regions of amplified product for all samples included in our local study into A flavus were done seeking for detection similarity difference and presence of any mutation within these sequence related to regulatory of aflatoxin B1 were assembled to yield the entire aflR gene sequence the aflR sequences of each strain were aligned using CLUSTAL X[ ]. Alignment of AFLAR1Gene af all samples with data published for known sequence seeking for enough homology Ahomology with AFLAR1Gene of Aspegillus flavus from Gene Bank was done Conclusion from the results of sequenxing analysis its necessory to stud gen expression of aflatoxin b1 regulatory gen . Paper reference: SPS1266-14 Phenotypic and genotypic diagnosis of Gaucher disease in Algeria *Dr. HALLAL SIHAM

[email protected] Algeria

CHU MUSTAPHA ALGERIA

Abstract Gaucher disease is the most common lysosomal storage in our population, it is due to a deficiency of β –glucosidase acid. The enzyme deficiency causes a pathological accumulation of undegraded substrate in lysosomes. This metabolic overload is responsible for a multisystemic disease with

hepatosplenomegaly, anemia , thrombocytopenia, and bone involvement . Neurological involvement is rare. The laboratory diagnosis of Gaucher disease consists of ; phenotypic diagnosis by determining the enzymatic activity of β - glucosidase by fluorimetric method , a study by genotypic diagnosis in the GBA gene , limiting the search recurrent mutations ( N370S , L444P , 84 GG) ; PCR followed by an enzymatic digestion . Abnormal profiles were verified by sequencing. Monitoring of treated patients is provided by the determination of chitotriosidase . Our experience spans a period of 7 years (2007-2014) has enabled us to diagnose 78 patients out of a total of 328 requests from the various departments of pediatrics, internal medicine, neurology. Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha , which help define the clinical form ; or 5 of them had type III disease , carrying the L444P mutation in the homozygous state . Three others were composite ( N370/L444P ).( N370S / other unintended mutation in our study) , and only one family no recurrent mutation has been found. This molecular study permit screening of heterozygous essential for genetic counseling . Background Gauche disease, mutation, B-glucosidase acid, N370S, L444P Materials and Methods PCR Sequencing Results Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha , which help define the clinical form ; or 5 of them had type III disease , carrying the L444P mutation in the homozygous state . Three others were composite ( N370/L444P ).( N370S / other unintended mutation in our study) , and only one family no recurrent mutation has been found. Conclusion Our experience spans a period of 6 years (2007-2014) has enabled us to diagnose 78 patients Paper reference: SPS1264-14 Profiling of the bacteria responsible for sepsis in dogs by 16S rRNA gene metagenomic sequencing Dr. Ho-Seong CHO

[email protected]

South Korea - 한국

Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National Univ.

Abstract In summary, understanding microbial community dynamics and their impact on the functional potential of microbial systems has become increasingly important in diagnostics of animal and human diseases. The introduction of low cost, scalable and rapid NGS technologies such as the PGM platform provide small to medium laboratories with the high throughput capabilities

which allow a fundamental basis to these studies, as well as affording highly replicated and longitudinal studies in a cost effective way. Background Blood sepsis also known as septicemia is a consequence of a long lasting infection that is not being treated. The bacteria enter the blood stream and the affects the entire system of animals and humans. Here we profiled the bacterial composition in canine blood by 16S ribosomal RNA (rRNA) gene based on next-generation sequencing (NGS) technology to identify etiological agents of sepsis and to provide information of their 16S rRNA sequences for application to DNA-based techniques in animals. Materials and Methods Bacterial S rRNA genes were amplified by PCR from DNA samples using a range of V3 and V6 oligonucleotide primers specific for domain bacteria. Templated - ISPs were sequenced on “ ” micro - chips using the Ion Torrent Personal Genome Machine for cycles ( flows ), resulting in an expected average read length of > 100 b.p. for Ion Express Template 100 chemistry. After sequencing, the individual sequence reads were filtered within the PGM software to remove low quality and polyclonal sequences. Results Metagenomic sequencing of 16S rRNA gene showed that 85% of the blood samples contained single or multiple genera of known bacteria such as Escherichia coli, Streptococcus and unassigned Enterobacteriaceae. E. coli and Streptococcus were predominantly found in the canine blood samples. Conclusion We demonstrated that the occurrence of sepsis was associated with four known bacterial genera as well as unassigned bacteria and that 16S rRNA gene sequencing was more useful than conventional culture methods for accurate identification of bacterial pathogens from canine blood. Paper reference: SPS1262-14 Synthesis and characterization of metal nanoparticles using extracts of indigenous plants of UAE and delivery of anticancer bioflavonoids to MCF-7 breast cancer cell lines *Dr. RENUKA SEENIVASAN

[email protected]

Dr. HUSSAINA BANU MALKHAN Dr. SAAD SULTAN KHAN Dr. KAVYA NARAYNAN Dr. ALMA RAHEEM Dr. SAJIDAH HASHIM Dr. GEETHA VASANTHAKUMAR

UAE UAE UAE UAE UAE UAE

UAE

MANIPAL UNIVERSITY

MANIPAL UNIVERSITY, DUBAI MANIPAL UNIVERSITY, DUBAI MANIPAL UNIVERSITY, DUBAI MANIPAL UNIVERSITY, DUBAI MANIPAL UNIVERSITY, DUBAI Holistic International Testing Services FZ LLC

Abstract Cancer is becoming the leading cause of death in most of the countries. During the last few decades, several approaches have been made to control cancer including the use of

chemotherapeutic agents, radiation and/or surgery. However, controlling and curing cancer still remains to be a challenge to the entire scientific community. Nanotechnology appears to be a promising field in the area of medicine. Nanoparticles have been explored as a drug delivery vehicle to deliver diagnostic and therapeutic agents to the directly to the specific cell. Nanoparticles, particularly metal nanoparticles, can also be used as a tool to cause photothermal ablation of the cancer cells as they have the potential to absorb light energy and deliver heat to the cancer cells. Date Palm Tree is of historical importance and is found to have several medicinal compounds in the various parts of the plant. Ghaf Tree, has antitumour activities and antimicrobial activities and is used a remedy for a variety of ailments. Miswak is highly recommended for its medicinal virtues coupled with its phytochemical make up and its capacity to act as a reducing agent. The above plants were used due to the presence of phytochemicals with reducing capacity to induce green synthesis of nanoparticles. We have synthesized silver and gold nanoparticles using the plant extracts, characterized the metal nanoparticles using techniques such as UV visible spectroscopy, scanning electron microscopy and energy dispersive spectroscopy for their size, shape and elemental analysis, and we have tested the delivery of anticancer bioflavonoids bound to the green synthesized metal nanoparticles as stabilizing and reducing agents into MCF-7 breast cancer cell lines. The overall results indicated that the possibility of using biologically synthesized silver and gold nanoparticles to reduce the growth of cancer cells and their cytotoxicity for potential treatments. Paper reference: SPS1260-14 Molecular and Cultural Characterization of Antibiotic Producing Soil Actinomycetes Recovered from Different Habitats in UAE *Dr. Ismail MK Saadoun

[email protected] UAE

Dr. Ban AL Joubori Dr. Mariam Al-Ali Dr. Aisha Al-Souqi Dr. Shahad Al-Nuaimi Dr. Moza Al-Kharje

UAE UAE UAE UAE UAE

University of Sharjah

University of Sharjah University of Sharjah University of Sharjah University of Sharjah University of Sharjah

Abstract A total of 19 different actinomycetal isolates were recovered from 6 soil samples that were collected from different cultivated/uncultivated soil habitats in the mid-region of the UAE. These were then characterized and assessed for their antagonistic activity against five clinical multi-drug resistant Gram-positive and negative pathogens. Results indicated that only one strain (strain 73) was active against all tested pathogens with an inhibition zone ranging between 10 and 15 mm in diameter. The most potent antibiotic-producing isolates were subjected to genomic DNA extraction in order to perform PCR amplification using specific primers targeting actinomycetes-specific sequences as well Streptomyces-specific sequence.

Overall, three isolates (43, 73, and 74) were confirmed to be actinomycetes which showed a single band of an approximate size of 350 bp, indicating that all 3 isolates have the actinomycetes 16S ribosomal DNA-specific sequence. Also, PCR results confirmed that two isolates (73 and 74) to be Streptomyces strains with an approximate size of 1000 bp. Background The detection and identification of possible strains from the genus Streptomyces is a valuable approach for finding novel drugs produced by new species as they are known to be the largest antibiotic-producing organisms and has been studied extensively for a very long period of time. Therefore, the main purpose for conducting this research is to describe the cultural and molecular characteristics of the recovered inhibitory bioactive compounds-producing actinomycetes from different terrestrial ecological habitats in UAE Materials and Methods Soil samples were collected from different cultivated uncultivated soil habitats in the mid region of the UAE. Streptomycetes were isolated by plate culture techniques. The recovered colonies were then characterized and assessed for their antagonistic activity against five clinical multi - drug resistant Gram - positive and negative pathogens. Genomic DNA extraction was conducted using Genomic DNA Minin Kit ( Invitrogen USA ) according to the manufacturer instructions. PCR amplification was carried out using iCycler thermocycler (Bio-Rad, USA). Results PCR reactions using AM45-F and AM45-R primers showed a single band for isolates 73 and 74 with an approximate size of 1000 bp, indicating that strains 73 and 74 both have the Streptomyces-specific sequence. By using Actino-16S-F (AO3) and Actino-16S-R (AO4) primers, data showed a single band with an approximate size of 350 bp, indicating that all 3 isolates have the Actinomycetes 16S ribosomal DNA-specific sequence. Conclusion The screening and isolation of actinomycetes from soil habitats performed in this work show that these microorganisms have a great potential to produce antimicrobial compounds. Further studies on optimization, purification and elucidation of chemical structure of active compound(s) in addition to the molecular identification of the distinct actinomycetes isolates are highly recommended.

Paper reference: SPS1259-14 Molecular and Cultural Characterization of Antibiotic Producing Soil Actinomycetes Recovered from Different Habitats in UAE *Dr. Ismail MK Saadoun

[email protected] UAE

Dr. Ban AL Joubori Dr. Mariam Al-Ali Dr. Aisha Al-Souqi Dr. Shahad Al-Nuaimi Dr. Moza Al-Kharje

UAE UAE UAE UAE UAE

University of Sharjah University of Sharjah University of Sharjah University of Sharjah University of Sharjah

University of Sharjah

Abstract A total of 19 different actinomycetal isolates were recovered from 6 soil samples that were collected from different cultivated/uncultivated soil habitats in the mid-region of the UAE. These were then characterized and assessed for their antagonistic activity against five clinical multi-drug resistant Gram-positive and negative pathogens. Results indicated that only one strain (strain 73) was active against all tested pathogens with an inhibition zone ranging between 10 and 15 mm in diameter. The most potent antibiotic-producing isolates were subjected to genomic DNA extraction in order to perform PCR amplification using specific primers targeting actinomycetes-specific sequences as well Streptomyces-specific sequence. Overall, three isolates (43, 73, and 74) were confirmed to be actinomycetes which showed a single band of an approximate size of 350 bp, indicating that all 3 isolates have the actinomycetes 16S ribosomal DNA-specific sequence. Also, PCR results confirmed that two isolates (73 and 74) to be Streptomyces strains with an approximate size of 1000 bp. Background The detection and identification of possible strains from the genus Streptomyces is a valuable approach for finding novel drugs produced by new species as they are known to be the largest antibiotic-producing organisms and has been studied extensively for a very long period of time. Therefore, the main purpose for conducting this research is to describe the cultural and molecular characteristics of the recovered inhibitory bioactive compounds-producing actinomycetes from different terrestrial ecological habitats in UAE Materials and Methods Soil samples were collected from different cultivated uncultivated soil habitats in the mid region of the UAE. Streptomycetes were isolated by plate culture techniques. The recovered colonies were then characterized and assessed for their antagonistic activity against five clinical multi - drug resistant Gram - positive and negative pathogens. Genomic DNA extraction was conducted using Genomic DNA Minin Kit ( Invitrogen USA ) according to the manufacturer instructions. PCR amplification was carried out using iCycler thermocycler (Bio-Rad, USA). Results PCR reactions using AM45-F and AM45-R primers showed a single band for isolates 73 and 74 with an approximate size of 1000 bp, indicating that strains 73 and 74 both have the Streptomyces-specific sequence. By using Actino-16S-F (AO3) and Actino-16S-R (AO4) primers, data showed a single band with an approximate size of 350 bp, indicating that all 3 isolates have the Actinomycetes 16S ribosomal DNA-specific sequence. Conclusion The screening and isolation of actinomycetes from soil habitats performed in this work show that these microorganisms have a great potential to produce antimicrobial compounds. Further studies on optimization, purification and elucidation of chemical structure of active compound(s) in addition to the molecular identification of the distinct actinomycetes isolates are highly recommended.

Paper reference: SPS1258-14 An integrated microsystem for piezo-resistive detection of mutations in Hemoglobinopathies Dr. Adarsh Venkataraman Ganesan

[email protected]

Dr. Reenu Anne Joy Dr. Kiran Menon Dr. Hardeep Kumar Dr. K.K. Singh Dr. Trupti Swaroop Gokhale Dr. Neeru Sood

BITS Pilani, Dubai Campus BITS Pilani, Dubai Campus BITS Pilani, Dubai Campus BITS Pilani, Dubai Campus BITS Pilani, Dubai Campus BITS Pilani, Dubai Campus

UAE UAE UAE UAE UAE UAE

UAE BITS Pilani, Dubai Campus

Abstract aborious, expensive and time consuming process in the healthcare sector. Due to these reasons, genetic counseling hasn’t gained popularity in most countries in spite of its possible life-saving implications. The advancements in molecular diagnostics have helped in better diagnosis and therapy of numerous diseases. MEMS based systems, which require low reagent volumes and provides results rapidly with high specificity and sensitivity have proven to be an attractive alternative. In this study, microcantilever based biosensor was designed and simulated for detection and screening of β-thalassemia, a prominent genetic disorder in the UAE. The genetic mutations associated with this disease vary between regions and ethnic backgrounds [1] [2]. This biosensor has been designed specifically to detect the mutations associated with β-Thalassemia among the local UAE population. The simulations were performed in COMSOL, based on the study of different mutations of the β-globin gene and the effect of immobilization of probes on a microcantilever. Background Hemoglobinopathies refer to genetic disorders caused as a result of structural defects in either of the two globin (α or β)chains of the hemoglobin molecule. Mutations in the β chain of hemoglobin results in reduced or lack of production of beta proteins or lead to the formation of abnormally structured β proteins. Various genetic disorders caused due to β- gene mutations include Thalassemias, Sickle cell anemia etc. Detection and diagnosis of β-thalassemia is a laborious, expensive and time consuming process. Materials and Methods The MEMS device has been for the detection of mutation in the β-globin gene. The theoretical model has been developed based on various physics involved in the system. COMSOL Multiphysics has been used to solve the theoretical background. Results The deflection properties of the cantilever were studied using COMSOL Multiphysics. The deflections were studied based on complete and incomplete hybridizations, depicting DNA hybridizations in a normal and affected patient. Conclusion MEMS based systems, which require low reagent volumes and provide results rapidly with high specificity and sensitivity have proven to be an attractive alternative. The fabrication of the MEMS device is an easy and cost effective affair. For the detection of β-Thalassemia, this model

can be used. The alternative options include the use of nanowires or other nanostructures which exhibit added sensitivity due to their similarity in size to biomolecules such as DNA. Paper reference: SPS1256-14 Autophagy retards inflammatory mRNA decay and elicits a white phenotype during adipocyte maturation *Dr. Jingxuan Shan

[email protected]

Dr. Andrea Worschech

Qatar

Dr. Remy Thomas

Qatar

Dr. Lotfi Chouchane

Qatar

Qatar

Weill Cornell Medical College in Qatar

Weill Cornell Medical College in Qatar Weill Cornell Medical College in Qatar Weill Cornell Medical College in Qatar

Abstract Background: Recently, the role of autophagy in glucose and lipid metabolism has been emerging. Mice experiments showed that autophagy deficiency could prevent diet-induced obesity, characterized by less fat and a browning phenotype of white adipocyte (WAT). However, the underlying molecular mechanism is not well explored and the data from human are limited. Method: The mRNA sequencing data of undifferentiated and differentiated human adipocyte cell lines, including two white adipocyte (WAT) and one brown adipocyte (BAT) were included in our analysis. Gene expression was reduced by RNA interference in human adipocyte and was enhanced by glucocorticoid, respectively. Qualifying the lipid droplet content and quantifying the adipolysis and differentiation marker expression were applied to evaluate WAT differentiation. LC3 was used as a marker to examine autophagy function of adipocyte. Result: We found a remarkable feature of adipocyte differentiation that inflammation signaling was significantly strengthened during WAT maturation, but not during BAT maturation. The alteration of Zinc Finger protein 36 (ZFP36), which mediates the decay of mRNA transcripts of inflammation molecules, obviously affected the phenotype of mature WAT: silencing of ZFP36 gene resulted in a more whitening phenotype and induction of ZFP36 resulted in a browning phenotype. ZFP36 activity was associated with p38 MAPK signaling that was regulated by autophagy. Conclusion: ZFP36 links autophagy to the determination of mature adipocyte phenotype. Therefore, ZFP36 is a potential target to prevent obesity and improve glucose and lipid metabolism. Background Recently, the role of autophagy in glucose and lipid metabolism has been emerging. Mice experiments showed that autophagy deficiency could prevent diet-induced obesity, characterized by less fat and a browning phenotype of white adipocyte (WAT). However, the underlying molecular mechanism is not well explored and the data from human are limited.

Materials and Methods The mRNA sequencing data of undifferentiated and differentiated human adipocyte cell lines, including two white adipocyte (WAT) and one brown adipocyte (BAT) were included in our analysis. Gene expression was reduced by RNA interference in human adipocyte and was enhanced by glucocorticoid, respectively. Qualifying the lipid droplet content and quantifying the adipolysis and differentiation marker expression were applied to evaluate WAT differentiation. LC3 was used as a marker to examine autophagy function of adipocyte. Results We found a remarkable feature of adipocyte differentiation that inflammation signaling was significantly strengthened during WAT maturation, but not during BAT maturation. The alteration of Zinc Finger protein 36 (ZFP36), which mediates the decay of mRNA transcripts of inflammation molecules, obviously affected the phenotype of mature WAT: silencing of ZFP36 gene resulted in a more whitening phenotype and induction of ZFP36 resulted in a browning phenotype. ZFP36 activity was associated with p38 MAPK signaling that was regulated by autophagy. Conclusion ZFP36 links autophagy to the determination of mature adipocyte phenotype. Therefore, ZFP36 is a potential target to prevent obesity and improve glucose and lipid metabolism.

Paper reference: SPS1255-14 Role of Chromosomal Translocations in Inducing Secondary Mutations Dr. Abdul Gafoor Puthiyaveetil Abdulkader

[email protected]

UAE American University of Ras Al Khaimah

Abstract Many hematological malignancies are generally characterized by Chromosomal translocations and collaborating mutations These secondary collaborating mutations can occur spontaneously or may be induced by the primary translocation itself Mutations can occur as a result of DNA damage and misrepair DNA double strand breaks ( DSBs)being the most serious types of cell damage. DSBs in somatic cells are repaired by the non - homologous end joining ( NHEJ ) mechanism, and impaired NHEJ may promote secondary mutations. We used transgenic mouse model expressing the myeloid leukemic fusion gene NUP98- HOXD13 ( NHD13 ). These mice develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations. Our studies have shown that when NHD13 is expressed pan - hematopoietically B lymphocyte development and class switch recombination are impaired. Class switch recombination in B lymphocytes includes physiological DSBs and breaks and NHEJ mediated break repair.Based on our initial finding we used in vitro class switch recombination ( CSR ) to delineate the DNA break induction and repair mechanisms in NHD13 B lymphocytes The DNA break induction pattern was determined using phosphorylated H2AX labeling combined with confocal microscopy and flow cytometry Our results showed that NHD13 B lymphocytes had a comparable break induction pattern but significantly reduced DNA repair Analysis of the cell cycle pattern of stimulated B cells at hour intervals showed cell cycle arrest at the G2 M phase

at hours following stimulation hallmark feature of impaired DNA break repair Gene expression analysis showed reduced expression of DNAPKcs Ligase and Xrcc4 and increased expression of alternative end joining factors in NHD13 B lymphocytes suggesting that cells failed to initiate NHEJ - mediated DNA repair.Our results suggest that a myeloid leukemic gene can impair the DNA repair mechanism and may indirectly promote mutations for malignant transformation. Background Hematological malignancies are generally characterized by abnormal changes in the DNA including Chromosomal translocations and secondary mutations. These secondary collaborating mutations can occur spontaneously, or may be induced by the primary translocation itself. Mutations can occur as a result of DNA damage and misrepair; DNA double strand breaks being one of the most serious types of cell damage. Double strand breaks in somatic cells are classically repaired by the non-homologous end joining (NHEJ) mechanism and impaired NHEJ may promote secondary mutations. Materials and Methods We used transgenic mouse model expressing the myeloid leukemic fusion gene NUP98-HOXD13 (NHD13).These mice develop Myelodysplastic syndrome and progress to acute leukemia after acquiring secondary mutations.We used in vitro class switch recombination assay, flow cytometry, confocal microscopy,cell cycle kinetics, reverse quantitative PCR and immunocytochemistry to determine the role of primary translocation in inducing secondary mutations. Results Our results showed that NHD13 B lymphocytes had a comparable break induction pattern but significantly reduced DNA repair. Analysis of the cell cycle pattern of stimulated B cells at 24 hour intervals showed cell cycle arrest at the G2 M phase following stimulation, a hallmark feature of impaired DNA break repair. Gene expression analysis showed reduced expression of DNAPKcs Ligase and Xrcc4 and increased expression of alternative end joining factors. Conclusion Our results showed that presence of a primary translocation can result in aberrant DNA repair mechanisms and help in the induction of secondary mutations. The classical DNA repair factors were suppressed and alternative repair mechanisms are promoted.

Paper reference: SPS1254-14 Multidrug resistance in Staphylococci isolated from Wastewater Treatment Plant in Dubai, UAE Dr. Munawwar Ali Khan

[email protected]

Dr. Husna Rasool Baksh UAE Dr. Javeria Mohsin UAE

UAE

Zayed University, Dubai

Zayed University, Dubai Zayed University, Dubai

Abstract Staphylococci species especially coagulase positive Staphylococcus aureus are responsible for a large proportion of infectious diseases around the world. During the last decade however, the

incidence of antibiotic resistance and multi drug resistance amongst these bacteria has increased dramatically. The engineered microbial ecosystems, such as those treating the domestic wastewater, represent important vehicles for the transfer of human-associated Staphylococci species to the environment. This study evaluated the prevalence of antibioticresistant Staphylococcus species isolated from a full scale municipal wastewater treatment plant in Dubai. Antibiotic susceptibility profile was determined using the disc diffusion method, and polymerase chain reaction (PCR) assay was employed for the detection of mec-A for methicillin resistance gene & NUC gene for the confirmation of Staphylococcus aureus species. A total of 57 Staphylococcus species were isolated from wastewater treatment plant and tested for antibiotic sensitivity against ten antibiotics. All 57 isolates were found to have NUC gene of size 270 bp which confirmed Staphylococcus aureus species. Out of 57 isolates, 26 were found positive for the presence of 310 bp mecA gene. Almost half the isolates (46%) were found resistant to oxacillin, 35 % to methicillin, and 12 % to vancomycin while about 23% of the isolates were resistant to streptomycin, gentamycin and other antibiotics. Out of the 57 isolates analysed 36 were found resistant to three or more antibiotics, which is 63% of the total isolates. In conclusion, a high incidence of multiple drug resistance observed in Staphylococcus aureus isolates from the wastewater environment, suggesting the wastewater treatment plant an important source of multidrug resistant bacteria in the UAE. Background Staphylococci species especially coagulase positive Staphylococcus aureus are responsible for a large proportion of infectious diseases around the world. During the last decade however, the incidence of antibiotic resistance and multi drug resistance amongst these bacteria has increased dramatically. The engineered microbial ecosystems, such as those treating the domestic wastewater, represent important vehicles for the transfer of human-associated Staphylococci species to the environment. This study evaluated the prevalence of antibioticresistant Staphylococcus species isolated from a full scale municipal wastewater treatment plant in Dubai. Materials and Methods Antibiotic susceptibility profile was determined using the disc diffusion method, and polymerase chain reaction (PCR) assay was employed for the detection of mec-A for methicillin resistance gene & NUC gene for the confirmation of Staphylococcus aureus species. Results A total of Staphylococcus species were isolated from wastewater treatment plant and tested for antibiotic sensitivity against ten antibiotics All isolates were found to have NUC gene of size bp which confirmed Staphylococcus aureus species Out of isolates were found positive for the presence of bp mecA gene Almost half the isolates ( %) were found resistant to oxacillin % to methicillin and % to vancomycin while about % of the isolates were resistant to streptomycin gentamycin and other antibiotics. Conclusion In conclusion, a high incidence of multiple drug resistance observed in Staphylococcus aureus isolates from the wastewater environment, suggesting the wastewater treatment plant an important source of multidrug resistant bacteria in the UAE.

Paper reference: SPS1253-14 Genome Wide Screening of Autism Spectrum Disorder in Western Saudi Arabia *Dr. Huda Abdul-Rahman Banni [email protected] KSA

Centre of Excellence in Genomic Medicine Research

Abstract Autism ( ASDs ) is heterogeneous group of neurodevelopmental disorders with a complex multifactorial etiology It is largely inherited disease with an estimated prevalence of – % in the general population The global prevalence of ASD is about according to a review in and the average ratio is about male - to - female respectively Studies on autism in Middle East are rare a recent study conducted in Saudi Arabia have estimated the autism frequency is slightly higher than the reported in developed countries Although it is quite complex however only – % of cases present with genetic component yet specific data on the contribution of each gene is still unclear Recently CNVs have been shown to contribute to over % of ASD cases Up - to - date strategies recommend chromosomal karyotype and fragile X DNA testing to exclude any other abnormality that may contribute to Autism. Seventeen patients were included in the study age ranged from - years Chromosomal karyotype and aCGH were done to test for sub - microscopic genomic deletions and duplications and to detect clinically significant CNVs Custom - designed oligonucleotide aCGH were used that includs exonic coverage of over genes of them known as autism candidate genes. Using the phenotypic examination database was generated Chromosomal analyses revealed one abnormal male karyotype whereas aCGH identified major aberrations deletions and duplications in all patients. Our results further confirm the diagnostic importance of aCGH in detecting the CNVs and demonstrate their importance with a variety of contributory genes It had the highest detection rate among clinically available genetic tests for ASD Interpretation of microarray data is complicated by the presence of both novel and recurrent CNVs of unknown significance despite these limitations aCGH should be considered as part of the initial diagnostic evaluation of patients with ASD. Paper reference: SPS1249-14 Succesful PGD for Spinal muscular atrophy (SMN 1) Dr. Ritu Nair

[email protected]

India

CRAFT hospital and research center

Abstract Single gene diseases are very common among populations because of consanguinous marriages in certain communities . Many couples who are unaware that they could be silent carriers of a single gene disease like SMN and thalassemia, they carry a very high risk of having an affected child. PGD being a very advanced technology is found to be very much clinically useful in avoiding the birth of an affected child in such couples. We report the success of PGD in one of the SMN 1 type case where parents were carriers of exon 7 deletion and had lost the previous children affected with SMN. IVF + PGD was offered to this couple and has successfully resulted in a normal pregnancy. Randomly other group of couples who are carriers for thalassemia gene and had their previous children affected with thalassemia major also opted for IVF + PGD for

saviour baby. HLA matching matching was also offered for these patients. Ongoing pregnancy reported. PGD is an ultimate option for such couples as genetic disorders cannot be treated but only can prevented through PGD. However embryo selection becomes crucial in certain cases. Background PGD has been rapidly utilized in IVF cases both to improve clinical pregnancies and also to prevent the birth of a chromosomally abnormal child in case of parents with a balanced translocation carrier status or affected with single gene disorders With the high number of cosanguinous marriages favoured in Indian community the risk of autosomal recessive single gene disorders increases Hence many parents who are known to be carriers of a single gene disease pass on the defective gene to their offspring PGD is the best option in such cases Materials and Methods Patients affected with single gene disorders (carriers) walked in for genetic counseling The single gene diseases were mostly spinal muscular atrophy(SMN) and thalassemia Thalassemia patients wanted a savior baby as thier first child was affected with major Patients who were carriers of SMN gene lost their previous children and wanted a child free from SMN Couples were subjected to molecular testing by real time PCR methods Thalassemia mutations were detected and also the exon deletions were detected in carriers This was used to perform PGD Results PGD was offered to SMN gene and thalassemia couples who underwent IVF and the embryos were tested for the detected genetic muataions and deletions . Embryos were biopsied on day 5 and TE cells were subjected to PGD specifically . Normal embryos free from the above abnormalities were transferred. Clinical pregnancy is reported in one of the SMN case and the child born is free from SMN. However the thalassemia case pregnancy is ongoing . Conclusion PGD is the best option to be offered to couples who are known to be carriers of any single gene disorder. PGD is the prefered option rather than medical termination of pregnancy . Lethal genetic disorders can be prevented as there is no as such treatment of genetic diseases till date. Moreover PGD give a ray of hope to couples who lost their children affected with single gene diseases, so that they can have a normal pregnancy without any complications Paper reference: SPS1248-14 Clinical pregnancy in 46,XX Male with his own sperms - Importance of Cytogenetics and Molecular Studies *Dr. Ritu Nair

[email protected]

India

CRAFT hospital and research institute

Abstract Testicular Disorder of Sexual Differentiation (DSD) is a rare condition characterized by a spectrum of clinical presentations, ranging from ambiguous to normal male genitalia.SRY ( sex determining region) plays a very important role in sex determination. On the basis of SRY detection 46,XX males can be classified as SRY positive and negative . We report two cases of 46,XX azoospermic males who are SRY positive and SRY negative confimed by molecular genetic studies . SRY negative male had a very small testes with low levels of testosterone , and was

referred for adoption . Whereas the SRY positive male had substantial levels of testosterone and testes size. Micro TESE was performed on this male and viable sperms were retrieved and clinical pregnancy reported in the case of male with 46, XX Sry Positive Translocation of Y chromosome including the SRY locus on X chromosome, which occurs due to recombination during paternal meiosis, can be easily demonstrated via molecular analyses in 90% of 46,XX male DSD cases. Hence Cytogenetic and Molecular genetic studies are very essential to know the reproductive status in infertile Men to provide correct diagnosis and treatment. Background Large number of chromosomal abnormalities are seen in infertile males majority of which disrupts the spermatogenesis process which leads to Azoospermia in males Most of them are diagnosed at puberty and confirmed diagnosis can be made at reproductive age Cytogenetic and molecular studies are very essential in diagnostic work up of infertile males to achieve clinical management of treatment Certain rare conditions like the XX Male syndrome should be accompanied by atleast SRY gene studies to reach a conclusion regarding offering the patient with correct reproductive options Materials and Methods Pateints with the 46,XX karyotype and others were selected as part of infertility treatment in our Institute . This selection was made out of random diagnosis by karyotyping studies . Total 300 infertile males were karyotyped and two were found to have the 46,XX karyotype . Rest were categorised in the other group with different chromosomal abnormalities. The 46XX was further subjected to SRY gene expression studies using taq man chemistry real time PCR. Control samples were also used along with these two cases . Results After the karyotype results Patients who require micro TESE was divided in seperate groups and males who have to undertake adoption as only option for reproduction were placed in different group. Considering the two XX cases .Presence of SRY gene was confirmed by using taq man probes in real time PCR .one case SRY negative and the other case was SRY positive Micro TESE was carried out in the SRY positive male after studying the hormonal parameters and viable sperms were retrieved and clinical pregnancy reported after ICSI. Conclusion Genetic studies form a very important part of diagnosis in infertility. Although many new technologies have evolved which rules out the standard traditional karyotyping studies but still it remains the gold standard in providing an accurate management and answer to many clinicians in dealing with infertile couples It is the dream of every parent to father their own child .Hence the importance of karyotype and SRY gene studies in XX males after Micro TESE has helped achieve pregnancy in 46, XX male .

Paper reference: SPS1247-14 The impact of genetic polymorphism of C285T of G protein β3 gene on body mass index (BMI) Dr. Jumana Al Zainal Dr. Shaima Khan

[email protected] UAE UAE

Dubai Medical College

Dubai Medical College

Dr. Sidiqa Rafiei

UAE

Dubai Medical College

Abstract Polymorphisms in genes, for proteins involved in G protein receptor signaling pathways, may alter corresponding hormone function. And therefore be involved in pathogenesis of endocrine disorder, including diabetes and obesity.The common C825T polymorphism of the gene that encodes G protein β3 subunit has shown to influence lipolysis in human adipocytes and is associated with body fat distribution and hypertension. Aim: To study the impact of genetic polymorphism of C825T of G protein β3 gene on body mass index Material and Method: 134 individuals with different BMIs were enrolled in the study. The β3 C825T genetic polymorphism was identified by the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) of peripheral blood DNA samples. Analysis of the data was done using SPSS program. Results: The frequency of CC genotype among individuals with the normal BMI was 32.6% as compared to 20.5% among individuals with BMI ≥ 25 kg/m2. The frequency of (CT and TT) genotype was 67.4% and 79.5% among individuals with the normal BMI and those with BMI ≥ 25 kg/m2 respectively. By statistical analysis it was found that carrier of T allele either in homozygote or heterzygote form were at risk of obesity (OR=1.9; 95% CI = 0.8- 4.2). Conclusion: T allele is a risk factor for obesity. Key words: BMI, G-Protein, G protein β3 polymorphism. Source of fund: Dubai Medical College Paper reference: SPS1246-14 The impact of genetic polymorphism of C285T of G protein β3 gene on body mass index (BMI) Dr. Jumana Al-Zainal Dr. Shaima Wasim Khan Dr. Sidiqa Rifai

[email protected] UAE UAE UAE

Dubai Medical College

Dubai Medical College Dubai Medical College

Abstract Background: Polymorphisms in genes, for proteins involved in G protein receptor signaling pathways, may alter corresponding hormone function. And therefore be involved in pathogenesis of endocrine disorder, including diabetes and obesity.The common C825T polymorphism of the gene that encodes G protein β3 subunit has shown to influence lipolysis in human adipocytes and is associated with body fat distribution and hypertension. Aim: To study the impact of genetic polymorphism of C825T of G protein β3 gene on body mass index Material and Method: 134 individuals with different BMIs were enrolled in the study. The β3 C825T genetic polymorphism was identified by the restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) of peripheral blood DNA samples. Analysis of the data was done using SPSS program.

Results: The frequency of CC genotype among individuals with the normal BMI was 32.6% as compared to 20.5% among individuals with BMI ≥ 25 kg/m2. The frequency of (CT and TT) genotype was 67.4% and 79.5% among individuals with the normal BMI and those with BMI ≥ 25 kg/m2 respectively. By statistical analysis it was found that carrier of T allele either in homozygote or heterzygote form were at risk of obesity (OR=1.9; 95% CI = 0.8- 4.2). Conclusion: T allele is a risk factor for obesity. Key words: BMI, G-Protein, G protein β3 polymorphism. Source of fund: Dubai Medical College

Paper reference: SPS1244-14 Follow Maus' Paws: Defining the Common genes in mitochondrial DNA control region of Modern Native Egyptian Mau Dr. Moataz AbdulGhaffar

[email protected]

Egypt

Faculty of Science, Alexandria University.

Abstract The purpose of this project is to compare the DNA of present day spotted cats in Egypt with Egyptian Mau, standard breds overseas, and ultimately with that from a mummified cat, to see if there are any markers in either or both of the modern day cats to the original Mau cats. The Egyptian Mau - Felis silvestris ornate- is the only spotted domestic cat that developed naturally. Egyptian Mau Cats are the oldest known descendents of domesticated cats in existence since Pharaonic time It Is probably recognized from 3500 B.C. This study was addressed the Egyptian Mau as a heritage and culture identity of Egypt. The experiment was designed to find an answer the question whether, the Egyptian Mau originally recognized at Egypt. The experiment was planned on three steps. Firstly, samples collected (hair shafts) that have been taken from cats in Egypt, United States of America and ancient cat mummy from the Egyptian Museum. Then step was through extracting the DNA from samples and amplifying certain gene by PCR technique. Finally, the third was done by determinating specific PCR product DNA sequence for the by using DNA sequencer. The results showed that DNA that isolated from native Egyptian Mau was compact band of 1600 bp. these purified DNA sequence were subjected to PCR reaction by specific primers to CR mtDNA region and yield an amplified 492 bp sequence which conducted to DNA sequencer. And also showed that we successfully isolated a pure DNA and CR mtDNA amplified sequence. Background Maus Nowadays are currently unrecognized, and often suffer from endemic feline diseases . In fact they are not protected animals or valued like they were in ancient times. Outside of Egypt, many Maus are also endangered due to interbreeding and lack of new bloodlines.And it is endangered not only because the gene pool of the breed outside Egypt weakening due to excessive in-breeding (little new blood), but in Egypt the increase of life threatening feline health Diseases threaten the continuing existence of this breed. Materials and Methods

The purpose of this study is; compare the DNA of present day spotted cats in Egypt to see if there are any markers or common genes of the modern day cats. It thought that this study could possible importance in upgrading the image of feral cats in Egypt. A DNA extraction was performed on 26 hair-shafts from pure bred of Maus, in a addition to 20 samples from feral cats. Results The data showed that RAPD profile with at least one lost or gained band. All random cats gained a band of 600, 300 and 200 while standard lost a band of 500 b.p Conclusion My study's results is not positive, it is believe that it came in the right time to prove that extinction of the Egyptian. And also to pay attention to many expressions which missed among Egyptians i.e. the biodiversity and rescuing the endangered animals. the similarities among the native Egyptian Mau only 30%. This will support the awareness public and the Egyptian citizen with the situation.

Paper reference: SPS1242-14 A novel MT-CO2 m.8249G>A pathogenic variation and the MT-TW m.5521G>A mutation in patients with mitochondrial myopathy *Dr. Emna MKAOUAR-REBAI

Dr. Afif BEN MAHMOUD Dr. Imen Chamkha Dr. Imen Chabchoub Dr. Thouraya Kammoun Dr. Mongia Hachicha Dr. Faiza Fakhfakh

[email protected]

Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA

Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA Service de Pédiatrie, C.H.U. Habib Bourguiba de Sfax, Tunisia Service de Pédiatrie, C.H.U. Habib Bourguiba de Sfax, Tunisia Service de Pédiatrie, C.H.U. Habib Bourguiba de Sfax, Tunisia Human Molecular Genetic Laboratory. Faculty of Medicine of Sfax. TUNISIA

Abstract Mitochondrial DNA defects were known to be associated with a large spectrum of human diseases and patients might present wide range of clinical features with various combinations. Mutations in mitochondrial tRNAs, rRNAs, and protein-coding genes or large-scale rearrangements have been implicated in several cytopathies. Mitochondrial myopathies, usually maternally inherited group of neuromuscular diseases caused by mitochondrial dysfunction occurring before the age of 20 and often begin with exercise intolerance, muscle weakness and neurodevelopmental retardation. We studied the mtDNA in 3 Tunisian patients with mitochondrial myopathy. The mutational analysis screening revealed the presence of 2 mitochondrial mutations : the m.5521G>A mutation in the D-stem region of the tRNATrp gene which could lead to a disruption of the secondary structure of this tRNA and affect the tRNA-

ribosome interaction with a consequent decrease in the rate of synthesis of mitochondrial proteins. The second mutation is the m.8249G>A (p.G222R) variation in the MT-CO2 gene which may affect the electrons transfer from cytochrome c to the bimetallic center of the catalytic subunit I.

Paper reference: SPS1240-14 Decreased Serum Paraoxonase and Arylesterase Activity in Familial Hypercholesterolemia Patients with Mutated LDLR Gene Dr. Akram Muhammad

[email protected] Pakistan

Department of Biosciences, Qaid-eAvenue University of Wah, Wah Cantt

Abstract Background Serum paraoxonase-1 (PON1) has recently been found to protect LDLR -/- mice against development and progression of atherosclerosis; however its role in LDLR mutated humans remains to be studied. There is no data available for PON1 genotype/ phenotype in familial hypercholesterolemia in Pakistan. Methods and Results Coding region SNPs of PON1 gene, serum arylesterase and paraoxonase activities were determined in a large Pakistani hypercholesterolemia family. All the patients of this family had an insertion mutation (c.2416_2417InsG) in exon 17 of the LDLR gene. Prevalence of PON1-55LM genotype showed minor differences between cases and controls. Majority of patients 8 (80%)) and controls 22 (91.7%) belonged to PON1-55MM genotype with 0% prevalence of PON1-55LL genotype. Prevalence of PON1-192QR genotype was different in cases as compared to controls. Most of patients 9 (90%)) belonged to PON1-192QQ genotype as compared to 10 (41.7%) of controls. Paraoxonase and arylesterase activities were significantly low in mutant patients (P = 0.001 and P = 0.002, respectively) as compared to the healthy controls. Conclusions Familial hypercholesterolemia patients with LDLR mutation might be susceptible to development of atherosclerosis due to reduced paraoxonase and arylesterase activity.

Paper reference: SPS1239-14 Isolation of Indigenous Brown Rot Fungi Coniophora puteana from Pakistan and their role in the Biodegradation of Disperse Textile Dyes Dr. Akram Muhammad

[email protected] Pakistan

Abstract Muhammad Akram1, R T. Mahmood1, M. Javaid Asad1

Department of Biosciences, Qaid-eAvenue University of Wah, Wah Cantt

Coniophora is a genus of fungi within the Boletaceae family with 20 different species one notable as Coniophora puteana which causes brown rot. Brown-rot fungus breaks down hemicellulose and cellulose which includes Serpula lacrymans, Fibroporia vaillantii and Coniophora puteana. Textiles dyes consist of stable aromatic compounds which show resistant towards degradation and are not easily eliminated from the environment. Dyes present in industrial effluents are carcinogenic when enter in food chain and also contaminate the environment. Currently many studies are focused on microorganisms having dyes degradation potential. Dyes degradation by using microbial enzymes is an efficient and eco-friendly process. In this study Coniophora puteana, brown rot fungus isolated on Malt Extract Agar and used for decolorization of six (D1,D2,D3,D4,D5 and D6) main disperse dyes. Extracellular enzymes involve in the dyes decolorization were identified by enzyme assay as laccases, maganse peroxidases and lignin peroxidases. The decolorization activities of reactive disperse dyes optimized condition of temperature and pH. The effect of pH and temperature tested that highest decolorization at acidic pH 5 and at temperature 28 0C. The results obtained that removal of dyes depend on initial dye concentration of the solution. The isolate of Coniophora puteana recorded the highest biomass accumulation after 8 days and it prove to be the most effiecient one in removal the four dyes (>80%) and two dyes ( Na > Ca > K The other parameters including pH TDS chlorides and phenol varied within the range of - - mg L - mg L and - µ g L respectively. Background In the current study the levels of water quality parameters were examined in ground and surface water of Rohri, Sukkur and Khairpur cities. The analytical investigation of liquid water generated by human activities within the vicinities of these cities were also carried out. For this purpose 73 sampling sites were selected in the study area which comprise of 5 from sewegae water (Sukkur and Rohri), 30 from surface water 20from ground water of Sukkur and Rohri cities, while 18 sampling sites from Khairpur city. Materials and Methods The main focus of the investigation was Arsenic which was determined by Atomic absorption coupled with As hydride generater. While other parameters which includes PH,TDS,Chlorides, phenols, Na,K,Ca,Mg were determined by using standard methods.The metal contents were determined by Atomic Absorption Spectrometry Results The result revealed that Arsenic contents in ground water and surface water varied within the range of - µ g L approximately fold higher than WHO limits while other metals were found within the levels of Pb ( - ) µ g L Zn ( - ) µ g L Cu ( - ) µ g L Ni ( - ), Cr ( - ), Co ( - ) µ g L Fe ( - ) µ g L Cd ( - ) µ g L Mn ( - ) µ g L Conclusion The observed results indicate that elevated levels of arsenic was of great concern and threat to the human health and concentration of some other water quality parameters were higher than the recommended limits of EPA Pakistan and WHO Contamination index ” Cd ” which determine the degree of contamination was also calculated for each sample and based on sum of the factor obtained from parameters exceeding upper permissible level.

Paper reference: SPS1225-14 Comparison of the ESE motifs binding to the SF2/ASF, SC35 and SRp40 SR proteins among human gene subgroups *Dr. Olfa SIALA

[email protected]

Dr. Ahmed REBAI Dr. Faouzi BAKLOUTI Dr. Faiza FAKHFAKH

Tunisia France Tunisia

Tunisia

Laboratoire de Génétique Moléculaire Humaine

CBS UNIVERSITE CLAUDE BERNARD LGMH

Abstract Exonic splicing enhancers ( ESEs ) take part in both alternative and constitutive splicing and act as binding sites for members of the SR protein family A considerable effort using empirical experiments and computational predictions has been undertaken to find the motifs characteristic of these splicing regulatory elements The motifs identified are short ( – nt ), degenerate and sometimes partially overlap All the previous studies were interested in comparing the different ESE motifs recognized by the different SR proteins ( SF2 ASF SRp40 SRp55 and SC35 ) ( Wang et al ). In our studies we undertake a large - scale genomic analysis in an attempt to uncover by computational using the ESEfinder program and statistical approaches if the exonic splicing enhancer motif binding to the SF2 ASF SC35 and the SRp40 SR protein is conserved among several groups of human genes Results showed that the same C A S A S G A ESE motif binding to the SF2 ASF is conserved between genes within the same chromosome within different chromosomes and between different levels of muscular cells differentiation However this motif displays subtle variations between genes expressed in different tissues accounting for the alternative splicing of several genes between tissues ( Siala et al ). The same Y C A C A G S SRp40 ESE motif was found in all the tested genes However subtle discrepancies were shown in the G G C C C C T G motif binding to the SC35 among human chromosomes This sequence flexibility can emphasize the presence of different translational isoforms of the SFRS2 gene encoding for the SC35 and the possibility that sequence context and structure are also very important for the recognition ESE - SC35 especially that SC35 is the only SR protein which have only one RNA recognition Background Exonic splicing enhancers ( ESEs ) take part in both alternative and constitutive splicing and act as binding sites for members of the SR protein family A considerable effort using empirical experiments and computational predictions has been undertaken to find the motifs characteristic of these splicing regulatory elements The motifs identified are short ( – nt ), degenerate and sometimes partially overlap All the previous studies were interested in comparing the different ESE motifs recognized by the different SR proteins ( SF2 ASF SRp40 SRp55 and SC35 ) ( Wang et Materials and Methods In our studies, we undertake a large-scale genomic analysis in an attempt to uncover by computational using the ESEfinder program and statistical approaches if the exonic splicing enhancer motif binding to the SF2/ASF, SC35 and the SRp40 SR protein is conserved among several groups of human genes.

Results Results showed that the same C A S A S G A ESE motif binding to the SF2 ASF is conserved between genes within the same chromosome within different chromosomes and between different levels of muscular cells differentiation However this motif displays subtle variations between genes expressed in different tissues accounting for the alternative splicing of several genes between tissues ( Siala et al ). The same Y C A C A G S SRp40 ESE motif was found in all the tested genes However subtle discrepancies were shown in Conclusion This sequence flexibility can emphasize the presence of different translational isoforms of the SFRS2 gene encoding for the SC35, and the possibility that sequence context and structure are also very important for the recognition ESE-SC35 especially that SC35 is the only SR protein which have only one RNA recognition motif, what requires high sequence specificity.

Paper reference: SPS1223-14 Association of ABO and Colton blood group gene polymorphisms with hematological traits variation *Dr. Shirin Shahbazi

[email protected] Iran

Dr. Reza Mahdian

Iran

Tarbiat Modares University

2Biotechnology Research Center, Molecular

Abstract Introduction: Hematological parameters are appraised routinely to determine overall human health and to diagnose and monitor certain diseases. In GWASs, more than 30 loci carrying common DNA polymorphisms have been identified related to hematological traits. In this study, we investigated the contribution of ABO rs2073823 along with AQP1 rs1049305 and rs10244884 polymorphisms in hematological traits variation in a cohort of Iranian healthy individuals. Methods: Genomic DNA was extracted from peripheral blood of 168 healthy volunteer. Genotyping was performed by ARMS-PCR or PCR-RFLP and confirmed by DNA sequencing. Statistical analysis was done by SPSS V16 software. Results: Significant association was observed between AQP1 rs1049305 and the hematological traits including hemoglobin, hematocrit and platelet count (p=0.012, p=0.008 and p=0.011 respectively). The AQP1 rs10244884 status was also significantly linked to hemoglobin and hematocrit levels in the study cohort (p=0.015 and p=0.041, respectively). Furthermore, ABO rs2073823 polymorphism was identified as a hemoglobin and hematocrit levels modifier (both with p=0.004). There was no major deviation from the expected Hardy–Weinberg equilibrium in the subject's allele frequencies. Conclusion: AQP1 and ABO variants appear to predict hemoglobin and hematocrit levels but not other erythrocyte phenotype parameters including red blood cell counts and red blood cell indices. Background

Hematological parameters are appraised routinely to determine overall human health and to diagnose and monitor certain diseases. In GWASs, more than 30 loci carrying common DNA polymorphisms have been identified related to hematological traits. In this study, we investigated the contribution of ABO rs2073823 along with AQP1 rs1049305 and rs10244884 polymorphisms in hematological traits variation in a cohort of Iranian healthy individuals. Materials and Methods Genomic DNA was extracted from peripheral blood of 168 healthy volunteer. Genotyping was performed by ARMS-PCR or PCR-RFLP and confirmed by DNA sequencing. Statistical analysis was done by SPSS V16 software Results Significant association was observed between AQP1 rs1049305 and the hematological traits including hemoglobin , hematocrit and platelet count ( p = p = and p = respectively ). The AQP1 rs10244884 status was also significantly linked to hemoglobin and hematocrit levels in the study cohort ( p = and p = respectively ). Furthermore ABO rs2073823 polymorphism was identified as a hemoglobin and hematocrit levels modifier ( both with p = ). Conclusion AQP1 and ABO variants appear to predict hemoglobin and hematocrit levels but not other erythrocyte phenotype parameters including red blood cell counts and red blood cell indices

Paper reference: SPS1221-14 Pectic acid from apple induced cell cycle arrest in MDA-MB-231 human breast cancer cells * Dr. Ladan Delphi

[email protected] Iran

Dr. Houri Sepehri

Animal Biology Department, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran Endocrinology and Metabolic Research Institute, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

Dr. Mohmmad Reza Khorramizadeh

Animal Biology Department, Faculty of Biology, College of Sciences, University of Tehran, Tehran, Iran

Abstract Background Breast cancer an estrogen - dependent disease is the most common cancer in women that starts in the cells of the breast Chemotherapy is used to treat breast cancer in which cancer cells direct to commit apoptosis Apart from the chemical drugs natural origin substances have also been noted for cancer therapy These substances are usually free from chemical drug side effects Pectin a product of plant cell walls is one natural substances extracted from the citrus and apple wastes Pectins include potential for apoptosis induction and anticancer effects Objective Here apple pectin ( pectic acid ) was used as an anticancer agent to inquire on a cellular model of breast cancer Methods MDA - MB - human breast cancer

cell line was cultured in RPMI medium supplemented with % fetal bovine serum The effect of pectic acid on cellular viability was determined by the MTT assay Morphological features of apoptotic cells were determined with AO EB followed by fluorescence microscopy The ratio of cells in the G0 G1 S and G2 M phases of cell cycle was determined by their DNA content using PI and flowcytometry analysis Results Cell viability of MDA - MB - in presence of pectic acid ( mg ml ) for h was concentration - dependent The high concentrations of pectic acid ( mg ml ) statistically reduced viable cell number compared to the untreated cells Also apoptotic features such as fragmented chromatin was determined with AO EB staining Cell - cycle analysis showed more than1mg ml pectic acid caused a large number of cells distributed in G0 region CONCLUSION Pectins such as pectic acid may contain great protective effects against cancer Our results indicated pectic acid induced a very strong sub - G1 arrest representing apoptosis in breast cancer cells. Background Breast cancer an estrogen-dependent disease is the most common cancer in women that starts in the cells of the breast. Chemotherapy is used to treat breast cancer in which cancer cells direct to commit apoptosis. Apart from the chemical drugs, natural origin substances have also been noted for cancer therapy. These substances are usually free from chemical drug side effects. Pectin a product of plant cell walls is one natural substances extracted from the citrus and apple wastes. Pectins include potential for apoptosis induction and anticancer effects. Materials and Methods MDA-MB-231 human breast cancer cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. The effect of pectic acid on cellular viability was determined by the MTT assay. Morphological features of apoptotic cells were determined with AO/EB followed by fluorescence microscopy. The ratio of cells in the G0/G1, S and G2/M phases of cell cycle was determined by their DNA content using PI and flowcytometry analysis. Results Cell viability of MDA-MB-231 in presence of pectic acid (0, 0.01, 0.1, 0.5, 1, 2.5, 5 mg/ml) for 24h was concentration-dependent. The high concentrations of pectic acid (1, 2.5, 5 mg/ml) statistically reduced viable cell number compared to the untreated cells. Also apoptotic features such as fragmented chromatin was determined with AO/EB staining. Cell-cycle analysis showed more than1mg/ml pectic acid caused a large number of cells distributed in G0 region. Conclusion Pectins such as pectic acid may contain great protective effects against cancer. Our results indicated pectic acid induced a very strong sub-G1 arrest representing apoptosis in breast cancer cells. Paper reference: SPS1213-14 Calpain 10 (CAPN10) SNP-44 and -19 (Ins/Del-19) Polymorhism in Type 2 Diabetes Patients in a Turkish Population Dr. NACİYE SELCEN BAYRAMCI

[email protected] Turkey - Türkiye GAZİOSMANPAŞA UNIVERSITY

Abstract Type diabetes ( T2DM ) is a complex metabolic disease in which genetic effects and metabolic and environmental factors contribute the pathogenesis CAPN10 gene which is located on chromosome q37 consists of exons spanning kb In this study we aimed to examine the role of CAPN10 SNP - and - polymorphisms in genetic susceptibility to T2DM in a Turkish population T2DM patients and non - diabetic control group were genotyped for CAPN10 SNP - and Mutagenically seperated PCR - RFLP method was used to examine SNP - whereas SNP polymorphism was genotyped by electrophoresis of the PCR product on % agorose gel with ethidium bromide No statistically significant difference was found between T2DM development and allele frequencies of CAPN10 SNP - and - polymorphisms CAPN10 gene SNP - del - allele and del del genotype and SNP - C - allele and TC genotype were found to be risk factors for T2DM development There were no significant difference between T2DM development and genotype frequencies of SNP - and - polymorphisms In the SNP - T2DM patients group patients had retinopathy patients had nephropathy and patients had neuropathy Likeweise in the SNP T2DM patients group patients had retinopathy patients had nephropathy and patients had neuropathy The presence of C - allele in SNP - TC genotype was found to be associated with fold increased risk of T2DM in overall T2DM patient population - fold in females and - fold in males and the presence of del - allele in SNP - del del genotype was found to be associated with - fold increased risk of T2DM in overall T2DM patient population - fold in females and - fold in males In addition T2DM patients who are homozygous for SNP - ins - allele had higher BMI than Background Type 2 diabetes (T2DM) is a complex metabolic disease in which genetic effects and metabolic and environmental factors contribute the pathogenesis. CAPN10 gene, which is located on chromosome 2q37.3, consists of 15 exons spanning 31 kb. In this study, we aimed to examine the role of CAPN10 SNP-44 and -19 polymorphisms in genetic susceptibility to T2DM in a Turkish population. Materials and Methods 125 T2DM patients and 112 non-diabetic control group were genotyped for CAPN10 SNP-44 and -19. Mutagenically seperated PCR-RFLP method was used to examine SNP-44 polymorphism, whereas SNP-19 polymorphism, which is the two-allele insertion/deletion (insdel) polymorphism containing two or three copies of the 32 bp repeated sequence, was genotyped by electrophoresis of the PCR product on 3% agorose gel with ethidium bromide. Results The presence of C-allele in SNP-44 TC genotype was found to be associated with 1,34-fold increased risk of T2DM in overall T2DM patient population, 3-fold in females and 5-fold in males and the presence of del-allele in SNP-19 del/del genotype was found to be associated with 1,30-fold increased risk of T2DM in overall T2DM patient population, 1,78-fold in females and 0,91-fold in males. Conclusion CAPN10 SNP-19 del-allele and del/del genotype and SNP-44 C-allele and TC genotype were found to be risk factors for T2DM development. There were no significant difference between T2DM development and genotype frequencies of SNP-44 and -19. In the SNP-44 T2DM patients group, 11 patients had retinopathy, 46 patients had nephropathy and 14 patients had

neuropathy. Likeweise in the SNP-19 T2DM patients group, 9 patients had retinopathy, 52 patients had nephropathy and 13 patients had neuropathy.

Paper reference: SPS1211-14 Genomic Variability by ERIC PCR and Biofilm Formation among Bacillus thuringiensis strains *Dr. Karina García Gutierrez Dr. Jorge Ibarra Dr. Alejandra Bravo Dr. Dafne Gutiérrez Dr. Díaz Javier Dr. Torres Patricia Dr. Patricia Gómez de León

[email protected] Mexico - México Mexico - México Mexico - México Mexico - México Mexico - México Mexico - México

Mexico - México

UNAM Faculty of Medicine

CINVESTAV IPN IBT, UNAM UNAM Faculty of Medicine UNAM Faculty of Medicine UNAM Faculty of Medicine UNAM Faculty of Medicine

Abstract Background. Bacillus thuringiensis (Bt) is of important agronomic and medical research interest. There are limitations with subspecies classification. Studies at phenotypic and genotypic levels are important to ascertain this variability. Diversity is one of the emerging themes in current biofilm research. The aim of this study was to evaluate the variability by using ERIC-PCR and biofilms formation among Bt strains from different localities in Mexico. Methods. The genomic relationships between forty environmental strains from CINVESTAVIrapuato and IBT-UNAM were evaluated. Genomic fingerprints were generated by PCR using the ERIC sequences. NTSYS-pc program was utilized to cluster strains. The UPGMA was used as the clustering algorithm. The biofilm-forming ability at 72 and 96 h of incubation was performed according to Wijman. Conclusions. A large intra-species genomic variability was observed among Bt isolates. Some strains isolated from the same locations were clustered in the phenogram together. At 96 h of incubation, most strains from the CINVESTAV collection showed moderate to strong biofilm forming ability, whereas those from IBT-UNAM collection were mainly weak biofilm producers. The repeated findings on the strong genetic diversity in Bt of are consistent with the results described here. Results. Thirty-nine fingerprinting patterns based on 24 polymorphic fragments of 139 -1468 bp were generated. Most of the Bt isolates did not group together. Strains form Morelos were more variable. Almost all strains (95%) showed biofilm formation. Depending on the OD620 values were stratified into 4 categories: 32.5% strong (OD620 >1.03), 35% moderate (OD620 1.03-0.52), 27.5% weak (OD620 0.51-0.27) and 5% null (OD620 ≤ 0.26). The strains from the CINVESTAV showed more heterogeneous biofilm-forming ability than strains from IBT-UNAM. Background Bacillus thuringiensis (Bt) is of important agronomic and medical research interest. There are limitations with subspecies classification. Studies at phenotypic and genotypic levels are

important to ascertain this variability. Diversity is one of the emerging themes in current biofilm research. The aim of this study was to evaluate the variability by using ERIC-PCR and biofilms formation among Bt strains from different localities in Mexico. Materials and Methods The genomic relationships between forty environmental strains from CINVESTAV-Irapuato and IBT-UNAM were evaluated. Genomic fingerprints were generated by PCR using the ERIC sequences. NTSYS-pc program was utilized to cluster strains. The UPGMA was used as the clustering algorithm. The biofilm-forming ability at 72 and 96 h of incubation was performed according to Wijman. Results Thirty-nine fingerprinting patterns based on 24 polymorphic fragments of 139 -1468 bp were generated. Most of the Bt isolates did not group together. Strains form Morelos were more variable. Almost all strains (95%) showed biofilm formation. Depending on the OD620 values were stratified into 4 categories: 32.5% strong (OD620 >1.03), 35% moderate (OD620 1.030.52), 27.5% weak (OD620 0.51-0.27) and 5% null (OD620 ≤ 0.26). The strains from the CINVESTAV showed more heterogeneous biofilm-forming ability than strains from IBT-UNAM. Conclusion A large intra-species genomic variability was observed among Bt isolates. Some strains isolated from the same locations were clustered in the phenogram together. At 96 h of incubation, most strains from the CINVESTAV collection showed moderate to strong biofilm forming ability, whereas those from IBT-UNAM collection were mainly weak biofilm producers. The repeated findings on the strong genetic diversity in Bt of are consistent with the results described here. Paper reference: SPS1204-14 The comorbidity of schizophrenia and tobacco dependence in a rodent model of psychosis: towards understanding of Water pipe addiction *Dr. wael Mohamed

[email protected]

Egypt

Menoufia medical school

Abstract Rationale and Objectives Schizophrenia is a neurodevelopmental disorder ( Khandaker et al ) and tobacco use in adolescence is a powerful predictor of tobacco dependence in adulthood ( DiFranza et al ).The aims of this proposal are to investigate the role of α and α β nAChRs in nicotine behavioral sensitization nicotine place conditioning and expression of BDNF and p CREB induced by nicotine exposure The second aim will investigate the role of α and α β nAChRs in the accumbal dopamine response to nicotine using the microdialysis technique Methods All animals will be given a single daily i p injection of either quinpirole ( mg kg ) or saline from PND1 - PND21 Beginning on PND33 animals will be i p injected with either nicotine ( or mg kg free base ) or saline and mins later placed into a square locomotor arena for a min behavioral testing session ( N = - per group ). Horizontal activity counts will be scored by an automated behavioral tracking system ( AnyMaze Stoelting Wood Dale IL ). This procedure will repeated every other day over a - day period ( from PND33 - ). Brain tissue from treated groups in addition to controls will be harvested for BDNF and pCREB Anticipated Results We

hypothesize that MLA ( Methyllycaconitine ) will block the enhanced nicotine sensitization and nicotine place conditioning in adolescent animals neonatally treated with quinpirole Ultimately nicotine sensitization and nicotine place conditioning of animals neonatally treated with quiniprole sensitized or conditioned to nicotine in adolescence and pretreated with MLA will be significantly below animals neonatally treated with saline sensitized or conditioned with nicotine and pretreated with MLA Conclusions and Implications This proposal is underway in collaboration with American team This proposal is focused on analyzing behavioral neurochemical

Paper reference: SPS1200-14 Markers of Neural and Muscular Regeneration in Duchenne Muscular Dystrophy *Dr. Iman Ehsan Abdel Meguid [email protected] Egypt Dr. Ekram Abdel-Salam Dr. Rania Shatla

Egypt Egypt

Faculty of Medicine - Cairo Univ

Faculty of Medicine - Cairo University Faculty of Medicine - Ain Shams University

Abstract Duchenne muscular dystrophy (DMD) is a severe and progressive disease characterized by muscle fiber degeneration and central nervous system disorders caused by mutations in the dystrophin gene. Abundant evidence suggests that neural circulating progenitor cells (NPCs) play an important role in mediating neural repair mechanisms and that the Nerve Growth factor (NFG) chemokine is responsible for both progenitor cell mobilization from the bone marrow to peripheral blood and homing to the sites of neural and tissue injury. Accordingly, the present study investigated levels of CD 45, nestin and NGF in an attempt to investigate makers of neural regeneration in DMD. Also, in muscle environment, Galactin (Gal-1) promotes myoblast fusion and axonal growth after muscle injury. nsulin-like growth factor-1 (IGF-1) is a multipotent growth factor involved in the growth, development and regulation of homeostasis in a tissue-specific manner. Results showed that NGF (165.8 ± 72 vs. 89.8 ± 35.9) and mononuclear cells expressing nestin (18.9 ± 6 vs. 9 ± 4), CD 45 (64 ± 5.4 vs. 53.3 ± 5.2) and CD34 (75 ± 6.2 vs. 60 ± 4.8) were significantly increased among DMD patients compared to controls. Similarly was Gal-1 and IGF-1. The significant increase plasma NFG and in the number of mononuclear cells bearing CD34, CD45 and nestin indicates that regeneration is an ongoing process in these patients. However, this regeneration cannot counterbalance the damage induced by dystrophine mutation. Paper reference: SPS1198-14 Arugula- The green solution for diabetics and cholesterol *Dr. SHAHEER MANJAKANDAN [email protected] India Uwin Life Sciences

Abstract Eruca Vesicaria sativa- A Global Anti-Lipidemic Agent M.K Shaheer*, A Finose and Dr. K.M Hashim Uwin life Sciences, Malappuram, Kerala, India-676505 Abstract: ¬- Eruca vesicaria sativa globally known as Arugula is a food supplement commonly used in Middle East along with the high fat content foods. We investigated the pharmacological activities on the plant and found that it is capable of lowering the lipid profile significantly. Statistical studies were carried out using patients of different age groups. Phytochemical studies were also performed in the plant for the detection of any anti-lipidemic compound. So far no chemical studies were reported in the plant so we advanced through the Liquid Chromatographic and Mass Spectroscopic studies and around 400 masses were found in which 120 were already known compounds. In-vitro anti-oxidant activity is also reporting in the plant for the first time. Bio-chemical parameters were also determined as a part of the plant characterization. The isolation of chemical constituents from the plant is under progress which may lead to the identification of new anti-lipidemic compounds. While concluding we recommend the plant in the daily diet of an age group above 40 years. Being a highly important plant, the daily usage will decrease the chances of other biological disorders too. Key words: Eruca vesicaria sativa, pharmacological, anti-lipidemic, Liquid Chromatography, Mass Spectroscopy *Correspondence: M.K Shaheer, Uwin Life sciences, V.T Towers, Malappuram, Kerala 676505 Tel: UAE: 0562828842, IND: 09995447424 e-mail: [email protected] Background M.K Shaheer*, A Finose and Dr. K.M Hashim Uwin life Sciences, Malappuram, Kerala, India676505 Materials and Methods Materials and methods Collection and Authentication: Eruca vesicaria (Arugula) were collected locally and the collected plant sample was authenticated from the Taxonomy department of Uwin Life Sciences, Malappuram Kerala. The voucher specimens of the plant sample were deposited in the Herbarium of Uwin Life Sciences, Malappuram. Results Results and discussions The phenolic contents of the plant show higher values The Flavonoids were also determined so as to sketch the F P ration of the plant The F P ratio is a measurement of the capacity of the sample to convert the Phenolics to Flavonoids which are highly potent8 The F P ratio of the plant was also higher which implies that this plant is highly potent The pharmacological effects were also supported the fact that the plant shows higher activity The combined nitric oxide and the DPPH Conclusion Conclusion All the results for the above mentioned parameter revealed that the Arugula is a highly potent anti - diabetic and anti - cholesterol plant as it contains a number of highly potent molecules The biochemical parameter F P ratio was also calculated and a higher value shows the efficacy of the plant to convert the Phenolics to Flavonoids The clinical trials were also revealed that this plant possesses the capacity to lower the blood glucose and cholesterol level by different mode of actions for all the age groups The

Paper reference: SPS1195-14 The insilico analyses revealed involvement of miR-146a in progress of Gastric cancer *Dr. Bahareh Adami

[email protected]

Dr. Kamran Ghaedi

Iran

Dr. Hosein Tabatabaeian

Iran

Dr. Mansoureh Azadeh

Iran

Dr. Ardeshir Talebi

Iran

Iran

Department of Microbiology,Faculty of Biological Science,Falavarjan Branch,Islamic Azad University, Isfahan, Iran

Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Isfahan Technical and Vocational training organization, Bionovin technology Institute, Isfahan, Iran Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Abstract Gastric cancer is the third common causes of death among Iranians.MicroRNAs regulate the expression of many genes that play important roles in production of cellular proteins in posttranscriptional mechanisms affecting levels of mRNAs. expression of MicroRNAs in cancer cell lines is different and wide variety of them shows themselves in many cancerous cells.Our analytical research showed that miRNA-146a has high level in stomach cancer and play as a regulator of the mammalian response to microbial infections were affected such as H.Pylori . Targets of miR-146a were gained from miRtarbase and miRwalk databases respectively. Then, their expressions were checked in gastrointestinal tumor using Unigene database. Finally, targetome which were expressed in gastric tissue were submitted into DAVID database for molecular pathway enrichment analysis.DAVID database disclosed many KEGG signaling pathways, including “TOLL-LIKE receptor signaling pathway” , “Insulin signaling pathway” , “Neurotrophin signaling pathway” , “Adipocytokin signaling pathway” as the most pertaining signaling pathways to miR-146a targetome. These signaling pathways leads to proliferation, differentiation and apoptosis by targeting signaling pathways.miR-146a also effects on proteasome and tight junctions.According to our computational studies, overexpression of miR146a may cause cell apoptosis, proliferation and differentiation by effecting on signaling pathway such as TOOL-LIKE receptor signaling pathway. Background Gastric cancer is the third common causes of death among Iranians.MicroRNAs regulate the expression of many genes that play important roles in production of cellular proteins in posttranscriptional mechanisms affecting levels of mRNAs. expression of MicroRNAs in cancer cell lines is different and wide variety of them shows themselves in many cancerous cells.Our analytical research showed that miRNA-146a has high level in stomach cancer and play as a regulator of the mammalian response to microbial infections were affected such as H.Pylori . Materials and Methods

Targets of miR-146a were gained from miRtarbase and miRwalk databases respectively. Then, their expressions were checked in gastrointestinal tumor using Unigene database. Finally, targetome which were expressed in gastric tissue were submitted into DAVID database for molecular pathway enrichment analysis. Results : DAVID database disclosed many KEGG signaling pathways, including “TOLL-LIKE receptor signaling pathway” , “Insulin signaling pathway” , “Neurotrophin signaling pathway” , “Adipocytokin signaling pathway” as the most pertaining signaling pathways to miR-146a targetome. These signaling pathways leads to proliferation, differentiation and apoptosis by targeting signaling pathways.miR-146a also effects on proteasome and tight junctions. Conclusion According to our computational studies, overexpression of miR-146a may cause cell apoptosis, proliferation and differentiation by effecting on signaling pathway such as TOOL-LIKE receptor signaling pathway. Paper reference: SPS1194-14 In-silico analysis predicated mir-222 in incidence of gastric cancer *Dr. Mina Noormohammad

Dr. Mehri Khatami Dr. Hosein Tabatabaeian Dr. Kamran Ghaedi Dr. Ardeshir Talebi Dr. Mohammad Mehdi Heidari

[email protected]

Department of Biology, School of Sciences, University of Yazd, Yazd, Iran

Department of Biology, School of Sciences, University of Yazd, Yazd, Iran Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Department of Pathology, school of Medicine, Isfahan University of Medical Science, Isfahan,Iran Department of Biology, School of Sciences, University of Yazd, Yazd, Iran

Abstract Gastric cancer is one of the most common diseases in the world. Chronic infection with Helicobacter pylori (H. pylori) is the strongest known risk factor for the development of gastric cancer. Among the mediators induced in response to the infection, microRNAs (miRNAs) have the potential to play a major impact on the outcome of the bacteria-host interaction. MicroRNAs are endogenous 22-25 nt RNAs plays an important regulatory roles in cellular and developmental processes. Recently, miRNAs have been remarkably used in treating many kinds of diseases. Recent studies have been shown that microRNA-222(mir-222) was up-regulated in H. pylori-infected gastric cancer. miR-222 is an oncogenic miRNA proposed as a possible biomarker of spread in gastric cancer. We have used bioinformatics databases to determine possible molecular function of miR-222, as a proposed prognostic biomarker in gastric cancer. According to our data, miR-222 correlates with proliferation, apoptosis and cell division through tight junction and MAPK signaling pathways.

Background Gastric cancer is one of the most common diseases in the world Chronic infection with Helicobacter pylori ( H pylori ) is the strongest known risk factor for the development of gastric cancer Among the mediators induced in response to the infection microRNAs ( miRNAs ) have the potential to play a major impact on the outcome of the bacteria - host interaction MicroRNAs are endogenous - nt RNAs plays an important regulatory roles in cellular and developmental processes Recent studies have been shown that microRNA - ( mir Materials and Methods We have used bioinformatics databases to determine possible molecular function of miR-222, as a proposed prognostic biomarker in gastric cancer. miR-222 targetome (including its validated and predicted targets) were regained from miRTarbase and miRWalk databases. Their expression in gastric cancer was evaluated in Unigene database. At last stomach expressed targetome of miR-222 were entered into DAVID database for molecular pathway enrichment analysis. Results According to our data from In-silico study , miR-222 correlates with proliferation, apoptosis, invasion cell division through tight junction and MAPK signaling pathways. Conclusion DAVID database demonstrated that several KEGG signaling pathways including “pathways in cancer”, “gap junction”, “tight junction” and “chemokine signaling pathway” are the most correlated pathways with miR-222 targetome. Interestingly, in all this signaling pathways except “tight junction” mir-222 may leads to progression and invasiveness of gastric cancer cells by targeting MAPK signaling pathway.

Paper reference: SPS1193-14 The insilico analyses revealed involvement of miR-21in progress of Gastric cancer *Dr. Elnaz Dehdashtian

[email protected]

Dr. Kamran Ghaedi Dr. Hosein Tabatabaeian Dr. Mansoureh Azadeh

Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran Isfahan Technical and Vocational training organization, Bionovin technology Institue, Isfahan, Iran Department of Pathology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

Dr. Ardeshir Talebi

Iran

Department of Microbiology,Faculty of Biological Science,Falavarjan Branch,Islamic Azad University, Isfahan, Iran

Abstract Stomach cancer, refers to cancer arising from any part of the stomach. MicroRNAs are small, non-coding molecules that play important roles in a variety of normal and diseased biological processes by post-transcriptionally regulating the expression of target gene. miRNA analysis is a

powerful tool that can be used to improve diagnosis and prognosis of gastric cancer.miR-21 was also significantly overexpressed in H. pylori-infected gastric mucosa.Some bioinformatics steps were implicate to assign molecular role of miR-21 in gastric cancer. : Targets of miR-21 including validated and predicted were retrieved from miRtarbase and miRwalk databases respectively.Next,checked their expression in gastrointestinal tumor using Unigene database. Finally,targetom which were expressed in gastric tissue were submitted into DAVID database for molecular pathway enrichment analysis. DAVID database showed many KEGG signaling pathways, including “MAPK”, “JAK-STAT”, “Chemokine” signaling pathways and “Pathways in cancer” as the most pertaining signaling pathways to miR-21 targetome.It is notable to know that in all of the signaling pathways miR-21 leads to proliferation, differentiation, invasive cells and RNA degradation by targeting signaling pathways. According to our survey, overexpression of miR-21 may cause cell apoptosis and degradation in cell cycle and gene expression by effecting on signaling pathways. It may cause inflammatory effects by effecting on MAPK signaling pathway. Background Stomach cancer, refers to cancer arising from any part of the stomach. MicroRNAs are small, non-coding molecules that play important roles in a variety of normal and diseased biological processes by post-transcriptionally regulating the expression of target gene. miRNA analysis is a powerful tool that can be used to improve diagnosis and prognosis of gastric cancer.miR-21 was also significantly overexpressed in H. pylori-infected gastric mucosa.Some bioinformatics steps were implicate to assign molecular role of miR-21 in gastric cancer. Materials and Methods Targets of miR-21 including validated and predicted were retrieved from miRtarbase and miRwalk databases respectively.Next,checked their expression in gastrointestinal tumor using Unigene database. Finally,targetom which were expressed in gastric tissue were submitted into DAVID database for molecular pathway enrichment analysis. Results DAVID database showed many KEGG signaling pathways, including “MAPK”, “JAK-STAT”, “Chemokine” signaling pathways and “Pathways in cancer” as the most pertaining signaling pathways to miR-21 targetome.It is notable to know that in all of the signaling pathways miR-21 leads to proliferation, differentiation, invasive cells and RNA degradation by targeting signaling pathways. Conclusion According to our survey, overexpression of miR-21 may cause cell apoptosis and degradation in cell cycle and gene expression by effecting on signaling pathways. It may cause inflammatory effects by effecting on MAPK signaling pathway.

Paper reference: SPS1187-14 FOXP3 gene mutations in lung cancer *Dr. Ban Abbas Abdul-Majeed Dr. Suhad Faisal Hatem

Iraq

[email protected] University

Iraq

University

Dr. Khalid Tobal

UK

Hospital

Abstract Background and objectives: Several reports have documented the presence of FOXP3 positive regulatory T lymphocytes (T reg) within human tumor tissue. By this, they may play a major role in preventing the development of effective antitumor immunity. Cancer cells may show deletions and somatic mutations of the FOXP3 gene. This was proved in breast and prostate cancers. The aim of the present study was to look for possible FOXP3 gene mutation in lung cancer tissues by submitting the gene to sequencing analysis of exons 3, 6 and 7. Materials and methods: DNA was extracted from formalin fixed paraffin embedded tissue blocks of 26 cases of lung cancer and 8 cases of non-malignant lung diseases. Conventional PCR amplification products of exons 3,6 and 7 were submitted for sequencing using the ABI prism 3730 sequence detecting system. Results: Missense mutations of exon 3 were detected in 3 malignant and 1 benign lesion. Those of exon 7 were detected in 17 malignant and 7 benign lesions. None were found in exon 6. Silent mutations were detected in exon 3 of 2 malignant lesions, in exon 6 in 1 malignant and 1 benign lesions and none in exon 7. Intronic mutations were found in 2 malignant and 1 benign in exon 3, 3 malignant in exon6 and 1 benign in exon 7. Wild type exons were found in 11 malignant and 6 benign in exon3, 14 and 7 in exon 6 and 1 and 1 in exon 7 for malignant and benign lesions respectively. Significant statistical differences were found between malignant and benign lesions regarding the types of mutation. Conclusion: Mutations in FOXP3 gene in lung cancers have an important role in mediating immune evasion and contributing to bad prognosis. Paper reference: SPS1186-14 Performance Comparison of Commercial Kits for Bisulfite Conversion of DNA from Tissues, FFPE Tissues, Cytologic Specimens, and Liquid Biopsies *Dr. Ingo Manfred Jenneckens Dr. Emily Eva Holmes Germany Dr. Maria Jung Germany Dr. Glen Kristiansen Germany Dr. Dimo Dietrich Germany

[email protected]

Germany

Analytik Jena AG

University Hospital Bonn (UKB) University Hospital Bonn (UKB) University Hospital Bonn (UKB)

Abstract DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-

Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA MethylationGold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine. Background A commercial bisulfite kit for a specific research application should be chosen based on the specific performance requirements regarding the respective sample material. This study aimed to evaluate the performance of commercially available bisulfite kits. Materials and Methods EpiTect® Fast FFPE Bisulfite Kit, EpiTect® Bisulfite Kit, EpiTect® Fast DNA Bisulfite Kit (Qiagen), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluid Kit (Analytik Jena), EZ DNA Methylation-Gold™ Kit, EZ DNA Methylation-Direct™ Kit, EZ DNA Methylation-Lightning™ Kit (Zymo Research) were compared. The performance was evaluated with regard to DNA yield, DNA degradation, conversion efficiency, DNA purity, stability, versatility, and handling. Results Time-to-result was between min (innuCONVERT kits) and min (EpiTect® Bisulfite Kit). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit. The innuCONVERT Bisulfite All-In-One Kit allowed for the bisulfite conversion of extracted DNA tissues FFPE tissues cell lines urine Sediment, and cellular fractions of bronchial aspirates, ascites, pleural effusions. Conclusion All tested kits showed high performance when applying extracted DNA The innuCONVERT Bisulfite kits showed the highest versatility regarding clinically relevant sample input materials. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained with the EZ DNA Methylation-Gold™ Kit and the innuCONVERT Bisulfite kits. Conversion efficiency was above 98.7 % for all kits. Inappropriate conversion of methylated cytosines to thymines ranged from 0.9 % (innuCONVERT Bisulfite kits) to 2.7 % (EZ DNA Methylation-Direct™ Kit).

Paper reference: SPS1185-14 Exons targeted mutational analysis of EGFr in cancer patients from the Arabian Gulf populations *Dr. Maryam H Marzouq

[email protected]

Bahrain Arabian Gulf University-College of Medicine and Medical Sciences

Dr. Mohamed-Dahmani Fathallah

Bahrain Arabian Gulf University-College of Graduate Studies

Abstract The human epidermal growth factor receptor (EGFR) plays an important role in cancer cells development. EGFR mutation analysis and identification of genetic variants are not sufficiently investigated in the Arabian Gulf countries. In this work we investigated the EGFR gene mutations and polymorphism in exons 13 to16 coding for the CR2 functional domain of the molecule, in six types of cancers affecting the Arabian Gulf populations. We studied a total of 104 patients and six types of cancers with 114 healthy controls. Mutation screening of exons 13, 14, 15 and 16 of the EGFR gene was carried out by PCR /DNA Sequencing. Our data showed the following: Exon 13: A new SNP (C1782T transition) with a CT genotype frequency of 4.08% in colon and bladder cancers and 0.87% in healthy controls. The 3 reported SNP rs2227983, rs142429250 and rs17336800 were also observed. Exon 14: A novel transition (G1894A) yielding a missense mutation Val550Met. The AA genotype frequency was 5.97% in colon and ovary cancers whereas; the GA genotype frequency was 25.37% in 5 types of cancers. The reported SNP rs17290103was also found. Exon 16: A new SNP/Mutation (T2142A transition) was found exclusively in patients with the TA genotype occurring at 10.14% frequency. An additional rare novel SNP (C/T) was found exclusively in the control population at a frequency of 1.75%. The known rs2227984 SNP was also found. No mutation was observed in Exon15 in this study. Our findings point to the EGFR gene mutations identified in exon 14 and 16 and the new SNPs found in exon 13 and 16 as specific of cancer in the Arabian Gulf population. These data are among the first to document the presence of both novel or reported human EGFR mutations and SNPs in Arabian Gulf cancer patients.

Paper reference: SPS1181-14 Status of molecular genetics research in Pakistan. *Dr. M. Iqbal Choudhary

Dr. M. Kamran Azim

[email protected]

International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

International Center for Chemical and Biological Sciences, University of Karachi, Karachi, Pakistan

Abstract The CEMB is involved in genetic basis of vision and hearing impairment. Researchers in this center want to reveal underlying genetic causes of Vision and hearing impairment (Congenital Cataract, Retinitis Pigmentosa and glaucoma) in Pakistani population. The ICCBS has made considerable progress in the field of genomics and bioinformatics during last several years. Recently, ICCBS has announced completion of genome sequencing project of the first Pakistani individual. This project has been carried out in technical collaboration with Beijing Genomics Institute (BGI), Shenzhen, China. This research provided valuable data related to complete genome sequencing and variant analysis of the Pakistani individual in an Asian and global context. The Genomics laboratory of ICCBS is involved in the sequencing of genomes of a number of important fruit crops i.e. mango, date palm and jamun (Syzygium cumini). More recently, mango gtranscriptomic analysis has published from ICCBS (Azim et al., 2014). Moreover, wholegenome sequencing of two pathogenic bacteria belonged to Burkholderia and Pseudomonas species has been carried out in the ICCBS using next generation DNA sequencing technology. The genome-level sequence data has provided important insight related to pathogenesis of these bacteria. Background Considerable research on molecular genetics of human genetic diseases has been carried in several Universities and research centers of Pakistan including Center of Excellence in Molecular Biology (CEMB), University of Punjab, Lahore; NIBGE, Faisalabad; Quaid-e-Azam University, Islamabad; International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi.

Paper reference: SPS1174-14 Expression and characterization of human EGFR L2 and CR2 domains using Escherichia coli Origami B and BL21 Strains *Dr. Nariman A Al-Mustafa

[email protected]

Dr. Sonia Bourguiba-Hachemi Dr. Mohamed-Dahmani Fathallah

Bahrain Bahrain

Bahrain

Arabian Gulf UniversityCollege of Graduate Studies

Arabian Gulf University-College of Graduate Studies Arabian Gulf University-College of Graduate Studies

Abstract The Epidermal growth factor receptor ( EGFR ) is a cell surface protein composed of an extracellular ligand - binding domain trans membrane segment and an intracellular tyrosine kinase domain The EGFR plays a key role in many of the cellular processes involved in cancer development and has been proven to be one of the most important and rational target for anti - tumor strategies Monoclonal antibodies bind to the extracellular domain of the EGFR and inhibit ligand binding to the receptor inducing receptor dimerization and down regulation Therefore recombinant mAbs specifically targeting mutation in the extracellular domain of the EGFR would robustly enhance anti - tumor action and may perhaps become valuable

therapeutic reagents In this project we used the Escherichia coli ( E coli ) system of recombinant protein expression to produce the human native EGFR extracellular domains L2 and CR2 Synthetic L2 and CR2 sequences were cloned into pGEX4T - plasmid under the control of tac promoter Two different Escherichia coli strains Origami B and BL21 strains were used to produce the recombinant proteins each fused to Glutathione - S - Transferase ( GST ). Recombinant protein expression was analyzed by SDS - PAGE and western blot assay Our results show that the recombinant EGFR L2 and CR2 domains are expressed as soluble recombinant proteins However approximately 60 % of the protein produced is trapped in inclusion bodies The amount of soluble EGFR L2 and CR2 domains is higher in E coli BL21 strains (35 %) cultured at 30oC and induced using 1mM IPTG The soluble recombinant EGFR L2 and CR2 domains expressed in BL21 can be produced in a preparative scale purified and used to carry out structure function analysis and to develop functional monoclonal antibodies to EGFR.

Paper reference: SPS1173-14 Fungal Engineering Aspect in decoloration Technologies of dye wastewater *Dr. Rafia NA Azmat

[email protected]

University of Karachi, Karachi, Pakistan

Dr. Sidra NA Javed

University of Karachi, Karachi, Pakistan

Abstract Current age of rapid increase in dyes and pigment practices their used in printing, textile, paper, cosmetics, and food and pharmaceutical industries. About 10% of the used dye is discarded as waste in running streams without proper treatment. Most the dyes and pigments whether natural or lab synthesized are lethal for living organisms. A number of studies for removal of dyes from industrial waste have been done including physicochemical processes and microbial degradation. Adsorption methods are widely used for elimination of dyes from industrial effluent. During sorption studies of deletion of dyestuff by prepared potato peels, fungal growth on reaction mixture were witnessed. This fungal growth was found to have a potential role in the decoloration of dye pigment. Dye pigment was almost decolorized by this auto fungal growth. The fungus was then identified as Penicllium which is saprophytic fungus and grows on moist and dead organic biodegradable material. The grown and identified Penicillium sp. were then collected and introduced along potato peel surface in four different dyes, methylene blue, Reactive blue, Reactive red, and Reactive yellow dye separately. The same experiments were also performed without fungus. It was an interesting observation that almost complete color of dye was disappeared in all dyes system leaving the branching pattern of fungal hyphae development only. It was suggested that the core structure of penicllium play important role in dye degradation where it was recommended that oxygen atom attached, help in bioremediation and converting the dye into CO2 and other carbon product. Background In recent years various bioremediation methods were applied for treatment of complex industrial waste water. Microbial degradation of environmental pollutants such as dyes is a

cheaper and environmentally friendly alternative. Penicillium is a genus of ascomycetous fungi of major importance in the natural environment as well as food and drug production. The main aim of our project is to get water free from the dye. In this way we can utilize a large amount of used industrial wastewater which is usually discarded in the main stream. Materials and Methods Waste potato peels were utilized for removal of dye from water. The auto growth of Fungus was observed during the experiments. The fungus identifies as Penicillium sp. The grown and identified Penicillium sp. were then inoculated along potato peel surface with 4 different dyes, methylene blue, Reactive blue, Reactive red, and Reactive yellow dye in a separate beakers in three replicates. The same experiments were also performed without fungus. The extent of dyestuff decolorization was determined by measuring the absorbance of each dye by UV/Visible spectrophotometer. Results Potato peel has influenced the physical properties of the aquatic medium i.e. water such as decrease in surface tension and slight increase in viscosity and density of solution. Potato peel is a main source of starch which is a natural media for the auto growth of fungi. The fungi growing on potato peel is characterized and found to be helpful in removal of various reactive and basic dyes. Spectral analysis showed the shift in wavelengths of dyes after treatment with Penicillium and with potato peels. Conclusion This study shows that Penicillium can be used as a potential decolorizer for dye removal along with the adsorbent surface prepared by potato peels. Therefore it is established from the above examination that the wastewater containing dyes can be treated using microbial activity along with the adsorbent surface. This study provides an integrated method for the management of industrial waste along with the cosmopolitan waste. Future aspects of this study are the Eco toxicological testing of clear water via marine organisms, analysis and recycling of recovered surface and penicillium. Paper reference: SPS1171-14 Production of the human EGFR recombineant CR2 extracellular domain genetic variants in E.coli. *Dr. Maryam H Marzouq

[email protected] Bahrain

Dr. Sonia Bourguiba-Hachemi Dr. Mohamed-Dahmani Fathallah

Arabian Gulf University-College of Medicine and Medical Sciences

Bahrain Arabian Gulf University-College of Graduate Studies Bahrain Arabian Gulf University-College of Graduate Studies

Abstract The epidermal growth factor receptor ( EGFR ) is one of the most critical targets for diagnostic and treatment of cancers Monoclonal antibodies ( MAb ) to EGFR are being sucessfully used to treat a number of tumors In a Preliminary work aimed at generating anti EGFR MAb according

to the genetic makeup of cancer patients we mapped a number of mutations and SNPs in the EGFR of cancer patients from the Arabian Gulf region to the Cysteine - rich domain ( CR2 ) located in the extracellular domain of the receptor particularly the polymorphism [ R521K ] presents in exon Therefore we decided to clone express and purify recombinant wild type CR2 R and the most common CR2 - K variants of the CR2 sub domain and use it to produce anti - CR2 - R and anti - CR2 - K monoclonal antibodies A fusion expression vector pGEX4T -1 containing Glutathione S - transferases ( GST ) was constructed by inserting the wild type variant of CR2 - R domain and the mutated variant CR2 - K into pGEX - T1 between EcoRI and XhoI sites After transformation of E coli BL21 strain by electroporation, the expression and purification of the two engineered proteins were confirmed by SDS - PAGE and western blot Finally the endotoxin was removed using Ion Exchange columns Soluble CR2 - R and CR2 - K proteins were highly expressed by culturing E Coli clones at ˚ C for hours after induction with mM IPTG CR2 - R and CR2 - K proteins were detected by SDS - PAGE and by western blot The purified recombinant CR2 - R and CR2 - K proteins were used to generate specific MABsto be developed into a novel therapeutic tool for Cancer tumors.

Paper reference: SPS1169-14 Allelic frequency and concordance study in Kuwaiti population of two amplification PCR kit AmpFℓSTR® NGMSElect™ and PowerPlex® ESI17 *Dr. Jassim M Abdullah

[email protected]

Dr. Mohamed-Dahmani Fathallah

Bahrain

Arabian Gulf University-College of Medicine and Medical Sciences

Arabian Gulf University-College of Graduate Studies

Abstract We carried out concordance study to compare the performance of three different human identification commercial kits: the Life Technologies AmpFℓSTR® NGMSElectTM ,the Promega PowerPlex® ESI17 and the ESSplex SE plus (Qiagen) all three based on the typing of the same 17 loci short tandem repeat (STR). The study started with collecting blood from 343 unrelated Kuwaitis preparing the genomic DNA and analyzing the variation of the 17 STRs. The concordance was observed for 90.67% (9952 out of 10976) of the compared STR loci. In addition to concordance studies, allele frequencies and statistical forensic parameters were determined and used to evaluate suitability and robustness of the new kit for forensic genetic analysis. The combined matching probability (MP) score for the 16 loci was 0.0565625. Eleven genotyping discrepancies due to a dropout allele was observed at the D2S1338, D18S51, D19S433, D21S11, TH01 and SE33 locus for NGMSElectTM, on the other hand; twenty-seven genotyping was observed dropout at the D22S1045, D2S1338, TH01,FGA and SE33 locus for PowerPlex® ESI 17 kit. As a result, the new kit is a valuable forensic tool and is suitable to

extend the Kuwait population genetic data obtained with well-established polymerase chain reaction (PCR) multiplex-kits of the PowerPlex® ESI17 kit (Promega) and the AmpFℓSTR® NGMSElectTM (Life Technologies - LT). Keywords: DNA Profile, STR, NGMSElectTM, PowerPlex® ESI 17 Paper reference: SPS1168-14 Expression in Pichia pastoris of engineered soluble human IGg Fc upper-hinge isoforms with improved binding to Fcγ Receptors variants *Dr. Dana Ashoor

[email protected]

Bahrain Arabian Gulf University-College of Graduate Studies

Dr. Sonia Bourguiba-Hachemi Bahrain Arabian Gulf University-College of Graduate Studies Dr. Mohamed-Dahmani Fathallah Bahrain Arabian Gulf University-College of Graduate Studies Abstract The interaction between IgG antibodies and FcγRs (CD16a and CD32a) triggers vast immune responses including antibody-dependent cell mediated cytotoxicity (ADCC), antibody neutralization, phagocytosis, inflammation and even tissue injury in case of autoimmune diseases. Studies showed that the length and flexibility of the antibody hinge region strongly influence the stability of the immune complex. In this study we designed 10 different upperhinge mutated isoforms of human IgG1 Fc region. These isoforms along with the extracellular expression vector pPICZαA downstream of the Saccharomyces cerevisiae Alpha factor signal sequence and in phase with a c-myc epitope and an additional 6X Histidine (His) tag on the 3`end. The engineered plasmids were introduced in P. pastoris by electroporation and the recombinant clones selected on increasing concentrations of zeocin. Protein expression was under the control of tightly regulated alcohol oxidase 1 (AOX1) promoter and induced by different concentrations of methanol in an EnPresso® Yeast Defined Tablet Medium (BioSilta). The recombinant proteins were expressed as soluble proteins in the supernatant. The supernatants were collected and the proteins were purified by Ni+2 HiTrap Chelating HP columns and concentrated by Millipore Amicon ultra centrifugal filters. SDS-PAGE and western blot analysis confirmed the production of recombinant proteins with a molecular mass of ~45 affinities of each produced recombinant proteins are being measured using the “Biacore” Surface Plasmon Resonance “SPR” technology. Keywords: FcγRs, IgG1, ADCC, expression, Pichia, Biacore

Paper reference: SPS1163-14 TRANSCRIPTOME PROFILING OF HEAVY METALS TOLERANCE RELATED GENES Dr. Shameem Raja Dr. Asif Ali Khan

[email protected] University of Agriculture Faisalabad Pakistan University of Agriculture Faisalabad Pakistan

Dr. Masooma Naseer Cheema

University of Agriculture Faisalabad Pakistan

Abstract Water scarcity is one of the main constraints for crop production in many countries. Consequently, wastewater is often used for irrigation, especially in peri urban areas. One of the drawbacks in applying waste water is heavy metal pollution in water and soil along with their entrance in food chain. The studies were conducted to assess the main molecular mechanisms governing heavy metals tolerance in tomato, particularly in relation to the role of heat shock proteins and metallothionin (M. thio). Although the biochemical and molecular mechanisms involved in heavy metals tolerance are not well understood however the knowledge of the molecular mechanism for heavy metals tolerance would help plant breeders to develop low metal-accumulators genotypes of crops. This study found that the transcript accumulation of HSPs and M. thio genes increased many fold higher in tomato accessions i.e. PB-017906 and CLN-2418A (low metal-accumulators) and Riograndi (non-low metal-accumulators) under high concentrations of lead (Pb) and chromium (Cr) metals stress compared to control conditions. However, the expression of these genes was higher in low metal-accumulators genotypes than in high metalaccumulators ones. Transcriptome analysis of heavy metal tolerance genes i.e. HSP and M. Thio showed that tomatoes respond to high concentrations of heavy metals through increased transcription of the HSP and M. Thio genes. It was observed that under Pb and Cr stress HSP and M. Thio protein transcripts accumulated to levels many times higher in roots and leaves of control plants, reducing protein damage from heavy metals and sustaining cellular homeostasis.

Paper reference: SPS1160-14 DNA testing for meat adulteration raises a manufacturing process-related halal issue. *Dr. Sonia Bourguiba-Hachemi

Dr. Mohamed-Dahmani Fathallah

[email protected]

Bahrain

Arabian Gulf UniversityCollege of Graduate Studies

Bahrain Arabian Gulf University-College of Graduate Studies

Abstract In the aftermath of the globalized horse-meat scandal, control of processed meat products became a must do in all countries and particularly in the Arabian Gulf countries that highly rely on imported goods . We performed DNA testing for meat adulteration in 105 samples of imported raw and processed food products marketed in the Arabian Gulf region. We used the highly sensitive real time PCR technique and accredited ISO/IEC17025 commercial kits for horse and pork species detection. Our results showed the presence of horse and pork DNA in respectively 7% and 26% of tested samples. Of the pork-DNA-positive samples, few revealed a “CT” value higher than the 1% in the standard pork-in-beef mix reference, while most of these products showed traces of pork )T and another at 3' c. 1355 G>A alterations have been found for NKX 2.5 gene. Background Complex developmental sequence and genes regulation of heart is in exploration since last two decades with several cardiac manifestation genes identified Some genes are associated with specific defects some genes are part of pathways that are active in cardiogenesis The homeobox gene NKX2 is a precursor and also interacts with GATA4 and MYH6 Mutations of all these genes are reported with both ASD and VSD In addition NKX2 is associated with defects in the electrical conduction of the heart This study reveals the non-coding mutations in NKX 2.5 gene Materials and Methods This is a case - control study a cohort of patients TOF ( 149), PDA (50 ), DTGA ( 26) and controls (140 ) healthy unrelated individuals All patients were sporadic and non syndromic The study after formal approval recruited patients from NICVD in three years A detailed family history was taken to elucidate the genetic and environmental factors Pediatric cardiologist confirmed the diagnosis on the basis of all standard testing like chest X - ray CBC ECG ECHO cardiac catherization reports etc DNA extraction and sequencing was done. Results

Untranslated regions (UTRs)/ non coding regions may have important regulatory roles in gene expression process as these can regulate mRNA function and influence gene function. Two mutations in UTR, one at 5’ c. 145 C>T and another at 3' c. 1355 G>A alterations have been found for NKX 2.5 gene. Conclusion NKX 2.5 mutation in coding region is infrequent in our population while two novel mutations have been noticed in untranslated regions of this gene, which play potential role in heart development. NKX 2.5, 5'UTR mutation reporting first time in a PDA patient.

Paper reference: SPS1125-14 Differential expression levels of phase I & II enzymes, cell proliferation and apoptosis markers between histologically non-malignant and grade III ovarian tumour tissues: Key markers for early stage ovarian cancer detection *Dr. Nagini Siddavaram

[email protected]

India

Annamalai University

Dr. Kavisa Ghosh

[email protected]

India

Annamalai University

Abstract Ovarian cancers are predominantly hormone dependent. The aim of the present work was to evaluate the differential expression levels of phase I ( cytochrome P450 total and the CYP isoforms CYP1A1 CYP1A2 and CYP2B ) and phase II ( GST and QR ) xenobiotic - metabolizing enzymes which are involved in estrogen metabolism as well as of markers reflecting cell proliferation ( PCNA and cyclin D1 ) and apoptosis ( Bcl2 and Bax ) in histologically non malignant and grade III ovarian tumours. Eighteen histologically non - malignant and grade III epithelial ovarian tumours and their corresponding blood samples were used for biochemical western blot and RT - PCR analysis We found an increase in levels of phase I enzymes in ovarian tumour tissues which were not compensated by a corresponding increase in phase II enzymes. The expression levels of CYPIB1 protein was found to be significantly high in tumour tissues and patient blood when compared to CYP1A1 protein expression. These changes reflect altered estrogen metabolism resulting in CYP1B1 mediated metabolism of E2 and formation of - OHE2 a toxicologically active metabolite of estradiol. No expression of CYP1B1 was found in blood of control samples Analysis of Bcl - family members revealed down - regulation of Bax and up regulation of Bcl2 in tumour tissues indicating apoptosis evasion in tumour cells. This was also associated with increased levels of cyclin D1 and PCNA in tumour tissues. These changes reflect increased overexpression of cyclin D1 and up - regulation of PCNA and Bcl2 by E2 - dependent enhancer activity through ERE The present study results provided substantial proof that an imbalance in phase I and phase II enzymes coupled with an imbalance in cell proliferation and apoptosis are important contributors for the development of epithelial ovarian tumours. Background We hypothesised that correlating the levels of phase I and phase II biotransformation enzymes involved in estrogen metabolism and correlating it with cell proliferation and apoptosis evasion

would provide key hallmark traits for ovarian malignancy and key markers for early detection of ovarian tumours. Materials and Methods The present study evaluates the differential expression levels of phase I (cytochrome P450 total and the CYP isoforms CYP1A1 CYP1A2 and CYP2B ) and phase II (GST and QR ) xenobiotic metabolizing enzymes as well as expression of markers reflecting cell proliferation (PCNA and cyclin D1) and apoptosis ( Bcl2 and Bax ) in patients with epithelial ovarian cancer Eighteen histologically non - malignant and grade III ovarian tumours and their corresponding blood samples were used for biochemical western and RT - PCR analysis. Results The present study results provided substantial proof that an imbalance in phase I and phase II enzymes, coupled with an imbalance in cell proliferation and apoptosis, are important contributors for the development of epithelial ovarian cancer and are key markers for early detection of ovarian tumours. Conclusion The results of the present study demonstrate that the increase in phase I enzymes in ovarian tumour tissues are not compensated by a corresponding increase in phase II enzymes, indicating insufficient detoxification in the face of increased carcinogen activation. This could lead to generation of toxic electrophiles and ROS that can cause DNA damage and accumulation of mutations. Excessive cell proliferation, as seen by increased expression of PCNA and cyclin D1 accompanied by apoptosis evasion, as evidenced by an increase in Bcl2/Bax ratio substantiate these observations.

Paper reference: SPS1121-14 Cytogenetic and Cytotoxic Effects of Rubia cordifolia Alkaloids on Mice Cells. *Dr. Sallam Hasan Abdallah

[email protected]

Jordan

Jordan ubiversity

Abstract Rubia cordifolia ((‫ عروق الصباغين‬,‫ الفوه‬which belongs to Rubiacea family, is an important component of the ayurvedic system of medicine; it has a wide range of pharmacological properties, due to its ability to produce a various range of structurally novel bioactive molecules (natural products), which have preventive and curative properties for human diseases. In the present study R. cordifolia alkaloids was investigated for its suspected effects on the mitotic activity of Swiss mice bone marrow cells, different concentration of R. cordifolia alkaloids was used at different periods of time. The results showed a notable decrease in the mitoic index at all periods of time, and almost in all concentrations compared to the control. Moreover; R. cordifolia alkaloids induced a wide range of chromosomal aberrations, these include distributed, sticky, ring and break chromosomes. The percentage of these aberrations increased as concentration and period of time increase. Furthermore; cytotoxicity effects have been measured in a high degree of precision using MTT assay, which detect the living but not dead cells. The results showed the proliferation index increase up to a limit of exposure time, and then a remarkable decrease occurred.

Since the use of natural products, its therapeutic properties led to the development of numerous pharmaceutical products with high efficacy and low side effects. Our observations suggest that R. cordifolia alkaloids have a greater potential of anti-proliferative activity, at high doses and long duration, but it could be considered safe at low doses.

Paper reference: SPS1116-14 Differential expression levels of phase I & II enzymes, cell proliferation and apoptosis markers between histologically non-malignant and grade III ovarian tumour tissues: Key markers for early stage ovarian cancer detection *Dr. Kavisa Ghosh

[email protected]

India

Annamalai University

Dr. Nagini Siddavaram

[email protected]

India

Annamalai University

Abstract Ovarian cancers are predominantly hormone dependent. The aim of the present work was to evaluate the differential expression levels of phase I ( cytochrome P450 total and the CYP isoforms CYP1A1 CYP1A2 and CYP2B ) and phase II ( GST and QR ) xenobiotic - metabolizing enzymes which are involved in estrogen metabolism as well as of markers reflecting cell proliferation ( PCNA and cyclin D1 ) and apoptosis ( Bcl2 and Bax ) in histologically non malignant and grade III ovarian tumours. Eighteen histologically non - malignant and grade III epithelial ovarian tumours and their corresponding blood samples were used for biochemical western blot and RT - PCR analysis We found an increase in levels of phase I enzymes in ovarian tumour tissues which were not compensated by a corresponding increase in phase II enzymes. The expression levels of CYPIB1 protein was found to be significantly high in tumour tissues and patient blood when compared to CYP1A1 protein expression. These changes reflect altered estrogen metabolism resulting in CYP1B1 mediated metabolism of E2 and formation of - OHE2 a toxicologically active metabolite of estradiol. No expression of CYP1B1 was found in blood of control samples Analysis of Bcl - family members revealed down - regulation of Bax and up regulation of Bcl2 in tumour tissues indicating apoptosis evasion in tumour cells. This was also associated with increased levels of cyclin D1 and PCNA in tumour tissues. These changes reflect increased overexpression of cyclin D1 and up - regulation of PCNA and Bcl2 by E2 - dependent enhancer activity through ERE The present study results provided substantial proof that an imbalance in phase I and phase II enzymes coupled with an imbalance in cell proliferation and apoptosis are important contributors for the development of epithelial ovarian tumours. Background We hypothesised that correlating the levels of phase I and phase II biotransformation enzymes involved in estrogen metabolism and correlating it with cell proliferation and apoptosis evasion would provide key hallmark traits for ovarian malignancy and key markers for early detection of ovarian tumours. Materials and Methods The present study evaluates the differential expression levels of phase I (cytochrome P450 total and the CYP isoforms CYP1A1 CYP1A2 and CYP2B ) and phase II (GST and QR ) xenobiotic -

metabolizing enzymes as well as expression of markers reflecting cell proliferation (PCNA and cyclin D1) and apoptosis ( Bcl2 and Bax ) in patients with epithelial ovarian cancer Eighteen histologically non - malignant and grade III ovarian tumours and their corresponding blood samples were used for biochemical western and RT - PCR analysis. Results The present study results provided substantial proof that an imbalance in phase I and phase II enzymes, coupled with an imbalance in cell proliferation and apoptosis, are important contributors for the development of epithelial ovarian cancer and are key markers for early detection of ovarian tumours. Conclusion The results of the present study demonstrate that the increase in phase I enzymes in ovarian tumour tissues are not compensated by a corresponding increase in phase II enzymes, indicating insufficient detoxification in the face of increased carcinogen activation. This could lead to generation of toxic electrophiles and ROS that can cause DNA damage and accumulation of mutations. Excessive cell proliferation, as seen by increased expression of PCNA and cyclin D1 accompanied by apoptosis evasion, as evidenced by an increase in Bcl2/Bax ratio substantiate these observations.

Paper reference: SPS1115-14 Diabetic studies in aqueous extract of Musa spp (banana) in Polyinosinic-polycytidylic acid (PolyIC) intoxicated Swiss albino mice *Dr. MUHAMMED MUHSIN. V

[email protected]

India BHARATHIDASAN UNIVERSITYTIRUCHIRAPPALLI

Abstract Banana is the common name for monocarpic flowering plants of the genus Musa, for the species Ensete ventricosum, and for the fruit they produce. The core of the stem part in the banana is believed to be useful in stomach upset and diabetes. Diabetes is usually a lifelong (chronic) disease in which is a high level of sugar in the blood. The aim of this work is to study the Pancreatic properties of aqueous extract of Banana plant stem in mice.the mice diabetics induced by giving Polyinosinic-polycytidylic acid (PolyIC). The animals selected as four groups based on chemical treatment. •First group of mice (n=5) received Polyinosinic-polycytidylic acid (PolyIC) treatment at a dose of 7.5 μg/g body wt. for 8 -14 days and second group as a control. •Second group was orally the aqoeus banana plant stem extract at a dose 50 mg/kg body weight for 15 days. •The third group was given both the plant extract (Orally 15 days)and Polyinosinic-polycytidylic acid (PolyIC) for last 8 days. •The forth group was kept at control. Finally, A portion of the pancreata, thyroid, and salivary glands obtained from the mice was fixed in 10% formalin, paraffin-embedded, and stained with hematoxylin and eosin. Pancreatic

sections were microscopically examined for the presence of insulitis helps to predicts the cells are functioned or not.

Paper reference: SPS1107-14 QUANTIFICATION AND DETERMINATION OF ALPHA KETO ACIDS IN DIABETIC PATIENT SERUM SAMPLES USING HPLC TECHNIQUE *Dr. Khalida Perveen Mahar Dr. Mohammad Yar Khuhawar

[email protected] Pakistan

Pakistan

1Shah Abdul Latif Universirty

Institute of Advance Research Studies in Chemical Sciences

Abstract A new reversed phase high performance liquid chromatographic (RPHPLC) method is developed and validated for the determination of seven alpha keto acids pyruvic acid, oxobutyric acid, 3methyl2, oxobutyric acid, 2-oxoglutaric acid, 3-methyl 2oxovaleric acid, 4-methyl 2,oxovaleric acid and phenyl pyruvic acid using 1, 2-propylenediamine (PDA) as derivatizing reagent in the diabetic patients blood samples. The method employs a Zorbax 300 SB6 C18 (4.6 ×150 mm) column with a mobile phase comprised of a methanol / water / acetonitrile (40:58:2, v/v/v) at a flow rate of 1 mL/min, at 25°C. The decetion is performed with photodiode array (PDA) detection set at 210-400 nm. The chromatographic achieved in less than 14 min for seven alpha keto acids. The linearity is established over the concentration range of 0.160 g/mL (r2 >0.99) in each case. The mean RSD values for precision studies are ≤ 3%. The recoveries of keto acids are ranged between 99.5% and 99.8%. The limits of detection and quantitation are determined within the range of 0.040.13 g/mL and 0.120.39 g/mL, respectively Background The work reports the development of new reversed phase high performance liquid chromatographic (RPHPLC) method for the analysis α-keto acids using 1, 2-propylenediamine as derivatizing reagent from the diabetic serum samples. α-keto acids has clinical importance, because these are intermediates in a number of biochemical processes. Materials and Methods Seven α- keto acids ( ) pyruvic acid ( PYR ), ( ) - oxobutyric acid ( KB ), ( ) - oxoglutaric acid ( KG ) ( ) - methyl - - oxobutyric acid ( MKBA ), ( ) - methyl - - oxovaleric acid ( K3MVA ), ( ) - methyl - oxovaleric acid ( K4MVA ), ( ) phenyl pyruvic acid ( PPY ) were derivatized with ( PDA ) at pH and separated from HPLC column Zorbax C – and the detection is performed with photodiode array ( PDA ) set Results The linearity is established over the concentration range of g mL ( r2 > ) in each case The mean RSD values for precision studies are ≤ %. The recoveries of keto acids are ranged between % and %. The limits of detection and quantitation are determined within the range of g mL and g mL The chromatographic achieved in less than min for seven alpha keto acids respectively The method was applied for determination of α- keto acids from a pharmaceutical preparation human serum and of healthy volunteers

Conclusion The method reports quick and simple isocratic HPLC elution method for the separation and determination of α-keto acids for possible clinical analysis from the serum samples.

Paper reference: SPS1103-14 Isolation and identification of Archanobacterium pyogenes (Actinomyces pyogenes) from Arabian gazelles *Dr. Mohammed Alsaggaf

[email protected]

Saudi Arabia

Saudi Wildlife Authority

Abstract Archanobacterium pyogene ( Actinomyces pyogene ) is an opportunistic pathogen of economically important livestock such as dairy beef cattle and gazelles It is also a common inhabitant of the mucous membranes of these animals This study was aimed to investigate the epidemiology of A pyogenes in the infected Arabian gazelles kept at King Khalid Wildlife Research Center at Thumamah km north of Riyadh Saudi Arabia and the relationship between symptoms and infection by A pyogenes Samples were collected from pleural fluid of infected gazelles and from upper respiratory tract of Background Archanobacterium pyogene ( Actinomyces pyogene ) is an opportunistic pathogen of economically important livestock such as dairy beef cattle and gazelles It is also a common inhabitant of the mucous membranes of these animals This study was aimed to investigate the epidemiology of A pyogenes in the infected Arabian gazelles kept at King Khalid Wildlife Research Center at Thumamah km north of Riyadh Saudi Arabia and the relationship between symptoms and infection by A pyogenes Samples were collected from pleural fluid of infected gazelles and from upper respiratory tract of Materials and Methods MATERIALS AND METHODS Materials used include captive healthy gazelles at King Khalid Wildlife Research Center breeding pens infected gazelles pleural fluid gazelles syringes and transport media as well as specimens of upper respiratory tract of healthy gazelles Idmi and Reem gazelles Reagents and experimental instruments sterile saline % sheep blood agar medium and Brain heart infusion ( BHI ) agar media sterile loop % CO2 Trypticase Soy Broth medium ( TSB ), % fetal bovine serum % ( v v ) sterile glycerol API CORYNE kit ( Biom é rieux France Results Archanobacterium pyogene ( Actinomyces pyogene ) is an opportunistic pathogen of economically important livestock such as dairy beef cattle and gazelles It is also a common inhabitant of the mucous membranes of these animals This study was aimed to investigate the epidemiology of A pyogenes in the infected Arabian gazelles kept at King Khalid Wildlife Research Center at Thumamah km north of Riyadh Saudi Arabia and the relationship between symptoms and infection by A pyogenes Samples were collected from pleural fluid of infected gazelles and from upper respiratory tract of

Conclusion Archanobacterium pyogene ( Actinomyces pyogene ) is an opportunistic pathogen of economically important livestock such as dairy beef cattle and gazelles It is also a common inhabitant of the mucous membranes of these animals This study was aimed to investigate the epidemiology of A pyogenes in the infected Arabian gazelles kept at King Khalid Wildlife Research Center at Thumamah km north of Riyadh Saudi Arabia and the relationship between symptoms and infection by A pyogenes Samples were collected from pleural fluid of infected gazelles and from upper respiratory tract of

Paper reference: SPS1102-14 A SIMPLE METHOD FOR DETECTION OF KRAS AND BRAF HOTSPOTS MUTATIONS IN PATIENTS WITH COLORECTAL CANCER *Dr. Hajar JADDA

[email protected] Morocco

Dr. Elmostafa EL FAHIME

Morocco

Dr. Fouad KETTANI Dr. Hicham BELLAOUI

Morocco Morocco

Faculty of Sciences Mohammed V University Agdal, Rabat,

Technical Support Unit for Scientific Research, National Center for Scientific and Technological Research, Rabat, Center of Pathology Nations Unies, Rabat, Faculty of Sciences Mohammes V University Agdal, Rabat,

Abstract Objective: Accurate mutation detection assays for KRAS and BRAF genes in colorectal cancer are strongly needed. We describe a simple and reliable technique for determination of KRAS and BRAF mutational status, and we estimate the KRAS and BRAF mutations frequency in Moroccan patients with colorectal cancer. Methods: Forty-seven samples from patients with metastasic colorectal adenocarcinomas were studied for BRAF exon 15 and KRAS codons 12 and 13 mutations. Tumor tissue was removed by manual macrodissection from formalin-fixed paraffin-embedded tissues specimens. After DNA extraction, conventional PCR was performed and the DNA was analyzed by direct sequencing. Results: KRAS codon 12 or 13 mutations were present in 51% of patients. Gly12Val mutation was present in 21% of all patients, Gly12Asp in 15%, Gly13Asp in 6%, Gly12Arg in 4%, Gly12Cys in 2% and Gly12Ala in 2%. No BRAF mutation was detected. Conclusion: Our data suggest that KRAS mutations are more frequent than BRAF mutations in Moroccan patients with colorectal carcinomas. To our knowledge, we are the first to report such a high proportion (more than 50%) of potentially non responsive patients for the antiEGFR treatment in Morocco. These results show that the method we used was accurate, costeffective and time-efficient.

Paper reference: SPS1101-14 Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease *Dr. TANVEER MAJEED

[email protected] Pakistan

NIBGE, Faisalabad, PAKISTAN

Abstract A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant.Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market. Background Proteases are hydrolytic enzymes that degrade proteins into constituent peptides and amino acids These enzymes have wide industrial applications spanning detergents food leather pharmaceuticals and bioremediation We report the first expression and characterization of a recombinant C proteolyticus protease The enzyme is demonstrated to be a serine protease with an alkaline pH optimum and functions at an elevated temperature (60 ° C ). The protease also has the desirable property of retaining high activity in the presence of a wide variety of surfactants. Materials and Methods Got the Bacterial Strains, Plasmid, did Vector Construction and Expressed gene in in B. subtilis system. Optimized assay for protease activity and determined the optimum temperature, pH for activity. I checked thermal stability and effect of different surfactants and metal ions.All additives were used at a final concentration of 5 mM. All incubations occurred at pH 9. After pre-incubations, casein was added to all the enzymes, the reactions proceeded at 60°C, and residual protease activities were assayed. Results A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10). In addition, the enzyme had an elevated optimum temperature (60°C). The protease was also stable in the presence of many surfactants and oxidant.

Conclusion This is the first report of the biochemical characterization of a recombinant protease from the thermophilic C proteolyticus The enzyme was demonstrated to be an alkaline serine protease that was active at elevated temperatures and resistant to many surfactants thus indicating potential utility of this enzyme in detergent applications In addition the new protein sequence of this enzyme will be of great value in the continued efforts to develop protease activity improvements.

Paper reference: SPS1100-14 Emerging of Plasmid-Mediated Quinolone Resistance Associated with the qnr Genes in clinical isolates of Shigella spp. in Iraq

*Dr. Thanaa Rasheed Abdulrahman

[email protected] Iraq

Al-nahrain medical college

Abstract Although quinolone resistance results mostly from chromosomal mutations in Enterobacteriaceae, it may also be mediated by plasmid-encoded qnr determinants. Shigella harbouring the novel qnr plasmid-mediated mechanism of quinolone resistance has been described worldwide.This study aimed to understand the distribution of serogroup of Shigella spp and to investigate the plasmid mediated quinolone – resistant (qnr) genes in clinical isolates of Shigella spp. resistant to quinolone in Iraq. We collected 59 clinical isolates of Shigella spp. between (1 June 2010 to 31 May 2011) from two hospitals in Baghdad. Susceptibility tests were performed using disk diffusin test and MIC.The isolates were screened for the plasmid-mediated quinolone resistance genes of qnrA, qnrB, and qnrS by Multiplex PCR. Results showed that the isolation rate of Shigella spp. was (14%).The highest percentage was Sh.flexneri (54.2%) followed by Sh. sonnei (37.3%) then Sh. dysenteriae (8.5%). Antimicrobial susceptibility tests revealed that (54.23%, 32/59) and (49.2%, 29/59) of both Sh.flexneri and Sh. sonnei were resistant to Nalidixic acid and Ciprofloxacin respectively. The MIC value of resistant isolates of Sh. flexneri and Sh. sonnei ranged between (2-64 μg/ml) and (32- 512 μg/ml) for Cip and NA respectively. Multiplex PCR amplification of plasmid-borne qnr A,qnrB,qnrS genes revealed that, the overall percentage of qnr-genes were (52.94%) distributed as (29.4%) qnr A, (20.6%) qnr S and (2.94%) qnr B detected alone or in combination. The genes were identified in (44.1%, 15/34) of quinolone resistant Shigella isolates.This is the first report detected the qnr genes among Shigella isolates resistant to fluoroquinolone in Iraq which is indicated that plasmid-mediated quinolone resistance has emerged in Iraqi patients.

Paper reference: SPS1099-14 Potential protective role of a novel antioxidant in Streptozotocin-induced diabetic rats under oxidative stress of γ-irradiation *Dr. Enas Mahmoud Moustafa

[email protected]

Egypt

Radiation Biology Department, National Centre for Radiation Research, and Technology, Atomic Energy Authority.

Abstract Potential protective role of a novel antioxidant in Streptozotocin-induced diabetic rats under oxidative stress of γ-irradiation. EMAN NOAMAN1, IHSAN S. HEDAYAT1, and ENAS M.MOUSTAFA1 1- Radiation Biology Department, National Centre for Radiation Research, and Technology, Atomic Energy Authority, Egypt. ABSTRACT The present study was designed to illustrate the protective and chemopreventive effects of the synthetic stobadine compound (30mg/kg) in male rats exposed to fractionated doses of γ-irradiation (2Gy/day after day up to 6Gy) and /or subjected to a single dose of

streptozotocin (45mg/kg body weight) once after 24 hours fasting. The protective effect of such treatment was evaluated by measuring the activities of the antioxidant defense system including reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT), as well as plasma level of malondialdehyde (MDA). In addition, serum glucose, plasma insulin, aldose reductase (AR), sorbitol dehydrogenase activities (SDH), and lipid profile, which are biomarkers of diabetes mellitus. The results revealed that stobadine was able to scavenge hydroxyl, peroxyl, and alkoxyl radicals, to quench singlet oxygen, and to preserve oxidation of SH groups by one electron donation. These effects originated from its ability to form a stable nitrogencentered radical on indole nitrogen. Consequently, it was able to diminish lipid peroxidation and the activity of antioxidant enzymes impairment under oxidative stress. Stobadine improved animal survival rate and recovery, the pyridoindole stobadine was found to exert antioxidant activity and thus possesses the potential to protect various tissues against oxidative stress. Background The present study was designed to illustrate the protective and chemopreventive effects of the synthetic stobadine compound Materials and Methods Chemopreventive effects of the synthetic stobadine compound (30mg/kg) in male rats exposed to fractionated doses of γ-irradiation (2Gy/day after day up to 6Gy) and /or subjected to a single dose of streptozotocin (45mg/kg body weight) once after 24 hours fasting. Results The protective effect of such treatment was evaluated by measuring the activities of the antioxidant defense system including reduced glutathione ( GSH ), glutathione peroxidase ( GPx ), glutathione - S - transferase ( GST ), glutathione reductase ( GR ), superoxide dismutase ( SOD ), catalase ( CAT ), as well as plasma level of malondialdehyde ( MDA ). In addition serum glucose plasma insulin aldose reductase ( AR ), sorbitol dehydrogenase activities ( SDH ), and lipid profile which are biomarkers of diabetes mellitus. Conclusion Consequently it was able to diminish lipid peroxidation and the activity of antioxidant enzymes impairment under oxidative stress Stobadine improved animal survival rate and recovery the pyridoindole stobadine was found to exert antioxidant activity and thus possesses the potential to protect various tissues against oxidative stress The results revealed that stobadine was able to scavenge hydroxyl peroxyl and alkoxyl radicals to quench singlet oxygen and to preserve oxidation of SH groups by one electron donation These effects originated from its ability to form a stable nitrogen. Paper reference: SPS1097-14 microRNA control of NBS-LRR based immunity in cucumis sativus *Dr. Sarwat Zahoor

[email protected] Pakistan University of Agriculture,Faisalabad

Abstract Cucumber belongs to the family cucurbitaceae and is the fourth widely cultivated vegetable of the world. The availability of genome sequences and their annotation has made the cucumber a model plant of cucurbitaceae for a variety of studies. This study was designed to understand the interactome of miRNAs and resistance genes, which provoke the immune response against biotic stresses. Plants respond against biological enemies by activating their innate immune system. The plant genome contains large number of nucleotide binding sites-leucine rich repeats proteins encoded by R-genes; which recognize specific pathogen effectors and prompt immune response. Another component of the immune system is a class of miRNAs involved in the modulation of NBS-LRR genes expression. Cucumber miRNAs has been predicted to target NBS-LRR genes transcripts. The objective of the study was to determine the role of miRNAs in reprogramming these genes in the resistant cucumber genotypes upon infection by pathogens under controlled conditions. This was revealing an important aspect of immune system in cucumber. For this purpose commercially cultivated genotypes will be collected and grown in containment. Plants were inoculated with cucumber mosaic virus (CMV) and resistant and susceptible genotypes were identified. Transcriptome profiling of various classes of NBS-LRR genes were carried out in the identified resistant and susceptible genotypes by qPCR analysis to select a specific NBS-LRR. Plasmid vectors for transient transformation was constructed in which miRNAs target region of NBS-LRR was cloned under reporter gene. The synthesized miRNAs was used to study its interaction with NBS-LRR genes in transformed cucumber plants with and without infection. Transient coexpression analysis was carried out in infected and noninfected model plant (tobacco) using same constructs to further confirm their interaction. Paper reference: SPS1095-14 The Exposure Effect of Water Pipe Smoke (WPS) on the Total Count leukocyte, Mitotic Index, Micronucleus Formation and Chromosome Aberration in Albino Male Mice *Dr. Mohammad Mahmoud Al-Halbosiy

[email protected]

Iraq

Biotechnology Research center, Al Nahrain University

Dr. Rakad Mohamad Al-Jumaily

Iraq

University of Baghdad

Dr. Fadhel mohamad Lafta

Iraq

University of Baghdad

Abstract The present study was carried out with the aim to evaluate the hematological and cytogenetic effects of water pipe smoke (WPS) in albino male mice. The investigated parameters were total count of leukocytes (TLC), mitotic index (MI), micronucleus (MN) formation and chromosomal aberrations. In this research study, mice were exposed to WPS using a special inhalation glass chamber (whole body exposure). Mice exposed daily to 100 puffs of WPS on the bases of (1h exposure per day). The exposure experiment continued daily for the periods of 3, 5, 7 weeks consequently. The results revealed that the TLC significantly decreased in the second and third treatment (5930, 4120 cell/ cu. mm. blood) respectively, while the MI decreased in all 3

treatment (after 3, 5, 7 weeks) respectively. But both the MN cells and chromosomal aberration they remain the same as the control in the first treatment and then began to be higher than the control in the second and third treatment. The results indicated that the WPS has cytotoxic and mutagenic effects, as judged by the finding of MN cells and chromosomal aberration assays in the three types of treatment.

Paper reference: SPS1091-14 Allelopathic Effect of Different Weed Extracts on the Growth and Germination of Oryza sativa. L *Dr. Farooq Ahmad

[email protected] Pakistan

University of the Punjab

Abstract The germination and growth of different varieties of rice were analyzed after treating with the distilled water (control) and aqueous weed extract of different native weeds of upland areas of Pakistan; Cyperus rotundus, Trianthema aportulacastrum, Convolvulus arvensis and Parthenium hysterophorus. The whole weed plants from different rice fields were collected, shade dried and crushed. The crushed powder was soaked in the distilled water for 24 hrs, filtered and diluted in distilled water by making the final volume up to 100 ml (10% w/v). T1 was prepared by mixing equal amounts of Cyperus rotundus and Parthenium hysterophorus while T2 was prepared by mixing equal amounts of Convolvulus arvensis and Trianthema aportulacastrum. For germination test, germination plate method of seed germination was used and blotter paper was moistened with different combinations of weed extract and for control distilled water was used. Observations were recorded at each 24 hrs interval for up to 8 days. After germination, seedlings were allowed to grow for a week and then their radical and plumule length as well as fresh and dry weight was recorded. Results showed the more drastic effects of T2 on rice germination and seedling growth as compared to T1. Germination rate of rice was most severely affected by the T2 with the exception of IRRI-6 which was affected by T1 more adversely. These studies can be applied to encourage the weed removal practices to increase the rice production and sowing of resistant varieties, where the intensity of weeds would be high.

Paper reference: SPS1090-14 Metabolic Pathway Analysis Approach: Identification of Novel Therapeutic Target against Methicillin Resistant Staphylococcus aureus *Dr. Reaz Uddin

[email protected]

Pakistan

Dr. Panjwani Center for Molecular Medicine, University of Karachi

Abstract Methicillin Resistant S. aureus (MRSA) is one of the MDR pathogen notorious for its widespread infection around the world. The high resistance acquired by MRSA needs a serious concern and

efforts should be carried out for the discovery of better therapeutics. With this aim, we designed a computational biology approach to compare the metabolic pathways of the pathogen (i.e. MRSA) with the human host (i.e. Homo sapiens). We identified several metabolic pathways unique to MRSA (i.e. absent in human host). Furthermore, a subtractive genomics approach was applied in which proteins were retrieved from only the unique metabolic pathways. Subsequently, proteins of unique MRSA pathways were compared with the host proteins. As a result, we have shortlisted few unique and essential proteins that could act as drug targets against MRSA. We further assessed the druggability potential of the shortlisted targets by comparing them with the Drugbank database. The identified drug targets could be useful for an effective drug discovery phase. We also searched the sequences of unique yet essential enzymes from MRSA in Protein Databank (PDB). We shortlisted 12 enzymes for which there were no corresponding deposition in PDB, reflecting that their crystal structures are not solved yet! We selected Glutamate synthase out of those 12 enzymes owing to its participation in significant metabolic pathways of the pathogen e.g., Nitrogen metabolism. Due to the unavailability of any crystal structure of Glutamate synthase, we generate its 3D structure by Homology modeling. The active site of the Glutamate synthase was identified by the ParallelProBiS algorithm. It was concluded that the comparative metabolic analysis using Computational Biology methods provide an effective approach for the identification of novel antibiotic targets against MRSA.

Paper reference: SPS1079-14 Comparative evaluation of a multiple- antigen (GlcB, HspX, MPT51 ,Ag 85B and PstS1) based diagnostic protocol versus Polymerase Chain Reaction assays (qRT-PCR and gel-based duplex PCR) for Rapid and Efficient diagnosis of childhood tuberculous meningitis *Dr. Abhishek Mittal

[email protected] India

Faculty Of Medical Sciences,Delhi, India

Abstract Introduction Tuberculosis is a global public health hazard. Tuberculosis Meningitis (TBM) is the most severe form of extrapulmonary tuberculosis. Prompt diagnosis is crucial for successful TBM management ; the case fatality rate for untreated TBM is almost 100% and delay in treatment often leads to permanent neurological damage. PCR and ELISA based assays for detecting antigens are fast and reliable but have not yet been amalgamated into routine diagnostics. Thus there is an overall need for improving TB diagnostics by the development of cost - effective and robust tools. Materials & Methods 132 CSF samples collected from pediatric wards were included in the study. An ELISA - based assay was performed for detection of mycobacterial antigens and its efficacy was compared with quantitative real - time PCR and gel - based duplex PCR. All statistical analyses were performed with SPSS. Results GlcB and HspX ELISA had the highest sensitivities of 95% and 93 % respectively with specificities of 96% and 97%. The sensitivity of MPT51, Ag85B and PstS1 ELISA ranged between 90-93% with specificities ranging between 92-96%.

qRT - PCR was the best performing assay with sensitivity and specificity of 97% and 99% respectively. Gel - based duplex PCR had a diagnostic accuracy of 84% vs 98% for the qRT - PCR assay. All tests had area under ROC curve ranging from 0.94-0.99. Conclusion The diagnosis of pediatric TBM is a problem both by conventional microbiological methods and radiological techniques. Present study concludes that in addition to qRT - PCR, GlcB and HspX can serve as potentially useful markers for rapid, user friendly, inexpensive diagnosis of TBM. Since no commercial test is licensed for diagnosis of TBM, present study provides a better insight into development of rapid tests for better TBM diagnosis.

Paper reference: SPS1078-14 Dimensions of Biomedical Decision Dr. Mohamed Helmy El Batanouny

[email protected] Egypt

Emeritus Prof. of General and Vascular Surgery Faculty of Medicine Former head of Department of laser medical and Biological National Institute of laser enhanced Sciences

Abstract Fast progress in biomedical field is accompanied with increasing Ethical problems. This initiated the emergence of great need to solve problems and tackle the moral dimensions of biomedical decisions. This resulted in change of domain of philosophy towards applied physiology and also invited the stress of forming ethical committees in medical and scientific foundations. Importance of values needed in different moral decisions, according to individual situations. The ethical implications in dealing with patients in critical situations will be discussed. Very important are the ethical principles, which control the clinical trials of new medical medicines and surgical interferences are stressed. Scientific research and publications is a vital field raising moral dimensions in practice. Educating concerned personals in in this field in all countries is of utmost importance Paper reference: SPS1071-14 Cryptosporidium infection in calves in Tunisia : molecular characterization of species in a longitudinal study. Dr. Raya Soltane

Dr. karine Guyot

[email protected]

KSA

Faculty of Applied Science, Biology Department, Umm Al-Qura University, Makkah, Saudi Arabia

Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d'Infection et d'Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8402, Université Lille Nord de France

Dr. Eduardo Dei-Cas

Dr. Ali Ayadi

Biologie et Diversité des Pathogènes Eucaryotes Emergents (BDEEP), Centre d'Infection et d'Immunité de Lille (CIIL), Institut Pasteur de Lille, INSERM U1019, CNRS UMR 8402, Université Lille Nord de France Fungal and Parasitic Molecular Biology Laboratory, Faculty of Medicine, University of Sfax, Sfax, Tunisia

Abstract A longitudinal study was undertaken to determine the prevalence of Cryptosporidium in a dairy farm in Sfax, Tunisia. 480 faecal samples were obtained from 30 calves under one month of age. All faecal samples were analysed for Cryptosporidium oocysts by microscopic examination of smears stained by modified Ziehl Neelsen technique. The parasite was detected in 26 calves (86.7 %). Infection was significantly associated with diarrhoea. A molecular characterization, performed in seven calves, confirmed that isolates were C. parvum. This work is the first report on Cryptosporidium in calves in Tunisia. Background In light of the veterinary importance, causation of production losses and its zoonotical potential, more knowledge about the prevalence of the parasite was needed. The objective of the present study was to determine the prevalence of Cryptosporidium in calves in a dairy farm in Tunisia associating molecular characterization of few parasite isolates. Materials and Methods FAECAL SPECIMENS MICROSCOPIC EXAMINATION STATISTICAL ANALYSIS MOLECULAR ANALYSIS FOR GENOTYPING : a fragment of the 18S rRNA gene was amplified by nested PCR. Amplified products were sequenced. In addition to this genotyping technique, DNA amplification and restriction fragment length polymorphism (RFLP) at the Laxer locus were performed. Results The Cryptosporidium overall prevalence was 86.7 % (26/30). The oocyst excretion was first detected five days after birth with a prevalence of 10 %. The percentage of calves infected increased up to 70 % at the age of 13 days and then decreased to reach 10 % at the age of 28 days. The calf’s diarrhoea was associated (P < 10–7) with the Cryptosporidium infection. Sequences were identified as C. parvum. Moreover, all C. parvum were of the L1- subgenotype. Conclusion The present work is the first report on Cryptosporidium occurrence in calves in Tunisia associating molecular characterization of few parasite isolates. Since C. parvum from cattle can infect humans, further studies with genotyping of human and animal isolates are necessary to evaluate the public health significance and give further insights into the epidemiology of the infection in Tunisia.

Paper reference: SPS1069-14 No association between F8 gene mutations and inhibitor development in hemophiliac a patients from Algeria. *Dr. MERIEM ABDI

[email protected] Algeria

Laboratoire de Génétique Moléculaire et Cellulaire, Université des Sciences et de la Technologie d’Oran, Algeria

Abstract Neutralizing inhibitors development toward factor VIII is one of the most challenging complications in the treatment of hemophilia A. Several studies have suggested that genetic factors influence the development of factor VIII inhibitors such as mutations in the factor 8 gene. The aim of the present study was to analyze the relationship between inhibitor development and F8 gene mutations in a sample of hemophiliac a patients from West Algeria. To study the genetic predisposition for inhibitor development, we genotyped 24 hemophiliac patients with and without inhibitors. Inhibitor detection for all patients was performed using the Bethesda assay once every 3 months. A conventional Fisher’s exact test was used for statistical analysis; p-value T (p.R80*) and c.629_630delCT (p.S210*). In exon 9 we identified a frameshift mutation, c.1550_1551delGA (p.R517Ifs*3). Conclusion To date, twelve mutations in LCA5 have been published. Here we present three novel mutations in three unrelated LCA families. Our results suggest that LCA5 mutations are a common cause of LCA in Pakistan.

Paper reference: SPS1050-14 Evaluating Decreased Risk of Atherosclerosis in Down Syndrome Patients *Dr. Ekram Abdel Salam

[email protected]

Dr. Iman Ehsan Abdel Meguid

Dr. Soheir Saad Korraa

Egypt

Egypt

Department of Pediatrics, Faculty of Medicin, Cairo University

Egypt Department of Pediatrics, Faculty of Medicin, Cairo University National Center for Radiation Research and Technology Egyptian Atomic Energy Authority

Abstract Background: Down syndrome (DS) is postulated to be a systemic anti-angiogenesis disease model. The present study was carried out to investigate the biochemical condition in DS that leads to the decreased risk of atherosclerosis phenomena observed in DS patients compared to the general population.

Methods: Plasma stromal derived growth factor (SDF-1), vascular endothelial growth factor (VEGF), homocysteine (Hcy) and methylene tetrahydrofolate reductase (MTHFR) were measured By ELIZA in blood of 62 DS patients vs. 30 controls. Cystathionine beta synthase mRNA relative expression and Receptors for glycation end products mRNA expression (RAGEs mRNA) in circulating lymphocytes and the frequency of circulating endothelial progenitor cells (EPCs), which are involved in repair of blood vessels and whose surface markers expressed over circulating mononuclear cells are identified as CD34, CD133 and kinase domain receptor (KDR) were quantified. Results: Results indicated a significant increase in RAGE mRNA expression (0.9 ± 0.3 vs. 0.7 ± 0.02) and CBS mRNA relative expression (0.56 ± .06 vs. 0.32 ± .02), and plasma H2S (72 ± 8.5 vs. 50.8 ± 4.1) among DS patients compared to controls. MTHFR (5.6 ± 1.2 vs. 7.4 ± 1.1) Hcy (6.1 ± 1.6 vs.7.6 ± 0.4), SDF-1(1950 ± 360 vs. 2170 ± 430) and VEGF (127.9 ± 16.9 vs. 137.5 ± 12.6) were significantly lower among DS compared to controls. A significant decrease was also observed in circulating CD133 (72 ± 4.8 vs. 95 ± 7, p C, p.R177P). Conclusion: Here we introduce two novel mutations in the same gene identified in two LCA families using unique methods. Genetic investigation of LCA families provided an ideal opportunity to identify further patients for phenotype-genotype analysis, to identify new mutations to aid the functional characterisation of the LCA proteins, to molecularly characterise patients for future therapeutic clinical trials and to identify families for future gene identification studies. Background Leber ’ s congenital amaurosis ( LCA ) is the disease name given to cases of severe early onset inherited retinal dystrophy but it is clear that it actually encompasses a group of diseases with different underlying pathologies It is a rare form of retinal dystrophy that present in the first year of life Studies commenced into the molecular genetics of LCA revealed fourteen different LCA genes correlated to % of LCA cases. Determining the molecular defect is becoming increasingly important with the recent gene therapy trials Materials and Methods Two large consanguineous LCA families were ascertained from Pakistan and genomic DNA from family members was isolated using standard procedures For each family DNA samples from affected individuals were pooled and processed using Affymetrix SNP microarrays Regions of autozygosity were identified using IBDfinder software ( http dna leeds ac uk ibdfinder )( Carr et al ). Genotyping was performed using fluorescently labelled microsatellites on an ABI3130xl DNA analyser A shared region of homozygosity was identified among all the affected individuals A potential region of homozygosity at GUCY2D locus. Results SNPchip analysis of pooled affected family members DNA revealed a potential regions of homozygosity around GUCY2D on chromosome ( chr17 - ) in each family The region spanned over Mb and contained SNPS in family while in family it spanned over Mb and contained SNPS

Genotyping confirmed this region of autozygosity which is known to contain the GUCY2D gene Two homozygous GUCY2D highly conserved missense mutations in exon in both families were identified ( c G > C p W194C in Family1 while c G > C p R177P in family2 Conclusion GUCY2D accounts for 8% to 12% of all LCA cases published (Coppieters et al., 2010, Li et al., 2009). Here we introduce two novel mutations in GUCY2D identified in two LCA families using unique methods . Genetic investigation of LCA families provided an ideal opportunity to identify further patients for phenotype-genotype analysis, to identify new mutations to aid the functional characterisation of the LCA proteins, to molecularly characterise patients for future therapeutic clinical trials and to identify families for future gene identification studies. Paper reference: SPS1044-14 CHARACTERIZATION OF A QTL REGION AFFECTING SOMATIC CELL SCORE ON CHROMOSOME BTA4 and BTA26 IN FRIESIAN COWS *Dr. Ayman Fouad Ashour [email protected] Egypt

Dr. Hassan Ghazy El-Awady

Egypt

Dr. Arafa Atia Halawa

Egypt

Dr. Fekry Elsayed Elkeraby

Egypt

Biotechnology Department,Animal Production Research Institute, Agriculture research center

Animal Production Department, Faculty of Agriculture, Kafrelsheikh University Cattle Breeding Research Department,Animal Production Research Institute, Agriculture research center Biotechnology Department,Animal Production Research Institute, Agriculture research center

Abstract This study aimed to confirm previously quantitative trait loci (QTL) affecting somatic cell score (SCS) in dairy cattle on Bos taurus autosomes BTA26 and BTA4 chromosome and how can make selection by markers which linkage with QTL for SCS in the Friesian cattle population in Egypt. A granddaughter design with selective genotyping was implemented that included haf-sibs from 6 sire families of Friesian cows. The animals were genotyped for 5 microsatellites markers on BTA4 and 5 microsatellites markers on BTA26. Heterozygosity on each locus showed wide variation among ten microsatellite markers studied among families. Across 6 families, the most likely QTL positions for SCS on BTA4 and BTA26 were all mapped at 46 cM, close to BMS885 and at 27 cM close to TGLA429-BMS882, respectively. Results for trait SCS, QTL with chromosomewide significance within and across families studied identified the QTL allele substitution effects estimated for each family while fixing the QTL at the most likely position (46 cM) on BTA4 and (27 cM) on BTA26 in 95% confidence interval (CI) QTL position. Sire families found likely have significant QTL effect on SCS. The calculated overall QTL sire effect values across the six families BTA4 and BTA26 (-0.147 ± 0.428) and (0.399 ± 0.370) respectively, indicating decrease SCS among all families studied on BTA4 and increase SCS among all families studied on BTA26. The fact that such genotypes are found in relative high frequencies in Friesian cows may reflect the combined breeding goal that is characterized by selection for SCS to resistance mastitis. The

identification of these markers raises the possibility of overcoming the unfavourable genetic correlation between milk production, SCS and mastitis traits through marker-assisted selection.

Paper reference: SPS1042-14 Targeting viral antigens to CD11c on dendritic cells induces retrovirus-specific T cell responses. 

Dr. Asim Ejaz

[email protected]

Austria

University of Innsbruck

Abstract Dendritic cells (DC) represent the most potent antigen presenting cells and induce efficient cytotoxic T lymphocyte (CTL) responses against viral infections. Targeting antigens (Ag) to receptors on DCs is a promising strategy to enhance antitumor and antiviral immune responses induced by DCs. Here, we investigated the potential of CD11c-specific single-chain fragments (scFv) fused to an immunodominant peptide of Friend retrovirus for induction of virus-specific T cell responses by DCs. In vitro CD11c-specific scFv selectively targeted viral antigens to DCs and thereby significantly improved the activation of virus-specific T cells. In vaccination experiments DCs loaded with viral Ag targeted to CD11c provided improved rejection of FV-derived tumors and efficiently primed virus-specific CTL responses after virus challenge. Since the induction of strong virus-specific T cell responses is critical in viral infections, CD11c targeted protein vaccines might provide means to enhance the cellular immune response to prophylactic or therapeutic levels. Background Dendritic cells are the most potent antigen presenting cells They play a key role in induction of T cell immunity by sampling antigens and presenting them to CD4 and CD8 T cells Thirty years since its identification HIV is still one of most dangerous viral infections Data suggest that a potent T cell response can be a better tool to fight against this infection. We utilized complement receptor to target antigens to DCs which can be presented to specific T cell to induce a potent anti-viral response. Materials and Methods We have used Friend virus (FV), a mouse retrovirus in this study. Single chain antibody fragments (scFv) were generated recombinantly to target antigens to DCs. T cell responses upon targeting were analysed by FACS. Results In this study we generated CD11c - specific scFv ( CD11c - scFv ) fused to the immunodominant region ( IDR ) of FV gag containing a CD8 T cell epitope ( IDRgag ). Using DCs treated with CD11c scFv we detected significantly improved activation of FV - specific CD8 + T cells both in vitro and in vivo FV - specific cytotoxic T lymphocytes ( CTL ) activated by DCs treated with the CD11c scFv - IDRgag construct showed efficient rejection of FV - derived tumor. Conclusion we present first evidence for the improvement of DC vaccine induced retrovirus - specific CD8 + and CD4 + T cell response by targeting viral Ags to CD11c on DCs both in vitro and in vivo Thus CD11c targeted protein vaccines triggering cellular immunity might provide alternatives to

other vaccination strategies or enhance the effectiveness of current strategies Further investigations have to be focused on protein vaccinations targeting viral Ags to CD11c followed by virus - challenge in vivo to elucidate the therapeutical potential of this strategy.

Paper reference: SPS1037-14 MUTAGENIC AND ANTIMUTAGENIC POTENTIAL OF HELIANTHUS ANNUS, LINN FLOWER EXTRACTS *Dr. Faisal Gulzar

[email protected]

Dr. Muhammad Shoaib Akhtar

Department of Pharmacology, Faculty of Pharmacy, University of Sargodha, Sargodha-Pakistan National Institute for biotechnology and Genetic Engineering (NIBGE)

Dr. Qaiser Mahmood Khan

Department of Pharmacology, Faculty of Pharmacy, University of Sargodha, Sargodha-Pakistan

Abstract Background: Innumerable plants have been used since centuries in traditional medicine for treatment of various diseases in men and animals but little information is available regarding their screening of potential risks on DNA. Helianthus annus, Linn is one of such plants; its petals are prescribed for the treatment of bronchial, laryngeal and pulmonary affections, coughs and colds, etc. Aims: Present study was conducted to evaluate the phytochemical constituents of Helianthus annus flower extracts and to screen out its mutagenic and antimutagenic potential by the Ames test. Methods: Aqueous, methanolic, DMSO extracts of Helianthus annus flower were evaluated for the occurrence of antioxidant, flavonoids, glycodies, phenolic content, steroids, reducing agent and tannin. Mutagenicity and antimutagenicity of extracts was analyzed at three doses (25, 50 and 100 µg/plate) by plate incorporation method using Salmonella typhimurium TA 98 and TA 100 strains in presence and absence of rat liver fraction. Results: Flower extracts exerted non-mutagenic effect against both TA-98 and TA-100 strains because number of histidine+ revertants colonies did not increase significantly (p > 0.05) as compared to negative control values with and without rat liver fraction. However, virtually the methanolic extracts of flowers exhibited varying degrees of antimutagenic effect against known and positive mutagens. The inhibitory effect was observed in a dose dependent manner. It is therefore, conceivable that testing flower extracts have strong antimutagenic activity possibly exerted by their flavonoids or phenolic or antioxidants. Conclusion: The present investigation has confirmed the non-mutagenic but reasonably antimutagenic activities of the Helianthus annus extracts, supporting its current use in traditional medicines.

Paper reference: SPS1021-14 Designing universal primers for the isolation of DNA sequences encoding Proanthocyanidins biosynthetic enzymes in Crataegus aronia *Dr. Afnan Said Zuiter

[email protected]

Dr. Jammal Sawwan Dr. Ayed Al Abdallat

Jordan Jordan

Jordan

the jordan university

the jordan university the jordan university

Abstract Background Hawthorn is the common name of all plant species in the genus Crataegus, which belongs to the Rosaceae family. Crataegus are considered useful medicinal plants because of their high content of proanthocyanidins (PAs) and other related compounds. To improve PAs production in Crataegus tissues, the sequences of genes encoding PAs biosynthetic enzymes are required. Findings Different bioinformatics tools, including BLAST, multiple sequence alignment and alignment PCR analysis were used to design primers suitable for the amplification of DNA fragments from 10 candidate genes encoding enzymes involved in PAs biosynthesis in C. aronia. DNA sequencing results proved the utility of the designed primers. The primers were used successfully to amplify DNA fragments of different PAs biosynthesis genes in different Rosaceae plants. Conclusion To the best of our knowledge, this is the first use of the alignment PCR approach to isolate DNA sequences encoding PAs biosynthetic enzymes in Rosaceae plants. Conclusion Genetic information related to different genes encoding PAs biosynthetic enzymes from C aronia plant were obtained Such information can be used to clone the full - length gene sequences from C aronia The information can also be used to improve PAs production in C aronia using genetic engineering and tissue culture systems developed specifically for this plant species The designed primers showed high levels of sequence similarity with their corresponding genes in different Rosaceae plants therefore they could be used to isolate DNA fragments of PAs genes from different Rosacea

Paper reference: SPS1017-14 IMPACT OF CODIUM IYENGARII ON THE GROWTH AND BIOCHEMICAL ATTRIBUTES OF LEAD TREATED LEGUMINOUS SPECIES PHASEOLUS MUNGO AND LENS CULINARIS *Dr. SABA HAIDER

[email protected] Pakistan

Dr. Rafia Azmat

Pakistan

Jinnah University for Women

University of Karachi

Abstract Lead contamination is a serious environmental problem affecting both atmosphere and geosphere ultimately affected the whole ecosystem. All living organisms are adversely effecting by Lead pollution especially plant are significantly agitate by this stress. So in response to detoxify the Lead toxicity several conventional remediation technologies were proposed. In

spite of being efficient, these methods are expensive, time consuming, and environmentally devastating. Recently green technologies have been developed which is economical and environmentally friendly technology. In phytoremediation algae play an important role for detoxification of heavy metal by serving as biosorbent, sea weeds have the ability for absorption of heavy metals and taking up toxic elements from the environment has been recognized for many years. The present study is examined the detoxification of Lead by using the sea weeds (SW)Codium iyengarii and explored the impact of phytoremediation on two important food crops Phaseolus mungo and Lens culinaris. In a greenhouse study, Lead chloride with adverse concentration 250 ppm were used in addition to this various concentration of SW 0, 2,4,6,8 g/kg were applied in the soil as the treatment and 0 was considered as control experimental unit without any treatment. The plant species with this treatment showed the high ability to tolerate Lead toxicity and achieved the usual growth rate. Results illustrate that under this immune developing strategy the tremendous affirmative results were obtained and germination rate in both pulses significantly enhanced and the regression value is 0.753 in Phaseolus mungo and 0.984 in Lens culinaris. Physical features were represented considerable improvement in root, shoot and leave length of both pulses. Biochemical attributes were also showed encouraging outcome. Protein content gradually raise with the increasing the SW concentration, positive relation was observed in between photosynthetic pigments and SW dose in response to adopting this defensive technique in both leguminous species. Background Toxic metals effluents are continuously released into the environment and their removal is a very difficult task due to more expensive treatment So there is vital need to develop less expensive and sustainable strategies for detoxification of Lead Seaweeds have the ability to remove of heavy metals from water and soil. Recently algal research became the main focus point due to their capability to detoxified the heavy metal toxicity specially emphasis on Codium because of its wide distribution Codium iyengarii are abundantly found in Karachi Materials and Methods Codium iyengarii was harvested from the Karachi coastal beach Buleji, after sampling sea weeds were thoroughly washed, dried and then powered. Seed of Phaseolus mungo and Lens culinaris were sterilized. Forty seeds were sown in each pots containing I kg soil with Hoagland based 250 ppm Lead chloride and various dose of sea weeds 0, 2,4,6,8 gm/kg. Seed germination was recorded after 4th day. After passing 15 days plants were harvested. Different agronomic parameters were recorded. Determine the photosynthetic pigments concentration by spectrophotometer by using of Maclachlan& Zalik (1963) method. Results Biosorption of heavy metals using dried algal biomass has been extensively described is known as green technology C.iyengarii was recognized as a imperative biological matter which can be consume for adsorption of Lead ions that can be significantly detoxify the Lead toxicity in Phaseolus mungo and Lens culinaris Results illustrated that Germination rate physical features and biochemical attributes with of Lead stress were increased in response to Codium iyengarii application in both pulses gm of sea weeds dose was showed tremendous results as compared to all level of treatments Conclusion

It was concluded that green algae found to be effective in controlling mobility of Pb ion via surface complexion phenomena Paper reference: SPS1000-14 Mapping of a novel locus for an autosomal recessive form of palmoplantar keratoderma on chromosome 3q27.2-q29 *Dr. Saadullah Khan

[email protected] Pakistan

Quaid-i-Azam University Islamabad

Abstract Palmoplantar keratodermas (PPKs) are a group of highly heterogeneous diseases causing hyperkeratosis of the palms and soles. They can be inherited in an autosomal recessive, dominant, mitochondrial or possibly X-linked recessive fashion. The present study describes clinical and molecular genetic analysis of a consanguineous Pakistani family showing a severe form of PPK inherited in an autosomal recessive manner. To map the gene responsible for an autosomal recessive form of PPK. Human genome scan using polymorphic microsatellite markers was performed to localize the disease gene. Eleven candidate genes, located in the linkage interval, were screened to identify the potential sequence variants. All five affected members of the family showed severe bilateral involvement of palms and soles with minor nail involvement, severe fissuring with bleeding, painful walking, and problems in grasping. Linkage analysis in the family mapped a novel locus for the disease on chromosome 3q27.2-q29. The candidate region flanked by markers D3S1530 and D3S1272 spans 28Æ22 cM, which corresponds to a physical distance of 11Æ63 Mb. The maximum two-point LOD score of 3Æ13 at h = 0Æ00 was obtained with marker D3S2748 along the disease locus. DNA sequence analysis of 11 candidate genes, located in the linkage interval, failed to detect functional sequence variants. A novel locus for an autosomal recessive form of PPK was mapped on chromosome 3q27.2-q29 in a consanguineous Pakistani family. Failure to detect pathogenic sequence variants in the 11 candidate genes suggests either that the variants are located in the regulatory regions of the genes or that another unknown gene, responsible for the disease, is present in the region. Background Palmoplantar keratodermas (PPKs) are a group of highly heterogeneous diseases causing hyperkeratosis of the palms and soles. They can be inherited in an autosomal recessive, dominant, mitochondrial or possibly X-linked recessive fashion. The present study describes clinical and molecular genetic analysis of a consanguineous Pakistani family showing a severe form of PPK inherited in an autosomal recessive manner. Materials and Methods Human genome scan using polymorphic microsatellite markers was performed to localize the disease gene. Eleven candidate genes, located in the linkage interval, were screened to identify the potential sequence variants. Results

All five affected members of the family showed severe bilateral involvement of palms and soles with minor nail involvement severe fissuring with bleeding painful walking and problems in grasping Linkage analysis in the family mapped a novel locus for the disease on chromosome q27 - q29 The candidate region flanked by markers D3S1530 and D3S1272 spans Æ cM which corresponds to a physical distance of Æ Mb The maximum two - point LOD score of Æ at h = Æ was obtained with marker D3S2748 along the disease locus DNA Conclusion A novel locus for an autosomal recessive form of PPK was mapped on chromosome 3q27.2-q29 in a consanguineous Pakistani family. Failure to detect pathogenic sequence variants in the 11 candidate genes suggests either that the variants are located in the regulatory regions of the genes or that another unknown gene, responsible for the disease, is present in the region.

Paper reference: SPS1000-14 Genetic Susceptibility of HCV RNA and its genotypic distribution in the Punjab, Pakistan. Dr. Ghazala Rubi Javaid

[email protected]

Dr. Muhammad Aslamkhan Khan

Agha Khan University Hospital Pakistan

University of Health Sciences Pakistan

Abstract Hepatitis C, a widespread infectious disease targeting circa 130 million people worldwide, is caused by the Hepatitis C Virus (HCV). HCV genome, consisting of 9,600 nucleotides, is fully sequenced. Population specific high variation in HCV genome exists. HCV is classified into 7 different genotypes with several subtypes. Genotypes 1, 2 and 3 are found worldwide. Objective: To find out the ecology and genetics of susceptibility of HCV RNA in various isonym groups of the Punjab population Background Introduction and rationale: Hepatitis C, a widespread infectious disease targeting circa 130 million people worldwide, is caused by the Hepatitis C Virus (HCV). HCV genome, consisting of 9,600 nucleotides, is fully sequenced. Population specific high variation in HCV genome exists. HCV is classified into 7 different genotypes with several subtypes. Genotypes 1, 2 and 3 are found worldwide. Materials and Methods A sample of 349 chronic HCV hospital patients was studied who were already taking the treatment of standard therapy of Interferon+Ribavirin thrice a week. The sample was naturally divisible into three groups: Responder, the patients who received the standard therapy and recovered/cured; Relapser, the patients who after a course of therapy became negative for HCV RNA but after sometimes (6-18 months) became HCV positive again; Non-responder, the patients who did not show positive response to therapy. Results

It was found that HCV genotype a is very common ( %) among responders group while genotype a is more common in relapser ( %) and non - responders ( %). Five of the Six main genotypes namely a ( %), a ( %), b ( %), a ( %) and an Untypeable ( %) were found among the different castes tribes isonym ethnic groups Genotype was not found The HCV frequency in isonym groups is as follows Arain ( %), Gujjar ( %), Jutt ( %), Kashmiri ( %), Conclusion Human genetic susceptibility to HCV genotypes appears to be of importance in getting the infection. The study suggests that IL-10 and IL-28B interleukin genes are common in two major caste of the Punjab. A cohort study needs to be done for better understanding of human susceptibility to HCV infection and its management. Paper reference: SPS1000-14 Glucose Restriction Decreases Telomerase Activity and Enhances its Inhibitor Response on Breast Cancer Cells: Possible extra-telomerase role of BIBR 1532 *Dr. layal Ahmad wardi

[email protected] Lebanon Faculty of medicine, Saint joseph university, Beirut

Dr. George Hilal

faculty of medicine, Saint Joseph University, Beirut, Lebanon

Abstract Considerable progresses have been made to understand the association between lifestyle and diet in cancer initiation and promotion. Since excessive glucose consumption is a key metabolic hallmark of cancer cells, glucose restriction (GR) decreases proliferation, promotes differentiation, and transformation to quiescent cells. The immortality of cancerous cells is largely assured by the telomerase, an interesting target for inhibition by BIBR 1532. In this study, we investigated the effect of glucose restriction on telomerase activity and the response of its inhibitor BIBR 1532. Breast cancer MDA-MB 231 cells were cultured in DMEM (Dulbecco’s modified eagle’s media) with 0, 1 or 4.5 g/l of glucose. First, the telomerase activity was measured by quantitative RealTime PCR and the two telomerase subunits were semi quantified by RT-PCR. Then, the effect of glucose restriction and BIBR 1532 on proliferation test and mitochondrial metabolism were assessed by tetrazolium salt reduction and cell counts. Finally, the detection of apoptosis was evaluated by caspase-3 measurement and Annexin-V staining. The decrease in telomerase activity by more than 80% was associated with a significant reduction in the mRNA expression of its catalytic subunit hTERT (Reverse Transcriptase) and in the mitochondrial metabolism by more than 80% in restricted glucose condition. In addition, the effect of BIBR 1532 was more potentiated by glucose restriction. Glucose deprivation induces apoptosis by BIBR 1532- mediated telomerase inhibition in MDA-MB 231 cells as assessed by caspase-3 measurement and Annexin analysis. Taken together, our results demonstrated that GR and telomerase inhibitor work together to induce breast cancer cell death. Background

Considerable progresses have been made to understand the association between lifestyle and diet in cancer initiation and promotion. Since excessive glucose consumption is a key metabolic hallmark of cancer cells, glucose restriction (GR) decreases proliferation, promotes differentiation, and transformation to quiescent cells. The immortality of cancerous cells is largely assured by the telomerase, an interesting target for inhibition by BIBR 1532. In this study, we investigated the effect of glucose restriction on telomerase activity and the response of its inhibitor BIBR 1532. Materials and Methods Breast cancer MDA-MB 231 cells were cultured in DMEM (Dulbecco’s modified eagle’s media) with 0, 1 or 4.5 g/l of glucose. First, the telomerase activity was measured by quantitative RealTime PCR and the two telomerase subunits were semi quantified by RT-PCR. Then, the effect of glucose restriction and BIBR 1532 on proliferation test and mitochondrial metabolism were assessed by tetrazolium salt reduction and cell counts. Finally, the detection of apoptosis was evaluated by caspase-3 measurement and Annexin-V staining. Results The decrease in telomerase activity by more than 80% was associated with a significant reduction in the mRNA expression of its catalytic subunit hTERT (Reverse Transcriptase) and in the mitochondrial metabolism by more than 80% in restricted glucose condition. In addition, the effect of BIBR 1532 was more potentiated by glucose restriction. Glucose deprivation induces apoptosis by BIBR 1532- mediated telomerase inhibition in MDA-MB 231 cells as assessed by caspase-3 measurement and Annexin analysis. Conclusion Taken together, our results demonstrated that GR and telomerase inhibitor work together to induce breast cancer cell death.