Editorial 2 2 In the past few decades, as new medical technologies have been incorporated into the common medical practice around the world, new ethical challenges have been introduced in the realms of philosophy and ethics. These issues include: the consent of cell donors and subject of property rights in the field of tissue engineering and enhancement of human capabilities for longevity; cloning of animals and humans (potentially) in the future; genetic manipulation for diagnosis and treatment of diseases; use of stem cells and gene therapy for treatment of human diseases; genetic manipulation of plants to produce therapeutic drugs and food for people and many other related issues. The bioethical aspects of many of these new medical biotechnologies have been fervently debated for many years in private bioethics centers, universities and governments’ sponsored studies in many developed countries. However, little attention has been paid to these issues in the developing countries, perhaps due to limited application of such technologies in a given country. Fortunately, in the past few years, some national and international conferences on ethical aspects of using these new technologies have been held in Iran. Incidentally, later this year, in the month of November, two conferences are to be held in Iran which will focus on bioethics and new technologies. The international conference on this issue will be held by the National Research Center for Genetic Engineering and Biotechnology of Iran and the national conference will be held by the Avicenna Research Institute. I must mention that the conference held by the Avicenna Research Institute will focus on genetics as a central theme and will consider the legal, ethical and psychological aspects as they relate to genetics and human health. These two conferences will provide an excellent opportunity and should be very beneficial to those who are interested in bioethics, ethics, law, philosophy, sociology, psychology as they relate to human health. I encourage researchers, students, leaders of research institutions and government offices currently involved with issues related to medical biotechnology research, genetics and bioethics to participate in these conferences. 114 114 Ali M. Ardekani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 159.pdf ####

Teratogen Screening: State of the Art 2 2 Due to the number of new substances coming into use every year and the increasing amounts of chemicals, which are introduced into the environment, there is a high demand for a rapid, reliable and cost-effective method for detection of developmental toxicity. To meet this challenge various in vitro techniques have been established additional to in vivo animal testing. This review introduces the techniques in existence at the moment. Requirements on an ideal in vitro teratogenicity test system are stated, and the advantages and disadvantages of the present methods are discussed. 115 121 Julia Schumann Faculty of Veterinary Medicine, Institute of Physiological Chemistry, University of Leipzig Faculty of Veterinary Medicine, Institute of Physiological Chemistry, University of Leipzig Germany Animal testing alternativesCell culture techniquesTeratogens 38.pdf Walmod PS, Gravemann U, Nau H, Berezin V, Bock E. Discriminative power of an assay for automated in vitro screening of teratogens. Toxicol In Vitro 2004;18(4): 511-525. ##Kochhar DM. In vitro testing of teratogenic agents using mammalian embryos. Teratog Carcinog Mutagen 1981;1(1):63-74. ##Fantel AG. Culture of whole rodent embryos in teratogen screening. Teratog Carcinog Mutagen 1982;2(3-4):231-242. ##Wilson JG. Review of in vitro systems with potential for use in teratogenicity screening. J Environ Pathol Toxicol 1978;2(1):149-167. ##Pearson RM. In-vitro techniques: can they replace animal testing? Hum Reprod 1986;1(8):559-560. ##Bernardini G, Vismara C, Boracchi P, Camatini M. Lethality, teratogenicity and growth inhibition of heptanol in Xenopus assayed by a modified frog embryo teratogenesis assay-Xenopus (FETAX) procedure. Sci Total Environ 1994;151(1):1-8. ##Bantle JA, Finch RA, Burton DT, Fort DJ, Dawson DA, Linder G, et al. FETAX interlaboratory validation study: phase III-Part 1 testing. J Appl Toxicol 1996;16(6):517-528. ##Flint OP. In vitro tests for teratogens: desirable endpoints, test batteries and current status of the micromass teratogen test. Reprod Toxicol 1993;7(Suppl 1):103-111. ##Umansky R. The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dev Biol 1966;13(1):31-56. ##Paulsen DF, Solursh M. Microtiter micromass cultures of limb-bud mesenchymal cells. In Vitro Cell Dev Biol 1988;24(2):138-147. ##Flint OP, Orton TC, Ferguson RA. Differentiation of rat embryo cells in culture: response following acute maternal exposure to teratogens and non-teratogens. J Appl Toxicol 1984;4(2):109-116. ##Flint OP, Orton TC. An in vitro assay for teratogens with cultures of rat embryo midbrain and limb bud cells. Toxicol Appl Pharmacol 1984;76(2):383-395. ##Wise LD, Clark RL, Rundell JO, Robertson RT. Examination of a rodent limb bud micromass assay as a prescreen for developmental toxicity. Teratology 1990;41(3):341-351. ##Renault JY, Melcion C, Cordier A. Limb bud cell culture for in vitro teratogen screening: validation of an improved assessment method using 51 compounds. Teratog Carcinog Mutagen 1989;9(2):83-96. ##Doetschman TC, Eistetter H, Katz M, Schmidt W, Kemler R. The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 1985;87:27-45. ##Rohwedel J, Guan K, Hegert C, Wobus AM. Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol In Vitro 2001;15(6):741-753. ##Tillner J, Winckler T, Dingermann T. Developmentally regulated promoters from Dictyostelium discoideum as molecular markers for testing potential teratogens. Pharmazie 1996; 51(11):902-906. ##Tillner J, Nau H, Winckler T, Dingermann T. Evaluation of the teratogenic potential of valproic acid analogues in transgenic Dictyostelium discoideum strains. Toxicol In Vitro 1998;12 (4):463-469. ##Dannat K, Tillner J, Winckler T, Weiss M, Eger K, Dingermann T. Effects of medicinal compounds on the differentiation of the eukaryotic microorganism dictyostelium discoideum: can this model be used as a screening test for reproductive toxicity in humans? Pharmazie 2003;58(3):204-210. ##Cotter DA, Sands TW, Virdy KJ, North MJ, Klein G, Satre M. Patterning of development in Dictyostelium discoideum: factors regulating growth, differentiation, spore dormancy, and germination. Biochem Cell Biol 1992;70(10-11): 892-919. ##Loomis WF. Lateral inhibition and pattern formation in Dictyostelium. Curr Top Dev Biol 1993;28:1-46. ##Schaap P, Tang YH, Othmer HG. A model for pattern formation in Dictyostelium discoideum. Differentiation 1996;60(1):1-16. ##Stigson M, Kultima K, Jergil M, Scholz B, Alm H, Gustafson AL, et al. Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro. Altern Lab Anim 2007;35(3):335-342. ##Jergil M, Kultima K, Gustafson AL, Dencker L, Stigson M. Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects. Toxicol Sci 2009;108:132-148. ##Robinson JF, Guerrette Z, Yu X, Hong S, Faustman EM. A systems-based approach to investigate dose- and time-dependent methylmercury-induced gene expression response in C57BL/6 mouse embryos undergoing neurulation. Birth Defects Res B Dev Reprod Toxicol 2010;89(3):188-200. ##Sha K, Winn LM. Characterization of valproic acid-initiated homologous recombination. Birth Defects Res B Dev Reprod Toxicol 2010;89(2):124-132. ##Fisher JC, Bodenstein L. Computer simulation analysis of normal and abnormal development of the mammalian diaphragm. Theor Biol Med Model 2006;3(1):9. ##Rajnikant, Dinesh, Chand B. Biological activity predictions, crystallographic comparison and hydrogen bonding analysis of cholane derivatives. Indian J Biochem Biophys 2007;44(6):458-469. ##

Optimization of Gene Transfection in Murine Myeloma Cell Lines using Different Transfection Reagents 2 2 Purification and isolation of cellular target proteins for monoclonal antibody (MAb) production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines (SP2/0, NS0, NS1, Ag8, and P3U1) were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein (EGFP), were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP2/0, 55.7%, 21.1% and 9.3% for NS0, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NS0 and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NS0 cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production. 123 130 Mahdi Shabani 1. Department of Immunology, School of Medicine, Shiraz University of Medical Sciences Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR 1. Department of Immunology, School of Medicine, Shiraz University of Medical Sciences Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Shayda Hemmati Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Reza Hadavi Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Iran Zahra Amirghofran Department of Immunology, School of Medicine, Shiraz University of Medical Sciences Department of Immunology, School of Medicine, Shiraz University of Medical Sciences Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Immune and Gene Therapy Lab, Cancer Center Karolinska, Karolinska Hospital, Karolinska Institutet Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranSweden Hodjattallah Rabbani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical Sciences Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical Sciences IranIran Fazel Shokri Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical Sciences Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical Sciences IranIran Enhanced green fluorescent proteinFlow cytometryMonoclonal antibodyMyeloma cell linesTransfection 39.pdf Han SY, Gai W, Yancovitz M, Osman I, Di Como CJ, Polsky D. Nucleofection is a highly effective gene transfer technique for human melanoma cell lines. Exp Dermatol 2008;17(5):405-411. ##Dean DA. Nonviral gene transfer to skeletal, smooth, and cardiac muscle in living animals. Am J Physiol Cell Physiol 2005;289(2):C233-245. ##Colosimo A, Goncz KK, Holmes AR, Kunzelmann K, Novelli G, Malone RW, et al. Transfer and expression of foreign genes in mammalian cells. Biotechniques 2000;29(2):314-331. ##Li XG, Wang YX, Zhou BQ, Qi CH. The effect of VEGF-ASODN transfection on expression of VEGF and growth in tongue squamous cancer cell line Tca8113. Shanghai Kou Qiang Yi Xue 2009;18(5):509-514. ##Villarroel Dorrego M, Whawell SA, Speight PM, Barrett AW. Transfection of CD40 in a human oral squamous cell carcinoma keratinocyte line upregulates immune potency and costimulatory molecules. Br J Dermatol 2006;154(2):231-238. ##Halama A, Kulinski M, Librowski T, Lochynski S. Polymer-based non-viral gene delivery as a concept for the treatment of cancer. Pharmacol Rep 2009;61(6):993-999. ##Zhang H, Wang YS, Han G, Shi Y. TIMP-3 gene transfection suppresses invasive and metastatic capacity of human hepatocarcinoma cell line HCC-7721. Hepatobiliary Pancreat Dis Int 2007;6(5):487-491. ##Griesenbach U, Alton EW. Cystic fibrosis gene therapy: successes, failures and hopes for the future. Expert Rev Respir Med 2009;3(4):363-371. ##Nagata S, Salvatore G, Pastan I. DNA immunization followed by a single boost with cells: a protein-free immunization protocol for production of monoclonal antibodies against the native form of membrane proteins. J Immunol Methods 2003;280(1-2):59-72. ##Chowdhury PS, Pastan I. Analysis of cloned Fvs from a phage display library indicates that DNA immunization can mimic antibody response generated by cell immunizations. J Immunol Methods 1999;231(1-2):83-91. ##Chowdhury PS, Viner JL, Beers R, Pastan I. Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc Natl Acad Sci USA 1998;95(2):669-674. ##Marget M, Sharma BB, Tesar M, Kretzschmar T, Jenisch S, Westphal E, et al. Bypassing hybridoma technology: HLA-C reactive human single-chain antibody fragments (scFv) derived from a synthetic phage display library (HuCAL) and their potential to discriminate HLA class I specificities. Tissue Antigens 2000;56(1):1-9. ##Bard F, Barbour R, Cannon C, Carretto R, Fox M, Games D, et al. Epitope and isotype specificities of antibodies to beta-amyloid peptide for protection against Alzheimer's disease-like neuropathology. Proc Natl Acad Sci USA 2003;100(4):2023-2028. ##Qin S, Tang H, Zhao LS, He F, Lin Y, Liu L, et al. Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells. World J Gastroenterol 2003;9(5):1111-1113. ##Geisse S, Gram H, Kleuser B, Kocher HP. Eukaryotic expression systems: a comparison. Protein Expr Purif 1996;8(3):271-282. ##Dorai H, McCartney JE, Hudziak RM, Tai MS, Laminet AA, Houston LL, et al. Mammalian cell expression of single-chain Fv (sFv) antibody proteins and their C-terminal fusions with interleukin-2 and other effector domains. Nat Biotechnol 1994;12(9):890-897. ##Qian F, Xiao CZ, Gao LH, Zhang ZG, Guo ZX, Yu WY. Expression of prourokinase in different mammalian cells. Sheng Wu Gong Cheng Xue Bao 2000;16(3):349-352. ##Bestagno M, Sola I, Dallegno E, Sabella P, Poggianella M, Plana-Durán J, et al. Recombinant dimeric small immunoproteins neutralize transmissible gastroenteritis virus infectivity efficiently in vitro and confer passive immunity in vivo. J Gen Virol 2007;88(Pt 1):187-195. ##Zuo L, Cullen CM, DeLay ML, Thornton S, Myers LK, Rosloniec EF, et al. A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness. J Immunol 2002;168(5):2554-2559. ##Fermentas life sciences [Internet]. [unknown]: Thermo Fisher Scientific Inc. (NYSE: TMO); c2001-2010. TurboFect™ in vitro Transfection Reagent; [2009]; [cited 2010 Augusts 24]; [4 p.]. Available from: http://www.fermentas.com/templates/files/tiny_mce/media_pdf/broch_turbofect_P26.pdf. ##Eppendorf [Internet]. Hamburg: Eppendorf; [unknown]. Multiporator®: Sp20-Ag14 transfection protocol; 2001 November [cited 2010 Augusts 24]; [2 p.]. Available from: http://eppendorf-can.com/utilities/download_pdf. asp?DR=downloads%2Fpdf%2Fapplications%2FBS%2Fecet-protocols&FN=4308915-026.pdf ##OZ Biosciences: the art of delivery systems [Internet]. Marseille : OZ Biosciences; [unknown]. DreamFect ™ List of cells; 2010 May [cited 2010 Augusts 24]; [2 p.]. Available from: http://www.ozbiosciences.com/docman/dreamfect-cells/download-2.html. ##Maurisse R, De Semir D, Emamekhoo H, Bedayat B, Abdolmohammadi A, Parsi H, et al. Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 2010;10:9. ##Simões S, Slepushkin V, Gaspar R, Pedroso de Lima MC, Düzgünes N. Successful transfection of lymphocytes by ternary lipoplexes. Biosci Rep 1999;19(6):601-609. ##Bartlett RJ, Secore SL, Singer JT, Bodo M, Sharma K, Ricordi C. Long-term expression of a fluorescent reporter gene via direct injection of plasmid vector into mouse skeletal muscle: comparison of human creatine kinase and CMV promoter expression levels in vivo. Cell Transplant 1996;5(3):411-419. ##Rejman J, Bragonzi A, Conese M. Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 2005;12(3):468-474. ##Kichler A, Leborgne C, Coeytaux E, Danos O. Polyethylenimine-mediated gene delivery: a mechanistic study. J Gene Med 2001;3(2):135-144. ##Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 2001;272(1-2):149-156. ##Lin JH, Wang M, Andrews WH, Wydro R, Morser J. Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems. Gene 1994;147(2):287-292. ##Feng G, Hicks P, Chang PL. Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells. In Vitro Cell Dev Biol Anim 2003;39(10):420-423. ##Thomas P, Smart TG. HEK293 cell line: a vehicle for the expression of recombinant proteins. J Pharmacol Toxicol Methods 2005;51(3):187-200. ##Toyoda E, Kurosawa A, Kamekawa H, Adachi N. Topoisomerase IIalpha inhibition following DNA transfection greatly enhances random integration in a human pre-B lymphocyte cell line. Biochem Biophys Res Commun 2009;382(3):492-496. ##

Human Tissue Plasminogen Activator Expression in Escherichia coli using Cytoplasmic and Periplasmic Cumulative Power 2 2 Tissue plasminogen activator (tPA) is a serine protease, which is composed of five distinct structural domains with 17 disulfide bonds, representing a model of high-disulfide proteins in human body. One of the most important limitations for high yield heterologous protein production in Escherichia coli (E. coli) is the expression of complex proteins with multiple disulfide bridges. In this study the combination of two distinct strategies, manipulated cytoplasm and native periplasm, was applied to produce the functional full length tPA enzyme in E. coli. Using a PelB signal peptide sequence at 5' site of tPA gene, the expression cassette was prepared and subsequently was transformed into a strain with manipulated oxidizing cytoplasm. Then the induction was made to express the protein of interest. The SDS-PAGE analysis and gelatin hydrolysis confirmed the successful expression of functional tPA. The results of this study showed that complex proteins can be produced in E. coli using the cumulative power of both cytoplasm and periplasm. 131 136 Keivan Majidzadeh Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iranian Center for Breast Cancer (ICBC), ACECR IranIran Fereidoun Mahboudi Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Mahdi Hemayatkar Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Fatemeh Davami Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Farzaneh Barkhordary Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Ahmad Adeli Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Mohammad Soleimani Azad university Azad university Qom Noushin Davoudi Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Vahid Khalaj Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Cytoplasm<i>Escherichia coli</i>PeriplasmProteinsTissue Plasminogen Activator 40.pdf Pennica D, Holmes WE, Kohr WJ, Harkins RN, Vehar GA, Ward CA, et al. 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Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J 1992;11(1):57-62. ##Peek JA, Taylor RK. Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae. Proc Natl Acad Sci USA 1992;89(13):6210-6214. ##Hua Q, Yang C, Oshima T, Mori H, Shimizu K. Analysis of gene expression in Escherichia coli in response to changes of growth-limiting nutrient in chemostat cultures. Appl Environ Microbiol 2004;70(4):2354-2366. ##Mattes R. The production of improved tissue-type plasminogen activator in Escherichia coli. Semin Thromb Hemost 2001;27(4):325-336. ##Ausubel FM. Current protocols in molecular biology. New York: John Wiley & Sons; 1987. ##Snoek-van Beurden PA, Von den Hoff JW. Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors. 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Expression of high levels of human tissue plasminogen activator in yeast under the control of an inducible GAL promoter. Appl Microbiol Biotechnol 1992;37(5):604-608. ##Furlong AM, Thomsen DR, Marotti KR, Post LE, Sharma SK. Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector. Biotechnol Appl Biochem 1988;10(5):454-464. ##Steiner H, Pohl G, Gunne H, Hellers M, Elhammer A, Hansson L. Human tissue-type plasminogen activator synthesized by using a baculovirus vector in insect cells compared with human plasminogen activator produced in mouse cells. Gene 1988;73(2):449-457. ##Nakamoto H, Bardwell JC. Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm. Biochim et Biophysica Acta 2004;1694(1-3):111-119. ##Aslund F, Beckwith J. The thioredoxin superfamily: redundancy, specificity, and gray-area genomics. J Bacteriol 1999;181(5):1375-1379. ##Bessette PH, Aslund F, Beckwith J, Georgiou G. 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In Silico Design and Selection of Anti-fungal AmB-polyene-analog Lead Molecules by Virtual Screening Method 2 2 A major group of drugs that have been approved for the therapy of systemic fungal infections are polyene antibiotics. Amphotericin B (AmB), one of the polyene antibiotics, has been used to treat serious systemic fungal infections by binding to sterols such as ergosterol in fungal cells membrane, and is believed to form pores in the membrane and create a transmembrane ion-channel. Since all eukaryotic cells contain sterols, using AmB can cause toxicity in mammalian cells; this is the most serious unwanted side effect. Therefore, there is still a need to develop suitable antifungal compounds to be entered in the drug development pipeline. In this study, we report the screening of various compounds from the Enhanced NCI database against ergosterol and cholesterol as receptors. The strategy employed is divided into two categories, screening and docking, respectively. Screening was performed using structure search based on AmB and molecular constraints to filter compounds with physico-chemical properties similar to the polyene macrolid antibiotics. The selected compounds were docked and scored to identify structurally novel ligands that make similar interactions to AmB. Our screening approach identified several molecules with high ranking criteria mentioned above. Among these compounds, two molecules, NSC 89270 and NSC 62792 were tested for their bioactivity against three fungal strains using broth microdilution assay that presented to have moderate antifungal activity against tested fungi. Thus, they could be possible lead compounds that grant further research on them to improve their potency and compare their mechanism of action in comparison to AmB. 137 143 Marziyeh Ferdosiyan Department of Biochemistry, Science and Research Branch, Islamic Azad University Department of Biochemistry, Science and Research Branch, Islamic Azad University Iran Soroush Sardari Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center Drug Design and Bioinformatics Unit, Medical Biotechnology Department, Biotechnology Research Center Iran Amphotericin BAntifungal agentsCholesterolErgosterol 41.pdf García-Ruiz JC, Amutio E, Pontón J. Invasive fungal infection in immunocompromised patients. Rev Iberoam Micol 2004;21(2):55-62. ##Walsh TJ, Groll A, Hiemenz J, Fleming R, Roilides E, Anaissie E. Infections due to emerging and uncommon medically important fungal pathogens. 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Synchronous Comparison of Mycobacterium tuberculosis Epidemiology Strains by "MIRU-VNTR" and "MIRU-VNTR and Spoligotyping" Technique 2 2 Molecular epidemiology analyses are frequently used in determining epidemiology of tuberculosis. Recently, Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) and Spoligotyping has become an important method, as it allows high-through put, discriminatory and reproducible analysis of clinical isolate. The purpose of this study is to compare techniques of “MIRU-VNTR” versus “MIRU-VNTR and Spoligotyping” together for study of genetic pattern of Mycobacterium tuberculosis (M. tuberculosis) strains. Sixty M. tuberculosis (MTB) isolates were selected (30 susceptible, 30 multi-drug resistant) for this study. Thereafter, the "MIRU-VNTR and spoligotyping" were performed to identify their genetic patterns. The frequency of unknown genetic pattern of MTB was compared using technique of “MIRU-VNTR” alone versus “MIRU-VNTR and Spoligotyping” together. The MIRU-VNTR allelic diversity at each of the loci was calculated by Hunter – Gaston Discriminatory Index (HGDI). Based on differentiation index of all strains 10, 16, 26, 31 and 40 loci were identified as the most distinctive (HGI = 0.6) and 2, 4, 20 and 24 as the weakest distinctive locus (HGI = 0.3). By using MIRU-VNTR technique 38% (n= 23) of isolates could not be typed, whereas by applying "MIRU-VNTR and Spoligotyping" together only 15% (n= 9) of isolates remained unknown (p = 0.004). For sensitive strains, the difference was significant (67% vs. 90%, p = 0.028), but only marginally significant for drug resistant strains (57% vs. 80%, p = 0.052). The discrimination power of 12-locus MIRU-VNTR and Spoligotyping was equal to that of MIRU-VNTR analysis. If appropriate loci are added to the standard MIRU analysis, MIRU-VNTR genotyping could be a valuable tool for strain typing and epidemiological research of M. tuberculosis. With this approach a more clear understanding about genetic pattern of MTB can be achieved. 145 152 Mehdi Jafarian Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran Muayed Aghali-Merza Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran Parissa Farnia Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran Mojtaba Ahmadi Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Mycobacteriology Research Center, National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran Mohammad Reza Masjedi National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran Ali Akbar Velayati National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences National Research Institute of Tuberculosis and Lung Disease (NRITLD), Shahid Beheshti University of Medical Sciences Iran AllelesGenetic LociMolecular epidemiologyMycobacterium tuberculosisRestriction fragment length polymorphism 42.pdf World Health Organization. 2001. 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Mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis compared to IS6110 –based restriction fragment length polymorphism analysis for investigation of apparently clustered cases of tuberculosis. J Clin Microbiol 2003:41(8):3514-3520. ##Supply P, Mazars E, Lesjean S, Vincent V, Gicquel B, Locht C. Variable human mini satellite-like regions in the mycobacterium tuberculosis genome. Mol Microbiol 2000;36 (3):762-771. ##Kamerbeek J, Schouls L, Kolk A, van Agterveld M, van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997;35(4):907-914. ##Dale JW, Brittain D, Cataldi AA, Cousins D, Crawford JT, Driscohl J, et al. Spacer oligonucleotide typing of bacteria of the Mycobacterium tuberculosis complex: recommendation for standardized nomenclature. Tuber Lung Dis 2001;5(3):216-219. ##Sola C, Filliol I, Legrand E, Mokrousov I, Rastogi N. 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Association of CALHM1 Gene Polymorphism with Late Onset Alzheimer’s Disease in Iranian Population 2 2 Alzheimer's disease (AD) is a genetically heterogeneous neurodegenerative disease and Late-Onset type (LOAD) is the most common form of dementia affecting people over 65 years old. CALHM1 (P86L) encodes a transmembrane glycoprotein that controls cytosolic Ca2+ concentrations and Aß levels and P86L polymorphism in this gene is significantly associated with LOAD in independent case controls in a number of studies. This study was performed to determine whether this polymorphism contributes to the risk for LOAD in Iranian population. One hundred and forty one AD patients and 141 healthy controls were recruited in this study. After extraction of genomic DNA, the genotype and allele frequencies were determined in case and control subjects using PCR/RFLP method. The statistical analysis showed a significant difference in the heterozygote genotype frequency in case and control groups and polymorphic allele had a protective role between two groups. Also after stratifying the subjects by their APOE-e4 status, no significant association was observed. Our study suggests that P86L polymorphism could be a protective factor for late-onset Alzheimer's disease (LOAD) in Iranian population. However, to confirm these results, further study with a bigger sample size may be required. 153 157 Meysam Jafari Aqdam Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Koorosh Kamali Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Iran Mehdi Rahgozar Epidemiology and Biostatistics Department, University of Social Welfare and Rehabilitation Sciences Epidemiology and Biostatistics Department, University of Social Welfare and Rehabilitation Sciences Iran Mina Ohadi Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Mehdi Manoochehri Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Ali Tahami Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Iran Leila Bostanshirin Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Hamid Reza Khorram Khorshid Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Reproductive Biotechnology Research Centre, Avicenna Research Institute, ACECR IranIran Alzheimer’s diseaseGenetic association studyGenePolymorphismPopulation 43.pdf Dreses-Werringloer U, Lambert JC, Vingtdeux V, Zhao H, Vais H, Siebert A, et al. A polymorphism in CALHM1 influences Ca2+ homostasis, Aß levels, and Alzheimer’s disease risk. Cell 2008;133 (7):1149-1161. ##Jillian J. Alzheimer disease: Alzheimer’s disease neuropathology in the oldest old. Nat Rev Neurol 2009;5(8):411-412. ##Tuppo EE, Arias HR. The role of inflammation in Alzheimer’s disease. Int J Biochem Cell Biol 2005; 37(2):289-305. ##Pastor P, Goate AM. Molecular genetics of Alzheimer's disease. Curr Psychiatry Rep 2004;6 (2):125-133. ##Strittmatter WJ, Saunders AM, Schmechel D, Pericak-Vance M, Enghild J, Salvesen GS, et al. Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. PNAS 1993 30;90(5):1977–1981. ##Seshadri S, Fitzpatrick AL, Ikram MA, DeStefano AL, Gudnason V, Boada M, et al. Genome-wide analysis of genetic loci associated with Alzheimer disease. JAMA 2010; 303(18):1832-1840. ##Bertram L, Blacker D, Mullin K, Keeney D, Jones J, Basu S, et al. Evidence for genetic linkage of Alzheimer's disease to chromosome 10q. Science 2000;290 (5500):2302-2303. ##Gozalpour E, Kamali K, Mohammd K, Khorram Khorshid HR, Ohadi M, Karimloo M, et al. Association between Alzheimers disease and Apolipoprotein E polymorphisms. Iranian J Publ Health 2010;39(2):1-6. ##Shoji M, Kuwano R, Asada T, Imagawa M, Higuchi S, Urakami K, et al. Japanese Study Group Genome-wide screening for Genes associated Alzheimer's disease; Advanced Brain Science Project. A proposal for diagnostic and clinical assessment criteria for Alzheimer's disease. Rinsho Shinkeigaku. 2005;45(2):128-137. ##Inoue K, Tanaka N, Yamashita F, Sawano Y, Asada T, Goto Y. The P86L common allele of CALHM1 does not influence risk for Alzheimer disease in Japanese cohorts. Am J Med Genet B Neuropsychiatr Genet 2010;153B(2):532-535. ##Cui PJ, Zheng L, Cao L, Wang Y, Deng YL, Wang G, et al. CALHM1 P86L polymorphism is a risk factor for Alzheimer's disease in the Chinese population. J Alzheimer’s Dis 2010;19 (1):31-35. ##Boada M, Antúnez C, López-Arrieta J, Galán JJ, Morón FJ, Hernández I, et al. CALHM1 P86L polymorphism is associated with late-onset Alzheimer's disease in a recessive model. J Alzheimer’s Dis 2010;20(1):247-251. ##Bertram L, Schjeide BM, Hooli B, Mullin K, Hiltunen M, Soininen H, et al. No Association between CALHM1 and Alzheimer’s disease risk. Cell 2008;135(6):993–996. ##Minster RL, Demirci FY, DeKosky ST, Kamboh MI. No Association between CALHM1 variation and risk of Alzheimer disease. Hum Mutat 2009;30(4):E566–E569. ##Beecham GW, Schnetz-Boutaud N, Haines JL, Pericak-Vance MA. CALHM1 polymorphism is not associated with late-onset Alzheimer disease. Ann Hum Genet 2009;73(3):379-381. ##Tan EK, Ho P, Cheng SY, Yih Y, Li HH, Fook-Chong S, et al. CALHM1 variant is not associated with Alzheimer's disease among Asians. Neurobiol Aging 2009. [Epub ahead of print]. ##Nacmias B, Tedde A, Bagnoli S, Lucenteforte E, Cellini E, Piaceri I, et al. Lack of implication for CALHM1 P86L common variation in Italian patients with early and late onset Alzheimer's disease. J Alzheimer's Dis 2010;20(1):37-41. ##