Chairman’s statement 2 2 In recent decades, increasing advancement in biological sciences brings the need for reliable updated re-sources. Scientific research journals facilitate accessibility to comprehensive resources and lay a ground for dis-seminating the latest findings of research projects to achieve constructive experiences and satisfy the scientific community. Accordingly, Avicenna Research Institute has published Avicenna Journal of Medical Biotechnology (AJMB) since four years ago as a mission to promote infrastructure in biotech research. Now, we are very delighted to being indexed in PubMed after four years of working in a skilled group of staff and enjoying the cooperation of a competent editorial board, executive manager and specifically the editorial assistance of Dr. Nima Rezaei. It is my great pleasure to appreciate the invaluable efforts of Dr. Ali Motevalizadeh Ardakani, the former editor in chief of AJMB, in promoting the quality of the journal and an-nounce that Professor Shahin Akhondzadeh, the Iranian prominent scientist and neuropsychopharmacologist, has been appointed to the position for the next three years. I am hopeful that taking advantage of his inval-uable experiences in other journals would be a precious asset to AJMB’s promotion in line with modern ad-vances in biological and medical sciences. Hereby, I like to express my gratitude for the precious efforts of the previous editorial board members, Dr. Montazeri, Dr. Dinarvand, Dr. Bahram, Dr. Rafi, Dr. Vasei, and Dr. Zakeri and warmly send my greetings to the new board members. Finally, I would like to ask all experts, scientists, and intellectuals to join hands to support AJMB and improve its quality by their productive and brilliant ideas. 67 67 Mohammad Mehdi Akhondi Department of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Department of Immunology, Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 170.pdf ####

Non-Viral Transfection Methods Optimized for Gene Delivery to a Lung Cancer Cell Line 2 2 Background: Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. Methods: In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Results: Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (superfect), 34.1% (electroporation), and 40.1% (lipofection). Lipofection had the highest intense SDF-1 expression of the analyzed methods. Conclusion: This study has shown that the lipofection and electroporation methods were more efficient at gene delivery to Mehr-80 cells. The quantity of DNA per transfection, reagent concentration, and incubation time were identified as essential factors for successful transfection in all of the studied methods. 68 77 Loghman Salimzadeh Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Iran Mansooreh Jaberipour Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Iran Ahmad Hosseini Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Shiraz Institute for Cancer Research, Shiraz University of Medical Sciences Iran Abbas Ghaderi Shiraz Institute for Cancer Research, Shiraz University of Medical SciencesDepartment of Immunology, School of Medicine, Shiraz University of Medical Sciences Shiraz Institute for Cancer Research, Shiraz University of Medical SciencesDepartment of Immunology, School of Medicine, Shiraz University of Medical Sciences IranIran Cell lineGreen flourescent proteinsLung cancerTransfection 111.pdf Young BH, Peng H, Huard J. Muscle-based gene therapy and tissue engineering to improve bone healing. Clin Orthop Relat Res 2002;(403 Suppl):S243-251.##Lieberman JR, Ghivizzani SC, Evans CH. Gene transfer approaches to the healing of bone and cartilage. Mol Ther 2002;6(2):141-147.##Song L, Chau L, Sakamoto Y, Nakashima J, Koide M, Tuan RS. Electric field-induced molecular vibration for noninvasive, high-efficiency DNA transfection. Mol Ther 2004;9(4):607-616.##Kay MA, Glorioso JC, Naldini L. Viral vectors for gene therapy: the art of turning infectious agents into vehicles of therapeutics. Nat Med 2001;7(1):33-40.##Van Tendeloo VF, Van Broeckhoven C, Berneman ZN. Gene therapy: principles and applications to hematopoietic cells. Leukemia 2001;15(4):523-544.##Li S, Huang L. Nonviral gene therapy: promises and challenges. Gene Ther 2000;7(1):31-34.##Ledley FD. Nonviral gene therapy: the promise of genes as pharmaceutical products. Hum Gene Ther 1995;6(9):1129-1144.##Graham FL, van der Eb AJ. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973;52(2):456-467.##Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 1987;7(8):2745-2752.##Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, et al. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 1987;84(21):7413-7417.##Liu D, Ren T, Gao X. Cationic transfection lipids. Curr Med Chem 2003;10(14):1307-1315.##Wu GY, Wu CH. Receptor-mediated gene delivery and expression in vivo. J Biol Chem 1988;263(29):14621-14624.##Kichler A, Zauner W, Ogris M, Wagner E. Influence of the DNA complexation medium on the transfection efficiency of lipospermine/DNA particles. Gene Ther 1998;5(6):855-860.##Yang JP, Huang L. Time-dependent maturation of cationic liposome-DNA complex for serum resistance. Gene Ther 1998;5(3):380-387.##Sakurai F, Inoue R, Nishino Y, Okuda A, Matsumoto O, Taga T, et al. Effect of DNA/ liposome mixing ratio on the physicochemical characteristics, cellular uptake and intracellular trafficking of plasmid DNA/cationic liposome complexes and subsequent gene expression. J Control Release 2000;66(2-3):255-269.##Capecchi MR. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell 1980;22(2 Pt 2):479-488.##Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 1990;87(24):9568-9572.##Yang NS, Sun WH. Gene gun and other non-viral approaches for cancer gene therapy. Nat Med 1995;1(5):481-483.##Heller LC, Ugen K, Heller R. Electroporation for targeted gene transfer. Expert Opin Drug Deliv 2005;2(2):255-268.##Wong TK, Neumann E. Electric field mediated gene transfer. Biochem Biophys Res Commun 1982;107(2):584-587.##Nickoloff JA, Reynolds RJ. Electroporation-mediated gene transfer efficiency is reduced by linear plasmid carrier DNAs. Anal Biochem 1992;205(2):237-243.##Tsong TY. Electroporation of cell membranes. Biophys J 1991;60(2):297-306.##Rols MP, Golzio M, Delteil C, Teissie J. In vitro delivery of drugs and other molecules to cells. Methods Mol Med 2000;37:83-97.##Sharifzadeh S, Owji SM, Pezeshki AM, Malek-Hoseini Z, Kumar PV, Ghayumi SM, et al. Establishment and characterization of a human large cell lung cancer cell line with neuroendocrine differentiation. Pathol Oncol Res 2004;10(4):225-230.##Abul-Hassan K, Walmsley R, Boulton M. Optimization of non-viral gene transfer to human primary retinal pigment epithelial cells. Curr Eye Res 2000;20(5):361-366.##Armeanu S, Pelisek J, Krausz E, Fuchs A, Groth D, Curth R, et al. Optimization of nonviral gene transfer of vascular smooth muscle cells in vitro and in vivo. Mol Ther 2000;1(4):366-375.##Peters MT, Brigham KL, King GA, Meyrick BO, Gao X, Stecenko AA. Optimization of cationic liposome-mediated gene transfer to human bronchial epithelial cells expressing wild-type or abnormal cystic fibrosis transmembrane conductance regulator (CFTR). Exp Lung Res 1999;25(3):183-197. ##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 Biotechnology 2010;10:9.##Jordan ET, Collins M, Terefe J, Ugozzoli L, Rubio T. Optimizing electroporation conditions in primary and other difficult-to-transfect cells. J Biomol Tech 2008;19(5):328-334.##Mack KD, Wei R, Elbagarri A, Abbey N, McGrath MS. A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages. J Immunol Methods 1998;1(211):79-86.##Lopata MA, Cleveland DW, Sollner-Webb B. High level transient expression of a chloramphenicol acetyl transferase gene by DEAE-dextran mediated DNA transfection coupled with a dimethyl sulfoxide or glycerol shock treatment. Nucleic Acids Res 1984;12(14):5707-5717.##Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barriers to gene transfer by a cationic lipid. J Biol Chem 1995;270(32):18997-19007.##

Design of Small Molecules with HIV Fusion Inhibitory Property Based on Gp41 Interaction Assay 2 2 Background: Gp41 of HIV (Human Immunodeficiency Virus) is a protein that mediates fusion between viral and cellular membranes. The agent, T-20, which has been approved for HIV inhibition, can restrain Gp41 function in the fusion process; nevertheless, it has disadvantages like instability, high cost of production and injection form to be delivered twice a day. Methods: Several molecules like NB-2 and NB-64 have been discovered that can inhibit HIV infection. These molecules were used as template compounds to design and develop more effective small molecules functioning as HIV-1 fusion inhibitors targeting Gp41. The process included in silico docking protocols using HEX and ArgusLab applications. A multisource database was created, after choosing the best molecules; they were tested in vitro for inhibitory activity by HIV-1 single-cycle model, transfected in HEK cells (293T). Results: Computational analysis and experimental data were combined to explore molecular properties and the most potent ones were found, with the best suitable criteria for interaction with Gp41. Several examples (DAA-6, DAA-9 and DAA-12) could inhibit infection in vitro as effective as NB-2, NB-64. Conclusion: Since disadvantages of available fusion inhibitor (T-20), it seems necessary to find similar molecules to be approved and have small size providing suitable bioactivity profile. The molecules explored in this study can be good candidates for further investigations to be used as oral HIV fusion inhibitors in the future. 78 86 Soroush Sardari Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran Iran Kayhan Azadmanesh Department of Virology, Pasteur Institute of Iran Iran Fereidoun Mahboudi Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran Iran Asghar Davood Department of Medicinal Chemistry, Pharmaceutical Branch, Azad University Department of Medicinal Chemistry, Pharmaceutical Branch, Azad University Iran Ruhollah Vahabpour Department of Virology, Pasteur Institute of IranDepartment of Hepatitis and AIDS, Pasteur Institute of Iran IranIran Rezvan Zabihollahi Department of Virology, Pasteur Institute of IranDepartment of Hepatitis and AIDS, Pasteur Institute of Iran IranIran Hosna Gomari Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of IranKhatam University Khatam University IranIran Antagonists and inhibitorsDrug designFlow cytometryHIV fusion inhibitors 112.pdf Blanco J, Clotet-Codina I, Bosch B, Armand-Ugón M, Clotet B, Esté JA. Multiparametric assay to screen and dissect the mode of action of anti-human immunodeficiency virus envelope drugs. Antimicrob Agents Chemother 2005;49(9):3926-3929.##Teixeira C, Barbault F, Rebehmed J, Liu K, Xie L, Lu H, et al. Molecular modeling studies of N-substituted pyrrole derivatives-potential HIV-1 gp41 inhibitors. Bioorg Med Chem 2008;16(6):3039-3048.##Furuta RA, Nishikawa M, Fujisawa J. Real-time analysis of human immunodeficiency virus type 1 Env-mediated membrane fusion by fluorescence resonance energy transfer. Microbes Infect 2006;8(2):520-532.##Rutenber E, Fauman EB, Keenan RJ, Fong S, Furth PS, Ortiz de Montellano PR, et al. Structure of a non-peptide inhibitor complexed with HIV-1 protease. Developing a cycle of structure-based drug design. J Biol Chem 1993;268(21):15343-15346.##Melikyan GB, Platt EJ, Kabat D. The role of the N-terminal segment of CCR5 in HIV-1 Env-mediated membrane fusion and the mechanism of virus adaptation to CCR5 lacking this segment. Retrovirology 2007;4:55.##Liu S, Wu S, Jiang S. HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds. Curr Pharm Des 2007;13(2):143-162.##Jiang S, Lu H, Liu S, Zhao Q, He Y, Debnath AK. N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob Agents Chemother 2004;48(11):4349-4359.##Frey G, Rits-Volloch S, Zhang XQ, Schooley RT, Chen B, Harrison SC. Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion. Proc Natl Acad Sci USA 2006;103:13938-13943. ##Jiang S, Zhao Q, Debnath AK. Peptide and non-peptide HIV fusion inhibitors. Curr Pharm Des 2002;8(8):563-580.##He Y, Liu S, Jing W, Lu H, Cai D, Chin DJ, et al. Conserved residue Lys574 in the cavity of HIV-1 Gp41 coiled-coil domain is critical for six-helix bundle stability and virus entry. J Biol Chem 2007;282(35):25631-25639.##Wang H, Qi Z, Guo A, Mao Q, Lu H, An X, et al. ADS-J1 inhibits human immunodeficiency virus type 1 entry by interacting with the gp41 pocket region and blocking fusion-active gp41 core formation. Antimicrob Agents Chemother 2009;53(12):4987-4998.##Teissier E, Penin F, Pécheur EI. Targeting cell entry of enveloped viruses as an antiviral strategy. Molecules 2011;16:221-250.##Lee-Huang S, Huang PL, Zhang D, Lee JW, Bao J, Sun Y, Chang YT, et al. Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol: Part I. fusion [corrected] inhibition. Biochem Biophys Res Commun 2007;354(4):872-878.##Yao X, Chong H, Zhang C, Waltersperger S, Wang M, Cui S, et al. Broad antiviral activity and crystal structure of HIV-1 fusion inhibitor sifuvirtide. J Biol Chem 2012;287(9):6788-6796.##Jun Tan J, Kong R, Xin Wang C, Zu Chen W. Prediction of the binding model of HIV-1 gp41 with small molecule inhibitors. Conf Proc IEEE Eng Med Biol Soc 2005;5:4755-4758.##Wilton D. Computer simulation of molecular docking to screen compounds. Biosci Technol 2007.##Debnath AK, Radigan L, Jiang S. Structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1. J Med Chem 1999;42(17):3203-3209.##Davood A, Mansouri N, Rerza Dehpour A, Shafaroudi H, Alipour E, Shafiee A. Design, synthesis, and calcium channel antagonist activity of new 1,4-dihydropyridines containing 4-(5)-chloro-2-ethyl-5-(4)-imidazolyl substituent. Arch Pharm (Weinheim) 2006;339(6):299-304.##Si Z, Madani N, Cox JM, Chruma JJ, Klein JC, Schön A, et al. Small-molecule inhibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins. Proc Natl Acad Sci USA 2004;101(14):5036-5041.##Zabihollahi R, Sadat SM, Vahabpour R, Aghasadeghi MR, Memarnejadian A, Ghazanfari T, et al. Development of single-cycle replicable human immunodeficiency virus 1 mutants. Acta Virol 2011;55(1):15-22.##Lin PF, Blair W, Wang T, Spicer T, Guo Q, Zhou N, et al. A small molecule HIV-1 inhibitor that targets the HIV-1 envelope and inhibits CD4 receptor binding. Proc Natl Acad Sci USA 2003;100(19):11013-11018.##Jiang S, Lu H, Liu S, Zhao Q, He Y, Debnath AK. N-substituted pyrrole derivatives as novel human immunodeficiency virus type 1 entry inhibitors that interfere with the gp41 six-helix bundle formation and block virus fusion. Antimicrob Agents Chemother 2004;48(11):4349-4359.##Hanna GJ, Lalezari J, Hellinger JA, Wohl DA, Nettles R, Persson A, et al. Antiviral activity, pharmacokinetics, and safety of BMS-488043, a novel oral small-molecule HIV-1 attachment inhibitor, in HIV-1-infected subjects. Antimicrob Agents Chemother 2011;55(2):722-728.##Zhao Q, Ernst JT, Hamilton AD, Debnath AK, Jiang S. XTT formazan widely used to detect cell viability inhibits HIV type 1 infection in vitro by targeting gp41. AIDS Res Hum Retroviruses 2002;18(14):989-997.##Najmanovich RJ, Allali-Hassani A, Morris RJ, Dombrovsky L, Pan PW, Vedadi M, et al. Analysis of binding site similarity, small-molecule similarity and experimental binding profiles in the human cytosolic sulfotransferase family. Bioinformatics 2006;23:e104-109.##Seki J, Ikedac R, Hoshino H. Dimethyl sulfoxide and related polar compounds enhance infection of human T cells with HIV-1 in vitro. Biochem Biophys Res Commun 1996;227(3):724-729.##

Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate 2 2 Background: Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb. Methods: According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method. Results: Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l/day. According to the results, the produced mAb had similar affinity to HER2+ tumor cells to that of Herceptin. Conclusion: High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR− CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR. 87 95 Soudabeh Akbarzadeh-Sharbaf Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB) Iran Bagher Yakhchali Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB Iran Zarrin Minuchehr Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB) Iran Mohammad Ali Shokrgozar National Cell Bank, Pasteur Institute of Iran Iran Sirous Zeinali Department of Molecular Medicine, Biotechnology Research Center, Pasteur Institute of IranKawsar Human Genetics Research Center Kawsar Human Genetics Research Center IranIran Dihydrofolate reductase (DHFR)Monoclonal antibodyRecombinant proteinsTrastuzumab 113.pdf Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J. Recombinant humanized anti-HER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 1998;58(13):2825-2831. ##Pegram M, Hsu S, Lewis G, Pietras R, Beryt M, Sliwkowski M, et al. Inhibitory effects of combinations of HER-2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 1999;18(13):2241-2251.##Carter PJ. Potent antibody therapeutics by design. Nat Rev Immunol 2006;6(5):343-357.##Zhang H, Richter M, Greene MI. Therapeutic monoclonal antibodies for the ErbB family of receptor tyrosine kinases. Cancer Biol Ther 2003;2(4 Suppl 1):S122-126. ##Ross JS, Gray K, Gray GS, Worland PJ, Rolfe M. Anticancer antibodies. Am J Clin Pathol 2003;119(4):472-485.##Drebin JA, Link VC, Weinberg RA, Greene MI. Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen. Proc Natl Acad Sci USA 1986;83(23):9129-9133.##Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89(10):4285-4289. ##Slamon DJ, Romond EH, Perez EA. Advances in adjuvant therapy for breast cancer. Clin Adv Hematol Oncol 2006;4(3 Suppl 7):suppl 1, 4-9; discussion suppl 10; quiz 2 p following suppl.##Katsumata M, Okudaira T, Samanta A, Clark DP, Drebin JA, Jolicoeur P, et al. Prevention of breast tumour development in vivo by downregulation of the p185neu receptor. Nat Med 1995;1(7):644-648.##Romond EH, Perez EA, Bryant J, Suman VJ, Geyer CE, Jr., Davidson NE, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. N Engl J Med 2005;353(16):1673-1684. ##Simmons LC, Reilly D, Klimowski L, Raju TS, Meng G, Sims P, et al. Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies. J Immunol Methods 2002;263(1-2):133-147.##Xiong H, Ran Y, Xing J, Yang X, Li Y, Chen Z. Expression vectors for human-mouse chimeric antibodies. J Biochem Mol Biol 2005;38(4):414-419.##Chusainow J, Yang YS, Yeo JH, Toh PC, Asvadi P, Wong NS, et al. A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 2009;102(4):1182-1196.##Xiong KH, Liang QC, Xiong H, Zou CX, Gao GD, Zhao ZW, et al. Expression of chimeric antibody in mammalian cells using dicistronic expression vector. Biotechnol Lett 2005;27(21):1713-1717.##Jiang Z, Huang Y, Sharfstein ST. Regulation of recombinant monoclonal antibody production in chinese hamster ovary cells: a comparative study of gene copy number, mRNA level, and protein expression. Biotechnol Prog 2006;22(1):313-318.##Liu W, Wei H, Liang S, Zhang J, Sun R, Tian Z. A balanced expression of two chains of heterodimer protein, the human interleukin-12, improves high-level expression of the protein in CHO cells. Biochem Biophys Res Commun 2004;313(2):287-293.##Schlatter S, Stansfield SH, Dinnis DM, Racher AJ, Birch JR, James DC. On the optimal ratio of heavy to light chain genes for efficient recombinant antibody production by CHO cells. Biotechnol Prog 2005;21(1):122-133.##Urlaub G, Kas E, Carothers AM, Chasin LA. Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell 1983;33(2):405-412.##Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685. ##Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2[-Delta Delta C(T)] method. Methods 2001;25(4):402-408.##Kim NS, Byun TH, Lee GM. Key determinants in the occurrence of clonal variation in humanized antibody expression of cho cells during dihydrofolate reductase mediated gene amplification. Biotechnology Prog 2001;17(1):69-75. ##Lucas BK, Giere LM, DeMarco RA, Shen A, Chisholm V, Crowley CW. High-level production of recombinant proteins in CHO cells using a dicistronic DHFR intron expression vector. Nucleic Acids Res 1996;24(9):1774-1779.##Kim SJ, Lee GM. Cytogenetic analysis of chimeric antibody-producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng 1999;64(6):741-749. ##Mutskov V, Felsenfeld G. Silencing of transgene transcription precedes methylation of promoter DNA and histone H3 lysine 9. EMBO J 2004;23(1):138-149.##Richards EJ, Elgin SC. Epigenetic codes for heterochromatin formation and silencing: rounding up the usual suspects. Cell 2002;108(4):489-500.##Grohs BM, Niu Y, Veldhuis LJ, Trabelsi S, Garabagi F, Hassell JA, et al. Plant-produced trastuzumab inhibits the growth of HER2 positive cancer cells. J Agric Food Chem 2010;58(18):10056-10063.##Yusibov V, Streatfield SJ, Kushnir N. Clinical development of plant-produced recombinant pharmaceuticals: vaccines, antibodies and beyond. Human Vaccin 2011;7(3):313-321.##Pikaart MJ, Recillas-Targa F, Felsenfeld G. Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators. Genes Dev 1998;12(18):2852-2862.##

Preparation and Cytotoxic Evaluation of Magnetite (Fe3O4) Nanoparticles on Breast Cancer Cells and its Combinatory Effects with Doxorubicin used in Hyperthermia 2 2 Background: Magnetic nanoparticles in a variable magnetic field are able to produce heat. This heat (42-45°C) has more selective effect on fast dividing cancer cells than normal tissues. Methods: In this work magnetite nanoparticles have been prepared via co-precipitation and phase identification was performed by powder x-ray diffraction (XRD). Magnetic parameters of the prepared nanoparticles were measured by a Vibrating Sample Magnetometer (VSM). A sensitive thermometer has been used to measure the increase of temperature in the presence of an alternating magnetic field. To evaluate the cytotoxicity of nanoparticles, the suspended magnetite nanoparticles in liquid paraffin, doxorubicin and a mixture of both were added to the MDA-MB-468 cells in separate 15 ml tubes and left either in the RT or in the magnetic field for 30 min. Cell survival was measured by trypan blue exclusion assay and flow cytometer. Particle size distribution of the nanoparticles was homogeneous with a mean particles size of 10 nm. A 15°C temperature increase was achieved in presence of an AC magnetic field after 15 min irradiation. Results: Biological results showed that magnetite nanoparticles alone were not cytotoxic at RT, while in the alternative magnetic filed more than 50% of cells were dead. Doxorubicin alone was not cytotoxic during 30 min, but in combination with magnetite more than 80% of the cells were killed. Conclusion: It could be concluded that doxorubicin and magnetite nanoparticles in an AC magnetic field had combinatory effects against cells. 96 103 Hojjat Sadeghi-Aliabadi School of Pharmacy, Isfahan Pharmaceutical Research Center, Isfahan University of Medical Sciences School of Pharmacy, Isfahan Pharmaceutical Research Center, Isfahan University of Medical Sciences Iran Morteza Mozaffari Department of Physics, University of Isfahan Department of Physics, University of Isfahan Iran Behshid Behdadfar Department of Materials Engineering, Isfahan University of Technology Department of Materials Engineering, Isfahan University of Technology Iran Maryam Raesizadeh School of Pharmacy, Isfahan Pharmaceutical Research Center, Isfahan University of Medical Sciences School of Pharmacy, Isfahan Pharmaceutical Research Center, Isfahan University of Medical Sciences Iran Hamid Zarkesh-Esfahani Department of Biology, Faculty of Sciences, University of Isfahan Department of Biology, Faculty of Sciences, University of Isfahan Iran DoxorubicinFlow cytometryHyperthermiaMagnetite nanoparticles 114.pdf Goldman A. Modern ferrite technology. Pittsburgh: Springer, 2006. ##Bahadur D, Giri J, Nayak BB, Sriharsha T, Pradhan P, Prasad NK, et al. Processing, properties and some novel applications of magnetic nanoparticles. PRAMANA J Phys 2005;65(4):663-679. ##Jurgons R, Seliger C, Hilpert A, Trahms L, Odenbach S, Alexiou C. Drug loaded magnetic nanoparticles for cancer therapy. J Phys: Condensed Matter 2006;18(38):S2893-S2902.##Hergt R, Dutz S, Müller R, Zeisberger M. Magnetic particle hyperthermia: nanoparticles magnetism and materials development for cancer therapy. J Phys: Condens Matter 2006;18:S2919-S2934.##Babincova M, Leszczynska D, Sourivong P, Cicmanec P, Babinec P. Superparamagnetic gel as a novel material for electromagnetically induced hyperthermia. J Magn Magn Mater 2001;225(1-2):109-112.##Kalambur VS, Han B, Hammer BE, Shield TW, Bischof JC. In vitro characterization of movement, heating and visualization of magnetic nanoparticles for biomedical applications. Nanotechnology 2005;16:1221-1233.##Wust P, Hildebrandt B, Sreenivasa G, Rau B, Gellermann J, Riess H, et al. Hyperthermia in combined treatment of cancer. Lancet Oncol 2002;3(8):487-497. ##Kong G, Dewhirst MW. Hyperthermia and liposomes. Int J Hyperthermia 1999;15:345-370.##Ghasemi E, Mirhabibi A, Edrissi M. Synthesis and rheological properties of an iron oxide ferrofluid. J Magn Magn Mater 2008;320(28):2635-2639.##Cullity BD. Introduction to magnetic materials. New York: Addison Wisely; 1972.##Mouroi M, Street R, McCormic PG, Amighian J. Magnetic properties of ultrafine MnFe2O4 powders prepared by mechanochemical processing. Phys Rev B 2001;63:184414.##Berry CC, Curtis ASG. Functionalisation of magnetic nanoparticles for application in biomedicine. J Phys D: Appl Phys 2003;36:R198-R206.##Lu AH, Salabas EL, Schüth F. Magnetic nanoparticles: synthesis, protection, functionalization, and application. Angew Chem Int Ed Engl 2007;46:1222-1244.##Willis AL, Turro NJ, O'Brien S. Spectroscopic characterization of the surface of iron oxide nanocrystals. Chem Mater 2005;17(24):5970-5975.##Torchilin V. Multifunctional pharmaceutical nanocarriers (Fundamental Biomedical Technologies). New York: Springer; 2008.##Chen B, Sun Q, Wang X, Gao F, Dai Y, Yin Y, et al. Reversal in multidrug resistance by magnetic nanoparticle of Fe3O4 loaded with adriamycin and tetrandrine in K562/A02 leukemic cells. Int J Nanomedicine 2008;3:277-286.##Wang X, Zhang R, Wu C, Dai Y, Song M, Gutmann S, et al. The application of Fe3O4 nanoparticles in cancer research: a new strategy to inhibit drug resistance. J Biomed Mater Res A 2007;80:852-860.##Jayakumar OD, Ganguly R, Tyagi AK, Chandrasekharan DK, Krishnan N, Cherupally K. Water dispersible Fe3O4 nanoparticles carrying doxorubicin for cancer therapy. J Nanosci Nanotechnol 2009;9:6344-6348. ##Yazdi MH, Najafi ZN, Khorramizadeh MR, Amini M, Shahverdi AR. Fabrication of docetaxel surfaced Fe3O4 magnetite nanoparticles and their cytotoxicity on 4 T1 breast cancer cells. DARU J Pharm Sci 2012;20:15-19.##Gould p. Nanomagnetism shows in vivo potentials. Nanotoday 2006;1(4):34-39.##Babincová M, Sourivong P, Leszczynska D, Babinec P. Blood-specific whole-body electromagnetic hyperthermia. Med Hyptoth 2000;55:459-460.##

Comparison of Proliferative and Multilineage Differentiation Potential of Sheep Mesenchymal Stem Cells Derived from Bone Marrow, Liver, and Adipose Tissue 2 2 Background: Despite major progress in our general knowledge related to the application of adult stem cells, finding alternative sources for bone marrow Mesenchymal Stem Cells (MSCs) has remained to be challenged. In this study successful isolation, multilineage differentiation, and proliferation potentials of sheep MSCs derived from bone marrow, adipose tissue, and liver were widely investigated. Methods: The primary cell cultures were prepared form tissue samples obtained from sheep 30-35 day fetus. Passage-3 cells were plated either at varying cell densities or different serum concentrations for a week. The Population Doubling Time (PDT), growth curves, and Colony Forming Unit (CFU) of MSCs was determined. The stemness and trilineage differentiation potential of MSCs were analyzed by using molecullar and cytochemical staining approaches. The data was analyzed through one way ANOVA using SigmaStat (ver.2). Results: The highest PDT and lowest CFU were observed in adipose tissue group compared with other groups (p<0.001). Comparing different serum concentrations (5, 10, 15, and 20%), irrespective of cell sources, the highest proliferation rate was achieved in the presence of 20% serum (p<0.001). Additionally, there was an inverse relation between cell seeding density at culture initiation and proliferation rate, except for L-MSC at 300 cell seeding density. Conclusion: All three sources of fetal sheep MSCs had the identical trilineage differentiation potential. The proliferative capacity of liver and bone marrow derived MSCs were similar at different cell seeding densities except for the higher fold increase in B-MSCs at 2700 cells/cm2 density. Moreover, the adipose tissue derived MSCs had the lowest proliferative indices. 104 117 Banafsheh Heidari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Abolfazl Shirazi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRDepartment of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRDepartment of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University IranIran Mohammad Mehdi Akhondi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Hossein Hassanpour 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 Bahareh Behzadi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Naderi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Ali Sarvari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Sara Borjian Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran LiverMesenchymal stromal cellsSheep 115.pdf Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284(5411):143-147.##Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 2000;28(8):875-884.##Giovannini S, Brehm W, Mainil-Varlet P, Nesic D. Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation 2008;76(2):118-129. ##Rentsch C, Hess R, Rentsch B, Hofmann A, Manthey S, Scharnweber D, et al. Ovine bone marrow mesenchymal stem cells: isolation and characterization of the cells and their osteogenic differentiation potential on embroidered and surface-modified polycaprolactone-co-lactide scaffolds. In Vitro Cell Dev Biol Animal 2010;46(7):624-634.##Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Res Ther 2007;9(1):204.##Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7(2):211-228. ##In 't Anker PS, Scherjon SA, Kleijburg-van der Keur C, de Groot-Swings GM, Claas FH, et al. Isolation of mesenchymal stem cells of fetal or maternal origin from human placenta. Stem Cells 2004;22(7):1338-1345.##Malgieri A, Kantzari E, Patrizi MP, Gambardella S. Bone marrow and umbilical cord blood human mesenchymal stem cells: state of the art. Int J Clin Exp Med 2010;3(4):248-269.##Goodwin HS, Bicknese AR, Chien SN, et al. Multilineage differentiation activity by cells isolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant 2001;7(11):581-588.##Panepucci RA, Siufi JL, Silva WA Jr, Proto-Siquiera R, Neder L, Orellana M, Rocha V, et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. Stem Cells 2004;22(7):1263-1278.##Giovannini S, Brehm W, Mainil-Varlet P, Nesic D. Multilineage differentiation potential of equine blood-derived fibroblast-like cells. Differentiation 2008;76(2):118-129. ##Williams JT, Southerland SS, Souza J, Calcutt AF, Cartledge RG. Cells isolated from adult human skeletal muscle capable of differentiating into multiple mesodermal phenotypes. Am Surg 1999;65(1):22-26.##Alexanian AR, Sieber-Blum M. Differentiating adult hippocampal stem cells into neural crest derivatives. Neuroscience 2003;118(1):1-5.##Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 2009;88(9):792-806. ##Campagnoli C, Roberts IA, Kumar S, Bennett PR, Bellantuono I, Fisk NM. Identification of mesenchymal stem/progenitor cells in human first-trimester fetal blood, liver, and bone marrow. Blood 2001;98(8):2396-2402.##In't Anker PS, Noort WA, Kruisselbrink AB, Scherjon SA, Beekhuizen W, Willemze R, et al. Nonexpanded primary lung and bone marrow-derived mesenchymal cells promote the engraftment of umbilical cord blood-derived CD34(+) cells in NOD/SCID mice. Exp Hematol 2003;31(10):881-889.##Mensing N, Gasse H, Hambruch N, Haeger JD, Pfarrer C, Staszyk C. Isolation and characterization of multipotent mesenchymal stromal cells from the gingiva and the periodontal ligament of the horse. BMC Vet Res 2011;7:42.##Raoufi MF, Tajik1P, Dehghan MM, Eini F, Barin A. Isolation and differentiation of mesenchymal stem cells from bovine umbilical cord blood. Reprod Domest Anim 2011;46(1):95-99.##McCulloch EA, Till JE. The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice. Radiat Res 1960;13(1):115-125.##Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteogenesis in transplants of bone marrow cells. J Embryol Exp Morphol 1966;16(3):381-390. ##Friedenstein AJ. Stromal mechanisms of bone marrow: cloning in vitro and retransplantation in vivo. Haematol Blood Transfus 1980;25:19-29.##Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13(12):4279-4295.##Kern S, Eichler H, Stoeve J, Klüter H, Bieback K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006;24(5):1294-1301. ##Suzuki A, Zheng YW, Kondo R, Kusakabe M, Takada Y, Fukao K, et al. Flowcytometric separation and enrichment of hepatic progenitor cells in the developing mouse liver. Hepatology 2000;32(6):1230-1239.##Taniguchi H, Suzuki A, Zheng Y, Kondo R, Takada Y, Fukunaga K, et al. Usefulness of flowcytometric cell sorting for the enrichment of hepatic stem and progenitor cells in the liver. Transplant Pro 2000;32(2):249-251.##Fiegel HC, Lange C, Kneser U, Lambrecht W, Zander AR, Rogiers X, et al. Fetal and adult liver stem cells for liver regeneration and tissue engineering. J Cell Mol Med 2006;10(3):577-587.##Chen PM, Yen ML, Liu KJ, Sytwu HK, Yen BL. Immunomodulatory properties of human adult and fetal multipotent mesenchymal stem cells. J Biomed Sci 2011;18:49.##Moreno R, Martínez-González I, Rosal M, Farwati A, Gratacós E, Aran JM. Characterization of mesenchymal stem cells isolated from the rabbit fetal liver. Stem Cells Dev 2010;19(10):1579-1588.##Petersen BE, Bowen WC, Patrene KD, Mars WM, Sullivan AK, Murase N, et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284(5471):1168-1170.##Schwartz RE, Reyes M, Koodie L, Jiang Y, Blackstad M, Lund T, et al. Multipotent adult progenitor cells from bone marrow differentiate into functional hepatocyte-like cells. J Clin Invest 2002;109(10):1291-1302.##Wagner W, Horn P, Castoldi M, Diehlmann A, Bork S, Saffrich R, et al. Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 2008;3(5):e2213.##Eslaminejad MB, Falahi F, Nazarian H, Taghiyar L, Daneshzadeh MT. Differentiation potential and culture requirements of mesenchymal stem cells from ovine bone marrow for tissue regeneration applications. IJVS 2007;2(5):53-65.##Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, Prockop DJ. Expansion of human adult stem cells from bone marrow stroma:conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 2002;20(6):530-541.##Jung S, Panchalingam KM, Rosenberg L, Behie LA. Ex vivo expansion of human mesenchymal stem cells in defined serum-free media. Stem Cells Int 2012; ID 123030, doi:10.1155/2012/123030.##Solchaga LA, Penick K, Porter JD, Goldberg VM, Caplan AI, Welter JF. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J Cell Physiol 2005;203(2):398-409.##Qian L, Saltzman WM. Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture surface modification. Biomaterials 2004;25(7-8):1331-1337.##Miyazaki M, Hardjo M, Masaka T, Tomiyama K, Mahmut N, Medina RJ, et al. Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure. Stem Cells 2007;25(11):2855-2863. ##Peterbauer-Scherb A, van Griensven M, Meinl A, Gabriel C, Redl H, Wolbank S. Isolation of pig bone marrow mesenchymal stem cells suitable for one-step procedures in chondrogenic regeneration. J Tissue Eng Regen Med 2010;4(6):485-490.##Alge DL, Zhou D, Adams LL, Wyss BK, Shadday MD, Woods EJ, et al. Donor-matched comparison of dental pulp stem cells and bone marrow-derived mesenchymal stem cells in a rat model. J Tissue Eng Regen Med 2010;4(1):73-81.##Eslaminejad MB, Nazarian H, Falahi F, Taghiyar L, Daneshzadeh MT. Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow. Iran J Basic Med Sci 2009;12(2):70-79.##Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64(2):278-294.##Bianco P, Riminucci M, Gronthos S, Robey PG. Bone marrow stromal stem cells: nature, biology, and potential applications. Stem Cells 2001;19(3):180-192. ##Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I. Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol 2008;36(9):1176-1185.##Schmelzer E, Zhang L, Bruce A, Wauthier E, Ludlow J, Yao HL, et al. Human hepatic stem cells from fetal and postnatal donors. J Exp Med 2007;204(8):1973-1987. ##Eslaminejad MB, Mardpour S, Ebrahimi M. Mesenchymal stem cells derived from rat epicardial versus epididymal adipose tissue. Iran J Basic Med Sci 2010;14(1):25-34.##Burgener A, Butler M. Medium development for cell culture technology for pharmaceutical and cell-based therapies. Fla, USA: CRC Press, Boca Raton; 2006. P. 41-79. ##Mannello F, Tonti GA. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold!. Stem Cells 2007;25(7):1603-1609.##Wang JJ ,Ye F, Cheng LJ, Shi YJ, Bao J, Sun HG, et al. Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor. J Zhejiang Univ Sci B 2009;10(5):355-367. ##Friedman MS, Long MW, Hankenson KD. Osteogenic differentiation of human mesenchymal stem cells is regulated by bone morphogenetic protein-6. J Cell Bio 2006;98(3):538-554.##Eslaminejad MB, Nazarian H, Taghiyar L. Mesenchymal stem cells with high growth rate in the supernatant medium from rat bone marrow primary culture. J Babol Uni Med Sci 2008;10(2):12-22.##Eslaminejad MB, Nikmahzar A, Taghiyar L, Nadri S, Massumi M. Murine mesenchymal stem cells isolated by low density primary culture system. Dev Growth Differ 2006;48(6):361-370.##

Expression of Shigella flexneri ipaB Gene in Tobacco 2 2 Background: Shigellosis is a leading cause of diarrhea in many developing countries and although the disease can be controlled and managed with antibiotics, the constant emergence of resistant species requiring ever newer antibacterial drugs make development of an effective vaccine necessary. The bacteria are highly contagious and since immunity to Shigella is serotype-specific a multi-serotype vaccine is required for adequate protection. Proteins encoded by Shigella invasion plasmid, which are part of the Type Three Secretion System (TTSS) of this bacteria, are good candidate as vaccine targets since they are both immunogenic and conserved between different Shigella species. The advent of molecular farming, which is a low cost system, has opened up new venues for production of recombinant proteins. In view of the difficulties encountered in expressing IpaB in Escherichia coli (E. coli), the feasibility of the expression of this protein in tobacco has been investigated. Methods: The ipaB gene was cloned in place of the Hygromycin gene in pCambia1304 containing GFP as a reporter gene. The vector was then transferred into competent Agrobacterium tumefaciens (A. tumefaciens) strain LBA4404 which was used for agro-infiltration of Nicotiana tobaccum (N. tobaccum) leaves. Transformation was confirmed by expression of GFP. The gene was also cloned in pBAD/geneIII A and transformed E. coli host containing the construct was induced using different amounts of L-arabinose as inducer. Expression of IpaB gene by both hosts was determined by Western blotting using anti-IpaB monoclonal antibody. Results: The data obtained showed that IpaB was expressed in plant leaves but expression in E. coli was not detectable. Conclusion: This study showed that N. tobaccum is capable of expressing this protein without its specific chaperon and in levels detectable by Western blotting. 118 124 Mandana Ohadi Molecular Biology Unit, Pasteur Institute of Iran Iran Rahimeh Rasouli Molecular Biology Unit, Pasteur Institute of Iran Iran Elham Darzi-Eslam Molecular Biology Unit, Pasteur Institute of Iran Iran Anis Jafari Molecular Biology Unit, Pasteur Institute of Iran Iran Parastoo Ehsani Molecular Biology Unit, Pasteur Institute of Iran Iran <i>Escherichia coli</i>Recombinant proteinsShigellosisTobacco 116.pdf Niyogi SK. Shigellosis. J Microbiol 2005;43(2):133-143.##WHO. Shigellosis: disease burden, epidemiology and case management. Wkly Epidemiol Rec 2005;80:93-100.##Martinez-Becerra FJ, Kissmann JM, Diaz-McNair J, Choudhari SP, Quick AM, Mellado-Sanchez G, et al. Broadly protective Shigella vaccine based on type III secretion apparatus protein. Infect Immun 2012;80(3):1222-1231.##Fontaine A, Arondel J, Sansonetti PJ. Role of Shiga toxin in the pathogenesis of bacillary dysentery, studied by using a Tox- mutant of Shigella dysenteriae 1. Infect Immun 1988;56(12):3099-3109.##WHO Initiative for Vaccine Research (IVR). Diarrhoeal diseases. 2009. Available from: http://www.who.int/vaccine_research/diseases/diarrhoeal/en/index.html.##Wong MR, Reddy V, Hanson H, Johnson KM, Tsoi B, Cokes C, et al. Antimicrobial resistance trends of Shigella serotypes in New York City, 2006-2009. Microb Drug Resist 2010;16(2):155-161.##Ye C, Lan R, Xia S, Zhang J, Sun Q, Zhang S, et al. Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri. J Clin Microbiol 2010;48(2):419-426.##McKenzie R, Walker RI, Nabors GS, Van De Verg LL, Carpenter C, Gomes G, et al. Safety and immunogenicity of an oral, inactivated, whole-cell vaccine for Shigella sonnei: preclinical studies and a Phase I trial. Vaccine 2006;24(18):3735-3745.##Barnoy S, Jeong KI, Helm RF, Suvarnapunya AE, Ranallo RT, Tzipori S, et al. Characterization of WRSs2 and WRSs3, new second-generation virG (icsA)-based Shigella sonnei vaccine candidates with the potential for reduced reactogenicity. Vaccine 2010;28(6):1642-1654.##Katz DE, Coster TS, Wolf MK, Trespalacios FC, Cohen D, Robins G, et al. Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun 2004;72(2):923-930.##Kotloff KL, Pasetti MF, Barry EM, Nataro JP, Wasserman SS, Sztein MB, et al. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1trial of CVD 1204 and CVD 1208. J Infect Dis 2004;190:1745-1754.##Rahman KM, Arifeen SE, Zaman K, Rahman M, Raqib R, Yunus M et al. Safety, dose, immunogenicity, and transmissibility of an oral live attenuated Shigella flexneri 2a vaccine candidate (SC602) among healthy adults and school children in Matlab, Bangladesh. Vaccine 2011;29(6):1347-1354.##Kotloff KL, Losonsky AG, Nataro JP, Wasserman SS, Hale TL, Taylor ND, et al. Evaluation of the safety, immunogenicity, and efficacy in healthy adults of four doses of live oral hybrid Escherichia coli-Shigella flexneri 2a vaccine strain EcSf2a-2. Vaccine 1995;13(5):495-502.##Cohen D, Ashkenazi S, Green M, Lerman Y, Slepon R, Robin G, et al. Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers. Infect Immun 1996;64(10):4074-4077.##Passwell JH, Harlev E, Ashkenazi S, Chu C, Miron D, Ramon R, et al. Safety and immunogenicity of improved Shigella O-specific polysaccharide-protein conjugate vaccines in adults in Israel. Infect Immun 2001;69(3):1351-1357.##Fries LF, Montemarano AD, Mallett CP, Taylor DN, Hale TL, Lowell GH. Safety and immunogenicity of a proteosome-Shigella flexneri 2a lipopolysaccharide vaccine administered intranasally to healthy adults. Infect Immun 2001;69(7):4545- 4553.##Tribble D, Kaminski R, Cantrell J, Nelson M, Porter C, Baqar S, et al. Safety and immunogenicity of a Shigella flexneri 2a Invaplex 50 intranasal vaccine in adult volunteers. Vaccine 2010;28(37):6076-6085.##Picking WL, Mertz JA, Marquart ME, Picking WD. Cloning, expression, and affinity purification of recombinant shigella flexneri invasion plasmid antigens IpaB and IpaC. Protein Expr Purif 1996;8(4):401-408.##Daniell H, Khan MS, Allison L. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends Plant Sci 2002;7(2):84-91.##Mett V, Farrance CE, Green BJ, Yusibov V. Plants as biofactories. Biologicals 2008; 36(6):354-358.##Daniell H. Medical molecular farming: expression of antibodies, biopharmaceuticals and edible vaccines in plants. London: Kluwer Academic Publishers; 2003.##Sambrook J, Fritsch ER, Maniatis T. Molecular cloning, a laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2003. ##Sheerman S, Bevan MW. A rapid transformation method for Solanum tuberosum using binary Agrobactrum tumefaciens vectors. Plant Cell Rep 1987;7(1):13-16.##D'Aoust MA, Lavoie PO, Belles-Isles J, Bechtold N, Martel M, Vézina LP. Transient expression of antibodies in plants using syringe agroinfiltration. Methods Mol Biol 2009;483:41-50.##Jurado P, de Lorenzo V, Fernandez LA. Thioredoxin fusions increase folding of a single chain Fv antibodies in the cytoplasm of Escherichia coli: evidence that chaperone activity is the prime effect of thioredoxin. J Mol Biol 2006;357(1):49-61.##Lammeli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685.##MacRaea AF, Preiszner J, Ng S, Bolla RI. Expression of His-tagged Shigella IpaC in Arabidopsis. J Biotechnol 2004;112(3):247-253.##Maghari BM, Ardekani AM. Genetically modified foods and social concerns. Avicenna J Med Biotech 2011;3(3):109-117.##Ma JK, Drake WMP, Christou P, The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 2003;4(10):794-805.##Fischer R, Schillberg S. Molecular farming, plant made pharmaceuticals and technical proteins. Weinheim: Wiley-VCH Verlag GmbH & Co; 2004. ##Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res 1987;15(20):8125-8148.##

Retraction: Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos 2 2 Background: Detection of Circulating Fetal Trophoblastic Cells (CFTC) by single cell genotyping not only allows to identify fetal cells from maternal blood, but also to characterize their bi-parental genome. Methods: We have tested intact fetal trophoblastes recovered at 4th to 10th weeks of gestation (WG) from blood (10 ml per mother) of 13 women after In Vitro Fertilization (IVF) and transfer of one or several embryos. Large cells isolated from blood were individually microdissected and studied by genetic fingerprinting with a mean number of 3 Short Tandem Repeats (STR) markers, known to be informative by testing paternal and maternal blood DNA. Results: CFTC were found in all mothers starting from the 5th WG. A mean number of 2.5 CFTC per ml of blood was found in all the analyzed samples collected at the different terms of pregnancy. All mothers who received the transfer of two or three embryos, including one who delivered twins and one with vanishing twin (identified by ultrasounds), were found to have CFTC with two or three different bi-parental genotypes, belonging to different embryos derived from the same parents. Conclusion: CFTC circulation is detectable starting from the 5th WG. A "vanishing twin" phenomenon frequently develops after IVF and transfer of multiple embryos, being undetectable by ultrasounds and revealed by genetic CFTC fingerprinting. 125 132 Hussein Mouawia Department of Biology, Faculty of Science, Lebanese UniversityParis Descartes University Department of Biology, Faculty of Science, Lebanese UniversityParis Descartes University Lebanon France Circulating fetal stem cellsDNA fingerprintingPregnant womenSingle cell PCRTwin 117.pdf Mujezinovic F, Alfirevic Z. Procedure-related complications of amniocentesis and chorionic villous sampling: a systematic review. Obstet Gynecol 2007;110(3):687-694.##Bianchi DW, Flint AF, Pizzimenti MF, Knoll JH, Latt SA. Isolation of fetal DNA from nucleated erythrocytes in maternal blood. Proc Natl Acad Sci USA 1990;87(9):3279-3283.##Herzenberg LA, Bianchi DW, Schröder J, Cann HM, Iverson GM. Fetal cells in the blood of pregnant women: detection and enrichment by fluorescence-activated cell sorting. Proc Natl Acad Sci USA 1979;76(3):1453-1455.##Holzgreve W, Garritsen H S, Ganshirt-Ahlert D. Fetal cells in the maternal circulation. J Reprod Med 1992;37(5):410-418.##Simpson JL, Jama ES. Isolating fetal cells from maternal blood. Advances in prenatal diagnosis through molecular technology. JAMA 1993;270 (19):2357-61.##Bianchi DW, Simpson JL, Jackson LG, Elias S, Holzgreve W, Evans MI, Dukes KA, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood: analysis of NIFTY I data. National Institute of Child Health and Development Fetal Cell Isolation Study. Prenat Diag 2002;22(7):609-615.##Wright CF, Chitty LS. Cell-free fetal DNA and RNA in maternal blood: implications for safer antenatal testing. BMJ 2009;339:b2451.##Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62(4):768-775.##Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996;93(2):705-708.##Beroud C, Karliova M, Bonnefont JP, Benachi A, Munnich A, Dumez Y, et al. Prenatal diagnosis of spinal muscular atrophy by genetic analysis of circulating fetal cells. Lancet 2003;361(9362): 1013-1014.##Mouawia H, Saker A, Jais JP, Benachi A, Bussières L, Lacour B, et al. Circulating trophoblastic cells provide genetic diagnosis in 63 fetuses at risk for cystic fibrosis or spinal muscular atrophy. Reprod Biomed Online 2012;25(5):508-520.##Saker A, Benachi A, Bonnefont JP, Munnich A, Dumez Y, Lacour B, et al. Genetic characterisation of circulating fetal cells allows non-invasive prenatal diagnosis of cystic fibrosis. Prenat Diagn 2006;26(10):906-916.##Vona G, Beroud C, Benachi A, Quenette A, Bonnefont JP, Romana S, et al. Enrichment, immunomorphological, and genetic characterization of fetal cells circulating in maternal blood. Am J Pathol 2002;160(1):51-58.##Zhang L, Cui X, Schmitt K, Hubert R, Navidi W, Arnheim N. Whole genome amplification from a single cell: implications for genetic analysis. Proc Natl Acad Sci USA. 1992;89(13):5847-5851.##Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350(9076):485-487.##Lun FM, Tsui NB, Chan KC, Leung TY, Lau TK, Charoenkwan P, et al. Noninvasive prenatal diagnosis of monogenic diseases by digital size selection and relative mutation dosage on DNA in maternal plasma. Proc Natl Acad Sci USA 2008;105(50):19920-19925.##Covone AE, Mutton D, Johnson PM, Adinolfi M.Trophoblast cells in peripheral blood from pregnant women. Lancet 1984;2(8407):841-843.##Covone AE, Kozma R, Johnson PM, Latt SA, Adinolfi M. Analysis of peripheral maternal blood samples for the presence of placenta-derived cells using Y-specific probes and McAb H315. Prenat Diagn 1988;8(8):591-607.##Tjoa ML, Delli-Bovi L, Johnson KL, Bianchi DW. Antibodies to trophoblast antigens HLA-G, placenta growth factor, and neuroD2 do not improve detection of circulating trophoblast cells in maternal blood. Fetal Diagn Ther 2007;22(2):85-89.##Pinborg A, Lidegaard O, la Cour Freiesleben N, Andersen AN. Consequences of vanishing twins in IVF/ICSI pregnancies. Hum Reprod 2005;20(10): 2821-2829. ##De Sutter P, Bontinck J, Schutysers V, Van der Elst J, Gerris J, Dhont M. First-trimester bleeding and pregnancy outcome in singletons after assisted reproduction. Hum Reprod 2006;21(7):1907-1911. ##Pinborg A. IVF/ICSI twin pregnancies: risks and prevention. Hum Reprod Update 2005;11(6):575-593. ##

Culture of Ovine IVM/IVF Zygotes in Isolated Mouse Oviduct: Effect of Basal Medium 2 2 Background: The basal medium that supports Isolated Mouse Oviduct (IMO) is important for supporting embryo development and quality. Methods: The culture of ovine IVM/IVF zygotes was done in IMO using SOFaaciBSA and SOFaaBSA as basal medium of IMO and in SOFaaBSA alone as control. For preparation of IMO mature inbred strain C57BL/6 female mice were synchronized and mated with vasectomized males. The females with vaginal plug were sacriﬁced and the zygotes were transferred in to the isolated oviduct at 20 hpi. The oviducts were cultured with SOFaaciBSA and SOFaaBSA for 6 days. Another group of zygotes were cultured in SOFaaBSA alone as control. Results: Culture of zygotes in the IMO with SOFaaciBSA and SOFaaBSA, did not significantly affect the development and quality of embryos (p>0.05). The hatching rate, total and trophectoderm cells number in IMO groups’ blastocysts were significantly higher than SOFaaBSA alone. The morphological appearance of IMO blastocysts was superior to SOFaaBSA alone. When the quality of oocytes was poor, IMO could better support ovine embryo development either with SOFaaBSA or SOFaaciBSA than SOFaaBSA alone and there was a significant difference in blastocyst formation at day 6 with SOFaaBSA alone. Conclusion: The culture of ovine IVM/IVF zygotes in IMO using two highly efficient ruminant embryo culture media not only could support development of ovine embryos similar to the level in non IMO culture system (SOFaaBSA alone) but also could improve the quality of resulting embryos. Additionally, IMO could better support the development of ovine embryos derived from poor quality oocytes compared to the SOFaaBSA alone. 133 137 Abbas Farahavar Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Iran Abolfazl Shirazi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRResearch Institute of Animal Embryo Technology, Shahrekord University Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRResearch Institute of Animal Embryo Technology, Shahrekord University IranIran Hamid Khoram Department of Animal Science, Faculty of Agriculture and Natural Resources, University of TehranDepartment of Clinical Sciences, Faculty of Veterinary Medicine, University of Shahid Chamran Department of Animal Science, Faculty of Agriculture and Natural Resources, University of TehranDepartment of Clinical Sciences, Faculty of Veterinary Medicine, University of Shahid Chamran IranIran Ahmad Zareh Shahneh Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Iran Ali Sarvari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Naderi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Sara Borjian Boroujeni Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mahdi Zhandi Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Department of Animal Science, Faculty of Agriculture and Natural Resources, University of Tehran Iran EmbryoMiceOrgan culture techniqueOvine 118.pdf izos D, Ward F, Duffy P, Boland MP, Lonergan P. Consequences of bovine oocyte maturation, fertilization or early embryo development in vitro versus in vivo: implications for blastocyst yield and blastocyst quality. Mol Reprod Dev 2002;61(2): 234-248.##Whittingham D. Development of zygotes in cultured mouse oviducts. I. The effect of varying oviductal conditions. J Exp Zool 1968;169(4):391-397.##Minami N, Bavister BD, Iritani A. Development of hamster two-cell embryos in the isolated mouse oviduct in organ culture system. Gamete Res 1988;19(3):235-240.##Krisher RL, Petters RM, Johnson BH, Bavister BD, Archibong AE. Development of porcine embryos from the one-cell stage to blastocyst in mouse oviducts maintained in organ culture. J Exp Zool 1989;249(2):235-239.##Rizos D, Pintado B, de la Fuente J, Lonergan P, Gutiérrez-Adán A. Development and pattern of mRNA relative abundance of bovine embryos cultured in the isolated mouse oviduct in organ culture. Mol Reprod Dev 2007;74(6):716-723.##Sharif H, Vergos E, Lonergan P, Gallagher M, Kinis A, Gordon I. Development of early bovine embryos in the isolated mouse oviduct maintained in organ culture. Theriogenology 1991;35:270.##Sparks AE, Gwazdauskas FC, McGilliard ML. Culture of one-cell bovine embryos in explanted mouse oviduct and bovine oviductal epithelial cells. Theriogenology 1992; 37(3):587-594.##Goodridge A. Regulation of fatty acid synthesis in isolated hepatocytes. Evidence for a physiological role for long chain fatty acyl coenzyme A and citrate. J Biol Chem 1973;248(12):4318-4326.##Gray CW, Morgan PM, Kane MT. Purification of embryotrophic factor from commercial bovine serum albumin and its identification as citrate. J Reprod Fertil 1992;94(1):471-480.##Holm P, Booth PJ, Schmidt MH, Greve T, Callesen H. High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myoinositol with or without serum proteins. Theriogenology 1999;52(4):683-700.##Downes C, MacPhee CH. Myo-inositol metabolites as cellular signals. Eur J Biochem 1990;193(1):1-18.##Chiu T, Tam PP. A correlation of the outcome of clinical in vitro fertilization with the inositol content and embryotrophic properties of human serum. J Assist Reprod Genet 1992;9(6):524-530.##Lonergan P, Rizos D, Gutierrez-Adan A, Fair T, Boland MP. Oocyte and embryo quality: effect of origin, culture conditions and gene expression patterns. Reprod Domest Anim 2003;38(4):259-267.##Bindon B. Systematic studs’ of preimplantation stages of pregnancy in the sheep. Aust J Biol Sci 1971;24(1):131-147.##Yuan YQ, Van Soom A, Coopman FO, Mintiens K, Boerjan ML, Van Zeveren A, et al. Influence of oxygen tension on appoptosis and hatching on bovine embryos cultured in vitro. Theriogenology 2003;59(7):1585-1596.##Holm P, Booth PJ, Callesen H. Kinetics of early in vitro development of bovine in vivo- and in vitro-derived zygotes produced and/or cultured in chemically defined or serum-containing media. Reproduction 2002;123(4): 553-565.##Van Soom A, Bols PEJ, Vanroose G, de Kruif A. A comparison between in vivo and in itro produced bovine embryos: Scientific and practical implications. Procceding of the 12th International Congress on Animal Reproduction; 1992 Aug 23-27; The Hague, The Netherlands. 1531-1533 p.##Van Soom A, Boerjan M, Ysebaert MT, De Kruif A. Cell allocation to the inner cell mass and the trophectoderm in bovine embryos cultured in two different media. Mol Reprod Dev 1996;45(2):171-182.##Im GS, Seo JS, Hwang IS, Kim DH, Kim SW, Yang BC, et al. Development and apoptosis of pre-implantation porcine nuclear transfer embryos activated with different combination of chemicals. Mol Reprod Dev 2006;73(9): 1094-1101.##Viuff D, Hendriksen PJ, Vos PL, Dieleman SJ, Bibby BM, Greve T, et al. Chromosomal abnormalities and developmental kinetics in in vivo-developed cattle embryos at days 2 to 5 after ovulation. Biol Reprod 2001;65(1):204-208.##Knijn HM, Gjørret JO, Vos PL, Hendriksen PJ, van der Weijden BC, Maddox-Hyttel P, et al . Consequences of In Vivo Development and Subsequent Culture on Apoptosis, Cell Number, and Blastocyst Formation in Bovine Embryos. Biol Reprod 2003;69(4):1371-1378.##Thompson JG, Gardner DK, Pugh PA, McMillan WH, Tervit HR. Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol Reprod 1995; 53(6):1385-1391.##