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Ethical Issues in Medical Biotechnology 2 2 The extraordinary revolution in biotechnology has created new possibilities for curing disease and manipulating our genetic heritage. It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes provides methods to treat the disease 1. But it has also created numerous ethical problems that need close philosophical attention. In another words, since biotechnology involves modifying living things for human purposes, there is great potential for ethical concerns. The recent advances in biotechnology present both benefits and risks. They have revolutionized the process of drug manufacture, diagnosis and treatment and the production of animal models for human diseases. There is a tremendous potential for creating new drugs and treatment. This technology raises important ethical issues in the social structures including families, preventive medicine, employment, health insurance etc. We must interact with the general public, to educate them, and prepare them better for the impact of biotechnology. The scientific and medical communities and the public, in general, have to use these powerful tools responsibly, for the maximum benefit of mankind 2. 129 129 Shahin Akhondzadeh Tehran University of Medical Sciences Tehran University of Medical Sciences Iran Editorial 176.pdf Kuszler PC. Biotechnology entrepreneurship and ethics: principles, paradigms, and products. Med Law 2006;25(3):491-502. ##Persson A. Research ethics and the development of medical biotechnology.Xenotransplantation 2006;13(6):511-513. ##

Cytogenetic Alterations in Preimplantation Mice Embryos Following Male Mouse Gonadal Gamma-irradiation: Comparison of Two Methods for Reproductive Toxicity Screening 2 2 Background: Genome instability is a main cause of chromosomal alterations in both somatic and germ cells when exposed to environmental, physical and chemical genotoxicants. Germ cells especially spermatozoa are more vulnerable to suffering from DNA damaging agents during spermatogenesis and also more potent in transmitting genome instability to next generation. Methods: To investigate the effects of γ-rays on inducing abnormalities manifested as numerical Chromosome Aberrations (CA) and Micronucleus (MN) in preimplantation embryos, adult male NMRI mice were irradiated with 4 Gy of γ-rays. They were then mated at weekly intervals with superovulated, non-irradiated female mice in 6 successive weeks. About 68 hr post coitous, four to eight cell embryos were retrieved and fixed on slides using standard methods in order to screen for CA and MN. Results: In embryos generated from irradiated mice, the frequency of aneuploidy and MN increased dramatically at all post-irradiation sampling times as compared to the control (p<0.01). The frequency of embryos expressed MN was much higher than chromosomally abnormal embryos, although the trend of MN formation was similar to chromosomal abnormalities seen in corresponding sampling times. Conclusion: Irradiation of sperms at any stages of spermatogenesis may lead to stable chromosomal abnormalities affecting pairing and disjunction of chromosomes in successive preimplantation embryos that are expressed as MN. Although chromosome analysis of embryos showed various types of chromosomal abnormalities, MN assay provide a simpler and faster technique for investigating the genotoxicity of agents affecting embryos at preimplantation stages. 130 139 Mahdieh Salimi Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology Iran Hossein Mozdarani Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University Iran Elmina Nazari Department of Science and Research, Azad University and Royan Institute Department of Science and Research, Azad University and Royan Institute Iran Chromosomal aberrationsEmbryoGerm cellsIrradiationMicronuclei 177.pdf Russell L, Ettlin R, Sinha Hikim A, Clegg E. Histological and histopathological evaluation of the testis. 1st ed. Clearwater, FL: Cache River Press; 1990.##Meistrich ML. Effects of chemotherapy and radiotherapy on spermatogenesis. Eur Urol 1993;23(1):136-141.##Shetty G, Meistrich ML. Hormonal approaches to preservation and restoration of male fertility after cancer treatment. J Natl Cancer Inst Monogr 2005;(34):36-39.##Fosså S, Magelssen H. Fertility and reproduction after chemotherapy of adult cancer patients: malignant lymphoma and testicular cancer. Ann Oncol 2004;15(Suppl 4):259-265.##Oliva R. Protamines and male infertility. Hum Reprod Update 2006;12(4):417-435.##Hourcade JD, Pérez-Crespo M, Fernández-González R, Pintado B, Gutiérrez-Adán A. Selection against spermatozoa with fragmented DNA after postovulatory mating depends on the type of damage. Reprod Biol Endocrinol 2010;8:9.##Dubrova YE. Radiation induced transgenerational instability. Oncogen 2003;22(45):7087-7093.##Dasenbrock C, Tillmann T, Ernst H, Behnke W, Kellner R, Hagemann G, et al. Maternal effects and cancer risk in the progeny of mice exposed to x-rays before conception. Exp Toxicol Pathol 2005;56(6):351-360.##Arnon J, Meirow D, Lewis-Roness H, Ornoy A. Genetic and teratogenetic effects of cancer treatment of gametes and embryos. Hum Reprod Update 2001;7(4):394-403.##Palyga GF. Embryogenesis and early postnatal ontogenesis of posterity of two generations of female Wistar rats, depending on the time of their fertilization after low dose radiation exposure, Radiat Biol Radioecol 2002;42(4):390-394.##Nakamura N. Genetic effects of radiation in atomic-bomb survivors and their children: past, present and future. J Radiat Res 2006;47(Suppl B):B67-73.##Nomura T. Transgenerational effects of radiation and chemicals in mice and humans. J Radiat Res 2006;47(Suppl B):B83-97.##Tamminga J, Koturbash I, Baker M, Kutanzi K, Kathiria P, Pogribny IP, al. Paternal cranial irradiation induces distant bystander DNA damage in the germ line and leads to epigenetic alterations in the offspring. Cell Cycle 2008;7(9):1238-1245.##Ahmadi A, Ng SC. Fertilizing ability of DNA-damaged spermatozoa. J Exp Zool 1999;284(6):696-704.##Fatehi AN, Bevers MM, Schoevers E, Roelen BA, Colenbrander B, Gadella BM. DNA damage in bovine sperm does not block fertilization and early embryonic development but induces apoptosis after the first cleavages. J Androl 2006;27(2):176-188.##Estop A, Catala V, Santalo J. Chromosome constitution of highly motile mouse sperm. Mol Reprod Dev 1990;27(2):168-172.##Olson SB, Magenis RE. Preferential paternal origin of de novo structural chromosome rearrangements. In: Daniels A, (eds). Progress and topics in cytogenetics, The cytogenetics of mammalian autosomal rearrangements. New York: Liss; 1988, 585-599.##Liu DY, Baker HW. Human sperm bound to the zona pellucida have normal nuclear chromatin as assessed by acridine orange fluorescence. Hum Reprod 2007;22(6):1597-1602.##Hasegawa M, Wilson G, Russll LD, Meistrich ML. Radiation-induced cell death in the mouse testis: relationship with apoptosis. Radiat Res 1997;147(4):457-467.##Hugenholtz AP, Bruce WR. Radiation induction of mutations affecting sperm morphology in mice. Mutat Res 1983;107(1):177-185.##Hamer G, Roepers-Gajadien HL, Gademan IS, Kal HB, De Rooij DG. Intercellular bridges and apo¬ptosis in clones of male germ cells. Int J Androl 2003;26(6):348-353.##Oakberg EF. Sensitivity and time of degeneration of spermatogenic cells irradiation in various stages of maturation in the mouse. Radiat Res 1995;2(4):369-391.##Cai L, Wang P. Induction of a cytogenetic adaptive response in germ cells of irradiated mice with very low-dose rate of chronic gamma-irradiation and its biological influence on radiation induced DNA or chromosomal damage and cell killing in their male offspring. Mutagenesis 1995;10(2):95-100. ##Muller WU, Streffer C. Micronucleus assay. In: Bauchinger M, (eds). Advance in mutagenesis research. The University of Michigan: Springer-Verlag; 1990, 1-134. ##Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 1985;147(1-2):29-36.##Fenech M. The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 1993;285(1):35-44.##Fenech M, Holland N, Chang WP, Zeiger E, Bonnasi S. The Human Micronucleus Project-an international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans. Mutat Res 1999;428(1-2):271-283.##Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Eastmond D, Fenech M, et al. Report from the in vitro micronucleus assay working group. Mutat Res 2003;540(2):153-163.##Fomenko LA, Lomaeva MG, Bezlepkin VG, Gaziev AI. Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay. Radiats Biol Radioecol 2006;46(4):431-435.##Konopacka M, Rzeszowska-Wolny J. Modifying effect of vitamins C, E and beta-carotene against gamma-ray induced DNA damage in mouse cells. Mutat Res 1998;417(2-3):85-94.##Jacquet P, Adriaens I, Buset J, Neefs M, Vankerkom J. Cytogenetic studies in mouse oocytes irradiated in vitro at different stages of maturation by use of an early preantral follicle culture system. Mutat Res 2005;583(2):168-177.##Tusell L, Alvarez R, Caballin MR, Genesca A, Miro R, Ribas M, et al. Induction of micronuclei in human sperm-hamster egg hybrids at the two cell stage after in vitro gamma-irradiation of human spermatozoa. Environ Mol Mutagen 1995;26(4):315-323. ##Bang D, Lee J, Oh H, Kim S, Kim T, Lee Y, et al. Dose incidence relationships on the prenatal effects of gamma-radiation in mice. J Vet Sci 2002;3(17):7-11.##Dyban AP. Reliable techniques for chromosomal preparations from mammalian oocytes and preimplantation embryos. Preimplantation genetics. New York: Plenum Press; 1991, 293-298.##Pellestor F, Girardet A, Andreo B, Amal F, Humeau C. Relationship between morphology and chromosomal constitution in human preimplantation embryos. Mol Reprod Dev 1994;39(2):141-146.##Santalo J, Veiga A, Calafel JM, Calderon G, Vidal F, Barri PN, et al. Evaluation of cytogenetic analysis for clinical preimplantation diagnosis. Fertil Steril 1995;64(1):44-50.##Fenech M. The in vitro micronucleus technique. Mutat Res 2000;455:81-95.##Fenech M, Chang WP, Kirsch-Volders M, Holland N, Bonassi S, Zeiger E; Human micronucleus project. Human Project: Detailed description of the scoring criteria for the cytokinesis-block micronucleus assay using isolated human lymphocyte cultures. Mutat Res 2003;534(1-2):65-75.##Norpa H, Falck GC, What do human micronuclei contain? Mutagenesis 2003;18(3):221-233.##Huang CC, Lin DP, Tsao HM, Cheng TC, Liu CH, Lee MS. Sperm DNA fragmentation negatively correlates with velocity and fertilization rates but might not affect pregnancy rates. Fertil Steril 2005;84(1):130-140.##Ardon F, Helms D, Sahin E, Bollwein H, Topfer-Petersen E, Waberski D. Chromatin-unstable boar spermatozoa have little chance of reaching oocytes in vivo. Reproduction 2008;135(4):461-470.##Barratt CL, Kirkman-Brown J. Man-made versus female-made environment-will the real capacitation please stand up? Hum Reprod Update 2006;12(1):1-2.##Suarez SS, Pacey AA. Sperm transport in the female reproductive tract. Hum Reprod Update 2006;12(1):23-37.##Holt WV, Van Look KJ. Concepts in sperm heterogeneity, sperm selection and sperm competition as biological foundations for laboratory tests of semen quality. Reproduction 2004;127(5):527-535.##Kamiguchi Y, Tateno H. Radiation- and chemical-induced structural chromosome aberrations in human spermatozoa. Mutat Res 2002;504(1-2):183-191.##Mastuda Y, Tobari I, Yamada T. Studies on chromosome aberrations induced in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation. Mutat Res 1985;148(1-2):113-117.##Brezani P, Kalina I. Cytogenetic and genetic effects of continuous irradiation at a low exposure rat. Folia Biol 1980;26(5):298-303.##Searle AG, Beechey CV. Cytogenetic effects of protracted gamma exposures from conception of male mice. Mutat Res 1982;95(1):61-68.##Cordelli E, Fresegna AM, Leter G, Eleuteri P, Spano M, Villani P. Evaluation of DNA damage in different stages of mouse spermatogenesis after testicular X irradiation. Radiat Res 2003;160(4):443-451. ##Tsuchida RS, Uchida IA. Radiation-induced chromosome aberrations in mouse spermatocytes and oocytes. Cytogenet Cell Genet 1975;14(1):1-8. ##Mozdarani H, Nazari E. Frequency of micronuclei in 4-8 cell mouse embryos generated after maternal gamma-irradiation in the presence and in the absence of vitamin C. 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Male Pronuclear Formation using Dog Sperm Derived from Ectopic Testicular Xenografts, Testis, and Epididymis 2 2 Background: Testis tissue xenografting and the resultant sperm in a xenograft may provide a unique approach to rescue the genetic material of males that die prematurely and is a model for the study of human spermatogenesis and can represent an alternative approach for fertility preservation in cancer patients. This study was aimed to evaluate the xenogenic dog sperm in formation of male pronucleus following injection into the sheep oocytes. Methods: The in vitro matured slaughterhouse derived sheep oocytes were subjected to Intracytoplasmic Sperm Injection (ICSI) with epididymal, testicular, and xenogenic dog sperm. The ICSI was performed after scoring of the sperm midpiece using an IX71-Olympus inverted microscope with Nomarsky optics. Within 1 hr after injection, the injected oocytes in activated group were exposed to 5 µM ionomycin for 5 min. The data were analyzed by Chi-square and ANOVA using SigmaStat, version 3.5, and p<0.05 was considered significant. Results: The formation of female pronucleus after ICSI of xenogenic sperm was higher than epididymal and testicular sperm in non-activated oocytes. The corresponding rate in activated oocytes was higher or comparable with testicular and epididymal sperm. The rate of male pronucleus formation after ICSI of xenogenic sperm was comparable with injection of two other sperm sources. Oocyte activation had an inductive role in female and male pronuclear formation. Conclusion: Dog xenogenic sperm was capable to induce oocyte activation and proportion of male pronucleous formation was comparable to the testicular and epididymal sperm. 140 146 Abolfazl Shirazi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University IranIran Asma Khadivi Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Naser Shams-Esfandabadi Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran CanineIntracytoplasmic sperm injection (ICSI)PronucleusXenograft 178.pdf Honaramooz A, Snedaker A, Boiani M, Scholer H, Dobrinski I, Schlatt S. Sperm from neonatal mammalian testes grafted in mice. Nature 2002;418(6899):778-781.##Geens M, De Block G, Goossens E, Frederickx V, Van Steirteghem A, Tournaye H. Spermatogonial survival after grafting human testicular tissue to immunodeficient mice. Hum Reprod 2006;21(2):390-396. ##Schlatt S, Kim SS, Gosden R. Spermatogenesis and steroidogenesis in mouse, hamster and monkey testicular tissue after cryopreservation and heterotopic grafting to castrated hosts. Reproduction 2002;124(3):339-346. ##Honaramooz A, Li MW, Penedo MC, Meyers S, Dobrinski I. Accelerated maturation of primate testis by xenografting into mice. Biol Reprod 2004;70(5):1500-1503. ##Oatley JM, de Avila DM, Reeves JJ, McLean DJ. Spermatogenesis and germ cell transgene expression in xenografted bovine testicular tissue. Biol Reprod 2004;71(2):494-501.##Snedaker AK, Honaramooz A, Dobrinski I. A game of cat and mouse: xenografting of testis tissue from domestic kittens results in complete cat spermatogenesis in a mouse host. J Androl 2004;25(6):926-930. ##Shinohara T, Inoue K, Ogonuki N, Kanatsu-Shinohara M, Miki H, Nakata K, et al. Birth of offspring following transplantation of cryopreserved immature testicular pieces and in-vitro microinsemination. Hum Reprod 2002;17(12):3039-3045. ##Honaramooz A, Cui XS, Kim NH, Dobrinski I. Porcine embryos produced after intracytoplasmic sperm injection using xenogeneic pig sperm from neonatal testis tissue grafted in mice. Reprod Fertil Dev 2008;20(7):802-807. ##Schlatt S, Honaramooz A, Boiani M, Scholer HR, Dobrinski I. Progeny from sperm obtained after ectopic grafting of neonatal mouse testes. Biol Reprod 2003;68(6):2331-2335. ##Yanagimachi R. Intracytoplasmic injection of spermatozoa and spermatogenic cells: its biology and applications in humans and animals. Reprod Biomed Online 2005;10(2):247-288.##Yamazaki T, Yamagata K, Baba T. Time-lapse and retrospective analysis of DNA methylation in mouse preimplantation embryos by live cell imaging. Dev Biol 2007;304(1):409-419.##Aitken J. The biochemistry and physiology of human spermatozoa: consequences for fertility and sterility. In: Van Blerkom J, (eds). The biological basis of early human reproductive failure: Application to medically assisted conception and the treatment of infertility. New York: Oxford University Press; 1994, 252-282.##Dozortsev D, De Sutter P, Rybouchkin A, Dhont M. Timing of sperm and oocyte nuclear progression after intracytoplasmic sperm injection. Hum Reprod 1995;10(11):3012-3017.##Holt WV, North RD. Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa. J Reprod Fertil 1991;91(2):451-461. ##Giraud MN, Motta C, Boucher D, Grizard G. Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Hum Reprod 2000;15(10):2160-2164. ##Chatterjee S, Gagnon C. Production of reactive oxygen species by spermatozoa undergoing cooling, freezing, and thawing. Mol Reprod Dev 2001;59(4):451-458. ##Liu CH, Tsao HM, Cheng TC, Wu HM, Huang CC, Chen CI, et al. DNA fragmentation, mitochondrial dysfunction and chromosomal aneuploidy in the spermatozoa of oligoasthenoteratozoospermic males. J Assist Reprod Genet 2004;21(4):119-126.##Derijck AA, van der Heijden GW, Ramos L, Giele M, Kremer JA, de Boer P. Motile human normozoospermic and oligozoospermic semen samples show a difference in double-strand DNA break incidence. Hum Reprod 2007;22(9):2368-2376. ##Kobayashi H, Sato A, Otsu E, Hiura H, Tomatsu C, Utsunomiya T, et al. Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients. Hum Mol Genet 2007;16(21):2542-2551. ##Abrishami M, Abbasi S, Honaramooz A. The effect of donor age on progression of spermatogenesis in canine testicular tissue after xenografting into immunodeficient mice. Theriogenology 2010;73(4):512-522.##Neagu VR, Macıas Garcıa B, Salazar Sandoval C, Morillo Rodrıguez A, Ortega Ferrusola C, Gonzalez Fernandez L, et al. Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves post thaw sperm membrane integrity. Theriogenology 2010;73(5):645-650.##Shirazi A, Ostad-Hosseini S, Ahmadi E, Heidari B, Shams-Esfandabadi N. In vitro developmental competence of ICSI-derived activated ovine embryos. Theriogenology 2009;71(2):342-348.##Sakkas D, Urner F, Bianchi PG, Bizzaro D, Wagner I, Jaquenoud N, et al. Sperm chromatin anomalies can influence decondensation after intracytoplasmic sperm injection. Hum Reprod 1996;11(4):837-843.##García-Mengual EGarcía-Mengual E, García-Roselló EGarcía-Roselló E, Alfonso JAlfonso J, Salvador ISalvador I, Cebrian-Serrano ACebrian-Serrano A, Silvestre MASilvestre MASilvestre MA. Viability of ICSI oocytes after caffeine treatment and sperm membrane removal with Triton X-100 in pigs. TheriogenologyTheriogenology 2011;76(9):1658-1666.##Rho GJ, Kawarsky S, Johnson WH, Kochhar K, Betteridge KJ. Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intracytoplasmic sperm injection into bovine oocytes. Biol Reprod 1998;59(4):918-924.##Flaherty SP, Payne D, Swann NJ, Mattews CD. Aetiology of failed and abnormal fertilization after intracytoplasmic sperm injection. Hum Reprod 1995;10(10):2623-2629.##Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after intracytoplasmic sperm injection. Hum Reprod 1994;9(12):2374-2380.##Nakai MNakai M, Kashiwazaki NKashiwazaki N, Takizawa ATakizawa A, Maedomari NMaedomari N, Ozawa MOzawa M, Noguchi JNoguchi J, et al. Morphologic changes in boar sperm nuclei with reduced disulfide bonds in electrostimulated porcine oocytes. Reproduction 2006;131(3):603-611.##

Effect of Peptone Feeding on Transient Gene Expression Process in CHO DG44 2 2 Background: Transient Gene Expression (TGE) gained popularity over the last decade as a rapid method for the production of milligram to gram quantities of recombinant proteins for preclinical studies in biophama industry. Thereby, the optimization of the TGE technique for Chinese hamster ovary (CHO) as the dominant host for the production of biotherapeutics is of great interest to reach the values for Human Embryo Kidney-293 (HEK-293) cells in terms of transfection efficiencies and production titers. TGE efficiencies are cell line and vector dependant. Methods: In transfection efficiency optimization experiments, different starting cell densities, different amounts of plasmid DNA and PEI transfection reagent were investigated to achieve the best conditions leading to maximum transfection efficiencies. Furthermore, in order to investigate the effect of peptone feeding on transfection efficiency, three different sources of peptones with the greatest effect in the CD DG44 basal media were selected; Casein Tryptone N1, Soy petone A2SC and Soy peptone E110. Results: The transfection strategy performed here was able to make an outstanding increase in transfection efficiency of CHO DG44 cell line transfected with pTracer-SV40-mutated t-PA plasmid from 3.6% in our starting non-optimized condition to 66.93% in finally optimized situation. Moreover, peptone feeding strategy used here was successful to increase volumetric productivities up to 37%. In addition, the amounts of both PEI and plasmid DNA were reduced up to 66% and 25% respectively compared to our previous protocol. Conclusion: Here we described an optimization process for TGE in suspension-adapted CHO cells based on Polyethylenimine (PEI)/DNA concentration, DNA: PEI ratio, starting cell densities and peptone feeding strategy. 147 155 Fatemeh Davami Biotechnology Research Center, Pasteur Institute of Iran Iran Farnaz Eghbalpour Biotechnology Research Center, Pasteur Institute of IranIslamic Azad University of Arak Islamic Azad University of Arak IranIran Farzaneh Barkhordari Biotechnology Research Center, Pasteur Institute of Iran Iran Fereidoun Mahboudi Biotechnology Research Center, Pasteur Institute of Iran Iran Chinese hamster ovary cellsPeptonesPolyethylenimineProtein productionTransient gene expression 179.pdf Wulhfard S, Baldi L, Hacker DL, Wurm F. Valproic acid enhances recombinant mRNA and protein levels in transiently transfected Chinese hamster ovary cells. J Biotechnol 2010;148(2-3):128-132.##Sakai K, Hayashi C, Yamaji H, Fukuda H. Use of nonionic surfactants for effective supply of phosphatidic acid in serum-free culture of Chinese hamster ovary cells. J Biosci Bioeng 2001;92(3):256-261.##Butler M. Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol 2005;68(3):283-291.##Backliwal G, Hildinger M, Chenuet S, Wulhfard S, De Jesus M, Wurm FM. Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 2008;36(15):e96.##Wulhfard S, Tissot S, Bouchet S, Cevey J, De Jesus M, Hacker DL, et al. Mild hypothermia improves transient gene expression yields several fold in Chinese hamster ovary cells. Biotechnol Prog 2008;24(2):458-465.##Ye J, Kober V, Tellers M, Naji Z, Salmon P, Markusen JF. High-level protein expression in scalable CHO transient transfection. Biotechnol Bioeng 2009;103(3):542-551.##Merten OW. Safety issues of animal products used in serum-free media. Dev Biol Stand 1999;99:167-180.##Merten OW, Manuguerra JC, Hannoun C, van der Werf S. Production of influenza virus in serum-free mammalian cell cultures. Dev Biol Stand 1999;98:23-37.##Keen MJ, Rapson NT. Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line. Cytotechnology 1995;17(3):153-163.##Zoletto R, Gagliardi G. New media and their advantages in the production of suspended cells and foot--and--mouth disease virus. Dev Biol Stand 1976;35:27-31.##Jan DC, Jones SJ, Emery AN, al-Rubeai M. Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture. Cytotechnology 1994;16(1):17-26.##Sakai K, Matsunaga T, Yamaji H, Fukuda H. Effects of phospholipids on growth of Chinese hamster ovary cells in serum-free media. J Biosci Bioeng 1999;88(3):306-309.##Pham PL, Perret S, Doan HC, Cass B, St-Laurent G, Kamen A, et al. Large-scale transient transfection of serum-free suspension-growing HEK293 EBNA1 cells: peptone additives improve cell growth and transfection efficiency. Biotechnol Bioeng 2003;84(3):332-342.##Pham PL, Perret S, Cass B, Carpentier E, St-Laurent G, Bisson L, et al. Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis. Biotechnol Bioeng 2005;90(3):332-344.##Burteau CC, Verhoeye FR, Mols JF, Ballez JS, Agathos SN, Schneider YJ. Fortification of a protein-free cell culture medium with plant peptones improves cultivation and productivity of an interferon-gamma-producing CHO cell line. In Vitro Cell Dev Biol Anim 2003;39(7):291-296.##Kim SH, Lee GM. Development of serum-free medium supplemented with hydrolysates for the production of therapeutic antibodies in CHO cell cultures using design of experiments. Appl Microbiol Biotechnol 2009;83(4):639-648.##Muller N, Girard P, Hacker DL, Jordan M, Wurm FM. Orbital shaker technology for the cultivation of mammalian cells in suspension. Biotechnol Bioeng 2005;89(4):400-406.##Davami F, Sardari S, Majidzadeh-A K, Hemayatkar M, Barkhordari F, Enayati S, et al. A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments. BMB Rep 2011;44(1):34-39.##Davami F, Sardari S, Majidzadeh-AK, Hemayatkar M, Barkhrdari F, Omidi M, et al. Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments. J Biomed Biotechnol 2010;2010:108159.##Davami F, Barkhordari F, Alebouyeh M, Adeli A, Mahboudi F. Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors. J Microbiol Biotechnol 2011;21(12):1299-1305.##Geisse S. Reflections on more than 10 years of TGE approaches. Protein Expr Purif 2009;64(2):99-107.##Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol 2004;22(11):1393-1398.##Franek F. Gluten of spelt wheat (Triticum aestivum subspecies spelta) as a source of peptides promoting viability and product yield of mouse hybridoma cell cultures. J Agric Food Chem 2004;52(13):4097-4100.##Jan DC, Jones SJ, Emery AN, al-Rubeai M. Peptone, a low-cost growth-promoting nutrient for intensive animal cell culture. Cytotechnology 1994;16(1):17-26.##Keen MJ, Rapson NT. Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line. Cytotechnology 1995;17(3):153-163.##Rajendra Y, Kiseljak D, Baldi L, Hacker DL, Wurm FM. A simple high-yielding process for transient gene expression in CHO cells. J Biotechnol 2011;153(1-2):22-26.##Jordan M, Wurm F. Transfection of adherent and suspended cells by calcium phosphate. Methods 2004;33(2):136-143.##Tuvesson O, Uhe C, Rozkov A, Lüllau E. Development of a generic transient transfection process at 100 L scale. Cytotechnology 2008;56 (2):123-136.##

An Investigation on Mitochondrial DNA Deletions and Telomere Shortening during Multiple Passages of Adult Stem Cells 2 2 Background: Limited resources for adult stem cells necessitate their in vitro culture prior to clinical use. Investigating mitochondrial DNA (mtDNA) and telomere shortening has proved to be important indications of stem cell validity. This study was designed to investigate these indicators in multiple passages of three adult stem cell lines which were produced in our stem cell laboratory. Methods: In this study, Dental Pulp Stem Cells (DPSCs), Periapical Follicle Stem Cells (PAFSCs) and Human Foreskin Fibroblast (HFF) cell lines were expanded for 20 passages. After 1, 5, 10, 15 and 20 passages, expanded cells were harvested and DNA was extracted for further studies. Common mtDNA mutation was detected by multiplex PCR and telomere shortening was tested by Southern blot analysis. Results: The common deletion was not detected in any of the stem cells or cell lines after several passages. In addition, Southern blot analysis indicated that the mean difference of telomere length between first and last passage was 0.25 kb in DPSC, 0.1 kb in PAFSC and 0.32 kb in HFF which indicates that the mean telomere length in various passages of the samples showed insignificant changes. Conclusion: Absence of mtDNA mutations in adult stem cell lines indicates good mitochondrial function even after 20 passages. In addition, absence of telomere shortening indicates stem cells validity after multiple passages. It is hoped this information could pave the way for using in vitro expansion of adult stem cells for future clinical applications. 156 162 Farzaneh Fesahat Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Iran Mohammad Hasan Sheikhha Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Iran Azam Rasti Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Iran Fatemeh Sadeghian Nodoshan Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Research and Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences Iran Hadi Zare-Zardini Young Researchers and Elite Club, Yazd Branch, Islamic Azad University Young Researchers and Elite Club, Yazd Branch, Islamic Azad University Iran Ali Reza Navabazam Department of Oral and Maxillo-facial Surgery, School of Dentistry, Shahid Sadoughi University of Medical Sciences Department of Oral and Maxillo-facial Surgery, School of Dentistry, Shahid Sadoughi University of Medical Sciences Iran Adult stem cellsAgingMitochondrial deletionsTelomere shortening 180.pdf Zhang J, Li X, Mueller M, Wang Y, Zong C, Deng N, et al. Systematic characterization of the murine mitochondrial proteome using functional validated cardiac mitochondria. Proteomics 2008;8(8):1564-1575.##Chinnery PF, Samuels DC, Elson J, Turnbull DM. Accumulation of mitochondrial DNA mutations in ageing, cancer and mitochondrial disease: is there a common mechanism? Lancet 2002;360(9342):1323-1325.##Aral C, Akkiprik M, Kaya H, Ataizi-Çelikel C, Caglayan S, Ozisik G, et al. Mitochondrial DNA common deletion is not associated with thyroid, breast and colorectal tumors in Turkish patients. Genet Mol Biol 2010;33(1):1-4 .##Hsieh RH, Tsai NM, Au HK, Chang SJ, Wei YH, Tzeng CR. Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes. Fertil Steril 2002;77(5):1012-1017.##Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G, Schon EA. Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 1995;57(2):239-247.##Barritt JA, Brenner CA, Cohen J, Matt DW. Mitochondrial DNA rearrangements in human oocytes and embryos. Mol Hum Reprod 1999;5(10):927-933.##Brenner CA, Kubisch HM, Pierce KE. Role of the mitochondrial genome in assisted reproductive technologies and embryonic stem cell-based therapeutic cloning. Reprod Fertil Dev 2004;16(7):743-751.##Futyma K, Putowski L, Cybulski M, Miotla P, Rechberger T, Semczuk A. The prevalence of mtDNA 4977 deletion in primary human endometrial carcinomas and matched control samples. Oncol Rep 2008;20(3):683-688.##Mohamed SA, Hanke T, Erasmi AW, Bechtel MJ, Scharfschwerdt M, Meissner C, et al. Mitochondrial DNA deletions and the aging heart. Exp Gerontol 2006;41(5):508-517.##Gibson TG, Kubisch MK, Brenner CA. Mitochondrial DNA deletions in rhesus macaque oocytes and embryos. Mol Hum Reprod 2005;11(11):785-789.##Bavister BD, Wolf DP, Brenner CA. Challenges of primate embryonic stem cell research. Cloning Stem Cells 2005;7(2):82-94.##O'Callaghan NJ, Fenech M. A quantitative PCR method for measuring absolute telomere length. Biol Proced Online 2011;13:1-10.##Samsonraj RM, Raghunath M, Hui JH, Ling L, Nurcombe V, Cool SM. Cool Telomere length analysis of human mesenchymal stem cells by quantitative PCR. Gene 2013;519(2):348-355.##Navabazam AR, Sadeghian Nodoshan F, Sheikhha MH, Miresmaeili SM, Soleimani M, Fesahat F. Characterization of mesenchymal stem cells from human dental pulp, preapical follicle and periodontal ligament. Iran J Reprod Med 2013;11(3):235-242. ##Aflatoonian B, Sadeghian F, Fesahat F, Khorad-Mehr A, Janan A, Aflatoonian R, et al. The transdifferentiation of the human foreskin fibroblasts to form germ cells using retinoic acid. Poster session presented at: ISSCR 9th Annual Meeting, Thursday Poster Abstracts Germline Cells Poster; 2011Jun 15-18; Toronto, Ontario, Canada.##Houshmand M, Panahi MS, Nafisi S, Soltanzadeh A, Alkandari FM. Identification and sizing of GAA trinucleotide repeat expansion, investigation for D-loop variations and mitochondrial deletions in Iranian patients with Friedreich's ataxia. Mitochondrion 2006;6(2):87-93.##Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, Nikbin B. Aging of mesenchymal stem cell in vitro. BMC Cell Biol 2006;7:14.##Tsai HD, Hsieh YY, Hsieh JN, Chang CC, Yang CY, Yang JG, et al. Mitochondria DNA deletion and copy numbers of cumulus cells associated with in vitro fertilization outcomes. J Reprod Med 2010;55(11):491-497.##Bavister BD. The mitochondrial contribution to stem cell biology. Reprod Fertility Develop 2006;18(8):829-838. ##Beltrami AP, Cesselli D, Beltrami CA. Stem cell senescence and regenerative paradigms. Clin Pharmacol Ther 2012;91(2):21-29.##Garcia-Lavandeira M, Quereda V, Flores I, Saez C, Diaz-Rodriguez E, Japon MA, et al. A GRFa2/ Prop1/stem (GPS) cell niche in the pituitary. PLoS ONE 2009;4(3):e4815.##Bharadwaj S, Liu G, Shi Y, Wu R, Yang B, He T, et al. Multipotential differentiation of human urine-derived stem cells: potential for therapeutic applications in urology. Stem cells 2013;31(9):1840-1856.##Chen Z, Jadhav A, Wang F, Perle M, Basch R, Young BK. Senescence and longevity in amniotic fluid derived cells. Stem Cell Discovery (SCD) 2013;3(1):47-55.##Flores I, Benetti R, Blasco MA. Telomerase regulation and stem cell behaviour. Curr Opin Cell Biol 2006;18(3):254-260.##Flores I, Canela A, Vera E, Tejera A, Cotsarelis G, Blasco MA. The longest telomeres: a general signature of adult stem cell compartments. Genes Dev 2008;22(5):654-667.##Wang C, Jurk D, Maddick M, Nelson G, Martin-Ruiz C, von Zglinicki T. DNA damage response and cellular senescence in tissues of aging mice. Aging Cell 2009;8:311-323.##Gattermann N, Berneburg M, Heinisch J, Aul C, Schneider W. Detection of the ageing-associated 5-Kb common deletion of mitochondrial DNA in blood and bone marrow of hematologically normal adults. Absence of the deletion in clonal bone marrow disorders. Leukemia 1995;9(10):1704-1710.##Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells following in vitro expansion. Stem Cells 2004;22(5):675-682.##Guillot PV, Gotherstrom C, Chan J, Kurata H, Fisk NM. Human first trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells 2007;25(3):646-654.##Parsch D, Fellenberg J, Brümmendorf TH, Eschlbeck AM, Richter W. Telomere length and telomerase activity during expansion and differentiation of human mesenchymal stem cells and chondrocytes. J Mol Med 2004;82(1):49-55.##Bernardo ME, Zaffaroni N, Novara F, Cometa AM, Avanzini MA, Moretta A, et al. Human bone marrow-derived mesenchymal stem cells do not undergo transformation after long-term in vitro culture and do not exhibit telomere maintenance mechanisms. Cancer Res 2007;67(19):9142-9149.##Gannon HS, Donehower LA, Lyle S, Jones SN. Mdm2-p53 signaling regulates epidermal stem cell senescence and premature aging phenotypes in mouse skin. Dev Biol 2011;353(1):1-9.##Gupta PB, Chaffer CL, Weinberg RA. Cancer stem cells: mirage or reality? Nat Med 2009;15(9):1010-1012.##Flores I, Blasco MA. The role of telomeres and telomerase in stem cell aging. FEBS Lett 2010;584(17):3826-3830.##Wei YH. Mitochondrial DNA mutations and oxidative damage in aging and diseases: an emerging paradigm of gerontology and medicine. Proc Natl Sci Counc Repub China B 1998;22(2):55-67.##Williams SL, Mash DC, Züchner S, Moraes CT. Somatic mtDNA mutation spectra in the aging human putamen. PLoS Genet 2013;9(12):e1003990.##

Production of L-glutamic Acid with Corynebacterium glutamicum (NCIM 2168) and Pseudomonas reptilivora (NCIM 2598): A Study on Immobilization and Reusability 2 2 Background: L-glutamic acid is one of the major amino acids that is present in a wide variety of foods. It is mainly used as a food additive and flavor enhancer in the form of sodium salt. Corynebacterium glutamicum (C. glutamicum) is one of the major organisms widely used for glutamic acid production. Methods: The study was dealing with immobilization of C. glutamicum and mixed culture of C. glutamicum and Pseudomonas reptilivora (P. reptilivora) for L-glutamic acid production using submerged fermentation. 2, 3 and 5% sodium alginate concentrations were used for production and reusability of immobilized cells for 5 more trials. Results: The results revealed that 2% sodium alginate concentration produced the highest yield (13.026±0.247 g/l by C. glutamicum and 16.026±0.475 g/l by mixed immobilized culture). Moreover, reusability of immobilized cells was evaluated in 2% concentration with 5 more trials. However, when the number of cycles increased, the production of L-glutamic acid decreased. Conclusion: Production of glutamic acid using optimized medium minimizes the time needed for designing the medium composition. It also minimizes external contamination. Glutamic acid production gradually decreased due to multiple uses of beads and consequently it reduces the shelf life. 163 168 Rajaram Shyamkumar Department of Biotechnology, Kamaraj College of Engineering and Technology, Virudhunagar India Innasi Muthu Ganesh Moorthy Department of Biotechnology, Kamaraj College of Engineering and Technology, Virudhunagar India Karuppiah Ponmurugan Department of Botany and Microbiology, College of Science, King Saud University Department of Botany and Microbiology, College of Science, King Saud University Kingdom of Saudi Arabia Rajoo Baskar Department of Chemical Engineering, Kongu Engineering College, Perunduari, Erode India CorynebacteriumGlutamic acidImmobilization 181.pdf Maerz U. GA-103R World markets for fermentation ingredients. http://www. Bccrese-arch.com/food/GA103R.html; 2005.##Birnbaum J, Demain AL. Reversal by citrate of the lodoacetate and fluoride inhibition of glutamic acid production by Corynebacterium glutamicum. Appl Microbiol 1969;18(2):287-288.##Hermann T. Industrial production of amino acids by coryneform bacteria. J Biotechnol 2003;104(1-3):155-172.##Yoshioka T, Ishii T, Kawahara Y, Koyama Y, Shimizu E. Method for producing L-glutamic acid by continuous fermentation, United States patent US 5,869,300. 1999.##Choi SU, Nihira T, Yoshida T. Enhanced glutamic acid production by Brevibacterium sp. with temperature shift-up cultivation. J Biosci Bioeng 2004;98(3):211-213.##Amin GA, Al-Talhi A. Production of L-glutamic acid by immobilized cell reactor of the bacterium Corynebacterium glutamicum entrapped into carrageenan gel beads. World Appl Sci J 2007;2(1):62-67.##Das K, Anis M, Azemi BM, Ismail N. Fermentation and recovery of glutamic acid from palm waste hydrolysate by ion exchange resin column. Biotech Bioeng 1995;48(5):55l-555.##Jyothi AN, Sasikiran K, Nambisan B, Balagopalan C. Optimization of glutamic acid production from cassava starch factory residues using Brevibacterium divaricatum. Process Biochem 2005;40(11):3576-3579.##Tavakkoli M, Hamidi-Esfahani S, Azizi MH. Optimization of Corynebacterium glutamicum glutamic acid production by response surface methodology. Food Bioprocess Technol 2012;5(1):92-99.##Ikeda M, Katsumata R. Hyperproduction of tryptophan by Corynebacterium glutamicum with the modified pentose phosphate pathway. Appl Environ Microbiol 1999;65(6):2497-2502.##Hartmeier W, Doppner T. Preparation and properties of mycelium bound glucose oxidase coimmobilized with excess catalase. Biotechnol Lett 1983;5(11):743-748.##Jagannadha Rao K. Studies on coimmobilization of Micrococcus glutamicus and Pseudomonas reptilivora for the production of L-glutamic acid [master’s thesis]. [Andhra University]: Visakhapatnam, India; 1992.##Kumar RS, Moorthy IM, Baskar R. Modeling and optimization of glutamic acid production using mixed culture of Corynebacterium glutamicum NCIM2168 and Pseudomonas reptilivora NCIM2598. Prep Biochem Biotechnol 2013;43 (7):668-681.##Spies JR. Colorimetric procedures for amino acids. In: Colowick SP. Kaplan N.O. methods in enzymology, Vol. III. New York: Academic Press; 1957, 468-471.##Sunitha I, Subba Rao MV, Ayyanna C. Coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel for production of L-glutamic acid. Bioprocess Eng 1998;18(1):353-359.##Prasad MP, Gupta N, Gaudani H, Gupta M, Gupta G, Krishna V, et al. Production of glutamic acid using whole and immobilized cells of Corynebacterium glutamicum. Int J Microbiol Res 2009;1(1):8-13.##Shinmyo A, Kimura H, Okada H. Physiology of α-amylase production by immobilized Bacillus amyloliquefaciens. Eur J Appl Microbiol Biotechnol 1982;14(1):7-12. ##Nasri M, Dhouib A, Zourgauni F, Kriaa H, Ellouz R. Production of lysine by using immobilized living Corynebacterium sp. Cells. Biotechnol Lett 1989;11:856-870.##Yugandhar NM, Raju AI, Rao PJ, Jaya RK, Reddy DSR. Production of glutamic acid using Brevibacterium roseum with free and immobilized cells. Res J Microbiol 2007;2(7):584-589.##Nampoothiri KM, Panday A. Immobilization of Brevibacterium cells for the production of L-glutamic acid. Bioresour Technol 1998;63(1):101-106.##Baskar R, Anantharaman N, Babu JS, Sundaram S. L-glutamic acid production in a novel three phase fluidized bed reactor using co-immobilized bio-catalyst. Biomed Sci Instrum 2001;37:457-462.##Prabu N, Babu JS, Sundaram S. L-glutamic acid production in a continuous stirred tank bioreactor using coimmobilized bio catalyst using a fluorosensor. Biomed Sci Instrum 2002;38:495-500.##Li J, Ma C, Ma Y, Li Y, Zhou W, Xu P. Medium optimization by combination of response surface methodology and desirability function: an application in glutamine production. Appl Microbiol Biotechnol 2007;74(3):563-571.##Nakazawa H, Kawashima H, Inao O, Keiji I, Yoshio K. Method of producing L- Glutamic acid by fermentation. United States patent US 5,492, 818. 1996.##Shaik Yakub P, Mir Naiman A, Hajera T, Mazharuddin KM. Comparative studies on production of Glutamic acid using wild type, mutants, immobilized cells and immobilized mutants of Corynebacterium glutamicum. Int J Eng Sci Technol 2011;3(5):3941-3949.##

The Effect of Sortilin Silencing on Ovarian Carcinoma Cells 2 2 Background: Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin (NTR3), the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Methods: Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Results: Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis (27.5±0.48%) in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart (p<0.05). Conclusion: As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma. 169 177 Fatemeh Ghaemimanesh Department of Genetics, School of Biological Sciences, Tarbiat Modares UniversityMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Genetics, School of Biological Sciences, Tarbiat Modares UniversityMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Gholamreza Ahmadian Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology Iran Saeid Talebi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Amir-Hassan Zarnani Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Iran University of Medical Sciences Nanobiotechnology Research Center, Avicenna Research Institute, ACECRImmunology Research Center, Iran University of Medical Sciences IranIran Mehrdad Behmanesh Department of Genetics, School of Biological Sciences, Tarbiat Modares University Department of Genetics, School of Biological Sciences, Tarbiat Modares University Iran Shayda Hemmati Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Reza Hadavi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Maryam Farzi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Akhondi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Hodjattallah Rabbani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran ApoptosisCancerOvarySilencingsiRNASortilin 182.pdf Tinger A, Waldron T, Peluso N, Katin MJ, Dosoretz DE, Blitzer PH, et al. Effective palliative radiation therapy in advanced and recurrent ovarian carcinoma. Int J Radiat Oncol Biol Phys 2001;51(5):1256-1263.##Velasco AP, Herraez AC, Ruiperez AC, Rincon DG, Garcia EG, Martin AG, et al. Treatment guidelines in ovarian cancer. Clin Transl Oncol 2007;9(5):308-316.##Liu B, Nash J, Runowicz C, Swede H, Stevens R, Li Z. Ovarian cancer immunotherapy: opportunities, progresses and challenges. J Hematol Oncol 2010;3:7.##Oei AL, Sweep FC, Thomas CM, Boerman OC, Massuger LF. The use of monoclonal antibodies for the treatment of epithelial ovarian cancer (review). Int J Oncol 2008;32(6):1145-1157.##Vincent JP, Mazella J, Kitabgi P. Neurotensin and neurotensin receptors. Trends Pharmacol Sci 1999;20(7):302-309.##Mazella J, Vincent JP. Functional roles of the NTS2 and NTS3 receptors. Peptides 2006;27(10):2469-2475.##Willnow TE, Petersen CM, Nykjaer A. VPS10P-domain receptors - regulators of neuronal viability and function. Nat Rev Neurosci 2008;9(12):899-909.##Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, et al. Sortilin is essential for proNGF-induced neuronal cell death. Nature 2004;427(6977):843-848.##Petersen CM, Nielsen MS, Nykjaer A, Jacobsen L, Tommerup N, Rasmussen HH, et al. Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography. J Biol Chem 1997;272(6):3599-3605.##Donninger H, Bonome T, Radonovich M, Pise-Masison CA, Brady J, Shih JH, et al. Whole genome expression profiling of advance stage papillary serous ovarian cancer reveals activated pathways. Oncogene 2004;23(49):8065-8077.##Hemmati S, Zarnani AH, Mahmoudi AR, Sadeghi MR, Soltanghoraee H, Akhondi MM, et al. Ectopic Expression of Sortilin 1 (NTR-3) in Patients with Ovarian Carcinoma. Avicenna J Med Biotechnol 2009;1(2):125-131.##Dorsett Y, Tuschl T. siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 2004;3(4):318-329.##Kreuzer KA, Lass U, Landt O, Nitsche A, Laser J, Ellerbrok H, et al. Highly sensitive and specific fluorescence reverse transcription-PCR assay for the pseudogene-free detection of beta-actin transcripts as quantitative reference. Clin Chem 1999;45(2):297-300.##Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002;30(9):e36.##Dal Farra C, Sarret P, Navarro V, Botto JM, Mazella J, Vincent JP. Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 2001;92(4):503-509.##Fauchais AL, Lalloue F, Lise MC, Boumediene A, Preud'homme JL, Vidal E, et al. Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival. J Immunol 2008;181(5):3027-3038.##Vaegter CB, Jansen P, Fjorback AW, Glerup S, Skeldal S, Kjolby M, et al. Sortilin associates with Trk receptors to enhance anterograde transport and neurotrophin signaling. Nat Neurosci 2011;14(1):54-61.##Martin S, Navarro V, Vincent JP, Mazella J. Neurotensin receptor-1 and -3 complex modulates the cellular signaling of neurotensin in the HT29 cell line. Gastroenterology 2002;123(4):1135-1143.##Carraway RE, Plona AM. Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools. Peptides 2006;27(10):2445-2460.##Morinville A, Martin S, Lavallee M, Vincent JP, Beaudet A, Mazella J. Internalization and trafficking of neurotensin via NTS3 receptors in HT29 cells. Int J Biochem Cell Biol 2004;36(11):2153-2168.##Martin S, Dicou E, Vincent JP, Mazella J. Neurotensin and the neurotensin receptor-3 in microglial cells. J Neurosci Res 2005;81(3):322-326.##Martin S, Vincent JP, Mazella J. Involvement of the neurotensin receptor-3 in the neurotensin- induced migration of human microglia. J Neurosci induced migration of human microglia. J Neurosci 2003;23(4):1198-1205.##Demont Y, Corbet C, Page A, Ataman-Onal Y, Choquet-Kastylevsky G, Fliniaux I et al. Pro-nerve growth factor induces autocrine stimulation of breast cancer cell invasion through tropomyosin-related kinase A (TrkA) and sortilin protein. J Biol ChemJ Biol Chem 2012;287(3):1923-1931.##

Genetic Profile Variation in Vaccine Strains and Clinical Isolates of Bordetella pertussis Recovered from Iranian Patients 2 2 Background: Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between clinical versus vaccine strains. In the current study, the genetic profiles of clinical isolates and vaccine strains of Bordetella pertussis (B. pertussis) were assessed by using Pulsed Field Gel Electrophoresis (PFGE). Methods: Following phenotypic and molecular identification of isolates, XbaI-digested genomic DNA of 5 clinical isolates, 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Results: Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 clinical isolates, 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain, Tohama I had a distinct profile. Vaccine and clinical profiles had low similarity, with relatedness of approximately 40%. Conclusion: The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically, the profiles of isolates circulating in the population should be monitored over the course of the re-emergence. 178 184 Faezeh Haghighi Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Iran Fereshteh Shahcheraghi Department of Bacteriology and Microbiology, Pasteur Institute of Iran Iran Ebrahim Abbasi Department of Bacterial Vaccines, Razi Vaccine and Serum Research Institute Iran Seyed Saeed Eshraghi Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Iran Hojjat Zeraati Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences Iran Seyed Ali Javad Mousavi Department of Pulmonology, Hazrat Rasool Hospital, Iran University of Medical Sciences Department of Pulmonology, Hazrat Rasool Hospital, Iran University of Medical Sciences Iran Hossein Asgarian-Omran Department of Immunology, School of Public Health, Tehran University of Medical SciencesDepartment of Immunology, School of Medicine, Mazandaran University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical SciencesDepartment of Immunology, School of Medicine, Mazandaran University of Medical Sciences IranIran Masoumeh Douraghi Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Division of Microbiology, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Iran Fazel Shokri Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Bordetella pertussisPFGE profileVaccinationWhooping cough 183.pdf Miyaji Y, Otsuka N, Toyoizumi-Ajisaka H, Shibayama K, Kamachi K. Genetic analysis of Bordetella pertussis isolates from the 2008-2010 Pertussis epidemic in Japan. PLoS One 2013;8(10):e77165.##McIntyre P, Wood N. Pertussis in early infancy:disease burden and preventive strategies. Curr Opin Infect Dis 2009;22(3):215-223.##de Melker HE, Conyn-van Spaendonck MA, Rumke HC, van Wijngaarden JK, Mooi FR, Schellekens JF. Pertussis in The Netherlands: an outbreak despite high levels of immunization with whole-cell vaccine. Emerg Infect Dis 1997;3(2):175-178.##Preston A. Bordetella pertussis: the intersection of genomics and pathobiology. Can Med Assoc J 2005;173(1):55-62.##Mooi FR, Van Loo IH, King AJ. Adaptation of Bordetella pertussis to vaccination: a cause for its reemergence? Emerg Infect Dis 2001;7(3 Suppl):526-528.##Mooi FR, Hallander H, Wirsing von Konig CH, Hoet B, Guiso N. Epidemiological typing of Bordetella pertussis isolates: recommendations for a standard methodology. Eur J Clin Microbiol Infect Dis 2000;19(3):174-181.##http://apps.who.int/immunization_monitoring/globalsummary/timeseries/tscoveragedtp1.html.##http://www.who.int/gho/publications/world_health_statistics/EN_WHS2013_Full.pdf.##Eslamifar A, Aghakhani A, Banifazl M, Gachkar L, Khadem Sadegh A, Ramezani A. Seroprevalence of Bordetella pertussis antibodies in different age groups. Iran J Infect Dis Tropic Med 2010;15(49):43-47.##Zarei S, Jeddi-Tehrani M, Mehdi Akhondi M, Zeraati H, Ferydonfar AA, Nasernia J, et al. Immunogenicity and reactogenicity of two diphtheria-tetanus-whole cell pertussis vaccines in Iranian pre-school children, a randomized controlled trial. Hum Vaccin Immunother 2013;9(6):1316-1322.##Shahcheraghi F, Nakhost Lotfi M, Parzadeh M, Sadat Nikbin V, Shouraj F, Zahraei M. Isolation of Bordetella pertussis and Bordetella parapertussis from clinical specimens at different provinces of Iran in 2009-2010. J Mazand Univ Med Sci 2012;22(88):2-8.##Loeffelholz MJ. Bordetella. 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Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination 2 2 Background: One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. Methods: The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)–labeled casein-substrate. Results: The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. Conclusion: It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase. 185 189 Mohammad Rabbani Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Iran Safoura Soleymani Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Iran Hamid Mir Mohammad Sadeghi Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Iran Narjes Soleimani Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences Iran Fatemeh Moazen Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan Pharmaceutical Sciences Research Center, Isfahan University of Medical Sciences, Isfahan, Iran Iran aprE geneBacillus subtillisSubtilisins 184.pdf Harwood CR. 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