Editorial 2 2 Virtual Institute of Medical Biotechnology (VIMB) (http://vimb.ir) is a website designed and developed in Iran to dedicate itself to providing the latest world news related to medical biotechnology. This web site also has accumulated a wealth of information on: e-books, conferences, references, articles, dissertations, films and animations, journals and biotech companies in Iran. The website fills one of the major gaps in the area of medical biotechnology in the country and is now serving those interested in the subject, free of charge. Although over the past 20 years many institutes and organizations have established biotechnology laboratories and initiated hundreds of projects, unfortunately many were not aware of the activities or sometimes existence of each other in the country. With the help of Internet and dedicated staff at VIMB, all of us working in any organizations, societies, universities, and biotech companies in Iran can now visit this website and get many of the information needed instantly. Staff at the VIMB are truly doing a great job and I recommend all individuals working in the field of biotechnology/medical biotechnology to spend a little bit of time to discover this informative and helpful web site. Another exciting news in the field of Biotechnology in Iran has been the recent announcement of the first international biotechnology exposition “IranBiotech 2013” which is to be held in Tehran-Iran from 14-18 February 2013. The “IranBiotech 2013” has declared, for the first time, to bring all the major international and Iranian biotech companies, universities, financial organizations, governmental and private institutes in the field of biotechnology together. This is great news for all of us who are involved in research, education and business aspects of biotechnology in general and medical biotechnology in particular. For more detailed information, please see the website www.irbiotech.com. 159 159 Ali M. Ardekani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 168.pdf ####

Characterization and Functional Assessment of Mouse PPARγ1 Promoter 2 2 Background: Peroxisome Proliferator Activated Receptor gamma (PPARγ), a member of nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs, which are arisen by alternative promoter usage. There are two promoter regions upstream of PPARγ gene. A 3 kb fragment, containing several transcription factor binding sites, acts as PPARγ1 promoter region. Thus, expression pattern of PPARγ1 isoform is due to the potential transcription factors that could influence its promoter activity. PPARγ, Retinoid X Receptor (RXR) and Vitamin D Receptor (VDR), as nuclear receptors could influence PPARγ gene expression pattern during several differentiation processes. During neural differentiation, PPARγ1 isoform expression reaches to maximal level at neural precursor cell formation. Methods: A vast computational analysis was carried out to reveal the PPARγ1 promoter region. The putative promoter region was then subcloned upstream of an EGFP reporter gene. Then the functionality of PPARγ1 promoter was assessed in different cell lines. Results: Results indicated that Rosiglitazone increased PPARγ1 promoter regulated EGFP expression of neural precursor cells during Embryoid Body (EB) formation. Furthermore vitamin D reduced PPARγ1 promoter regulated EGFP expression of neural precursor cells during EB formation through binding to its receptor. Conclusion: This study suggests that there are potential response elements for PPAR/RXR and VDR/RXR heterodimers in PPARγ1 isoform promoter. Also VDR/RXR heterodimers may decrease PPARγ expression through binding to its promoter. 160 169 Liana Lachinani Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology Iran Kamran Ghaedi Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECRDepartment of Biology, School of Sciences, University of Isfahan Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECRDepartment of Biology, School of Sciences, University of Isfahan IranIran Somayeh Tanhaei Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Ahmad Salamian Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Fereshteh Karamali Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Abbas Kiani-Esfahani Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Farzaneh Rabiee Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Marjan Yaghmaei Department of Biology, Science and Research Branch, Islamic Azad University Department of Biology, Science and Research Branch, Islamic Azad University Iran Hossein Baharvand Department of Developmental Biology, University of Science and Culture, ACECRDepartment of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Department of Developmental Biology, University of Science and Culture, ACECRDepartment of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR IranIran Mohammad Hossein Nasr-Esfahani Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran PPAR gammaMouseGene expression 95.pdf Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass CK, Gonzalez FJ, et al. International Union of Pharmacology. LXI. Peroxisome proliferator-activated receptors. Pharmacol Rev 2006;58(4): 726-741.##Clarke SD, Thuillier P, Baillie RA, Sha X. Peroxisome proliferator-activated receptors: a family of lipid-activated transcription factors. Am J Clin Nutr 1999;70(4):566-571.##Berger J, Moller DE. The mechanisms of action of PPARs. Annu Rev Med 2002;53:409-435.##Fajas L, Debril MB, Auwerx J. Peroxisome proliferator-activated receptor-gamma: from adipogenesis to carcinogenesis. J Mol Endocrinol 2001;27 (1):1-9.##Ghoochani A, Shabani K, Peymani M, Ghaedi K, Karamali F, Karbalaei K, et al. The influence of peroxisome proliferator-activated receptor γ1 during differentiation of mouse embryonic stem cells to neural cells. Differentiation 2012;83(1):60-67.##Zhu Y, Qi C, Korenberg JR, Chen X, Noya D, Rao MS, et al. Structural organization of mouse peroxisome proliferator activated receptor γ (mPPARγ) gene: Alternative promoter use and different splicing yield two mPPARγ isoforms. Proc Natl Acad Sci USA 1995;92(17):7921-7925.##Fajas L, Auboeuf D, Raspe E, Schoonjans K, Lefebvre A, Saladin R, et al. The organization, promoter analysis, and expression of the human PPARγ gene. J Biol Chem 1997;272(30):18779-18789.##Baharvand H, Matthaei KI. Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains. In Vitro Cell Dev Biol Anim 2004;40(3-4):76-81.##Ostadsharif M, Ghaedi K, Nasr-Esfahani MH, Mojbafan M, Tanhaie S, Karbalaie K, et al. The expression of peroxisomal protein transcripts increased by retinoic acid during neuronal differentiation. Differentiation 2011;81(2):127-132.##Kapadia R, Yi JH, Vemuganti R. Mechanisms of anti-inflammatory and neuroprotective actions of PPAR-gamma agonists. Front Biosci 2008;13: 1813-1826.##Feige JN, Gelman L, Michalik L, Desvergne B, outputs: Peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions. Prog Lipid Res 2006;45(2):120-159.##Sertznig P, Seifert M, Tilgen W, Reichrath J. Activation of vitamin D receptor (VDR)- and peroxisome proliferator-activated receptor (PPAR)-signaling pathways through 1,25(OH)2D3 in melano ma cell lines and other skin-derived cell lines. Der-matoendocrinol 2009;1(4):232-238.##Levenson CW, Figueiroa SM. Gestational vitamin D deficiency: long-term effects on the brain. Nutr Rev 2008;66(12):726-729.##Garcion E, Wion-Barbot N, Montero-Menei CN, Berger F, Wion D. New clues about vitamin D functions in the nervous system. Trends Endocrinol Metab 2002;13(3):100-105.##Wood RJ. Vitamin D and adipogenesis: new molecular insights. Nutr Rev 2008;66(1):40-46.##

Production and Characterization of Mouse Monoclonal Antibodies Recognizing Human Pan-IgG Specific Conformational or Linear Epitopes 2 2 Background: Pan-IgG specific monoclonal antibodies (MAbs) are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. Methods: Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins (Igs) of other species was studied by indirect ELISA using serum samples collected from 9 animals. Results: Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity (100%) was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs cross reacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×108 M-1 and 0.96×107 M-1. Conclusion: Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels. 170 177 Fatemeh Hajighasemi Department of Immunology, School of Public Health, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Shahed University Department of Immunology, School of Public Health, Tehran University of Medical SciencesDepartment of Immunology, Faculty of Medicine, Shahed University IranIran Jalal Khoshnoodi Department of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, 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, Academic Center for Education, Culture and Research (ACECR) Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) IranIran Enzyme linked immunosorbent assayHybridomasImmunoglobulin isotypesMonoclonal antibody 96.pdf Abbas AK, Litchman AH, Pilla S. Cellular and molecular immunology. 6th ed. Philadelphia: WB Sanders; 2007##Burton DR, Gregory L, Jefferis R. Aspects of the molecular structure of IgG subclasses. Monogr Allergy 1987;19:7-35.##Bartel G, Wahrmann M, Exner M, Regele H, Schillinger M, Horl WH, et al. Determinants of the complement-fixing ability of recipient presensitization against HLA antigens. Transplantation 2007;83(6):727-733.##Kanapeckiene V, Kalibatas J, Redaitiene E, Ceremnych J. The association between cytomegalovirus infection and aging process. Medicina (Kaunas) 2007;43(5):419-424.##Iritani N, Seto T, Hattori H, Natori K, Takeda N, Kubo H, et al. Humoral immune responses against norovirus infections of children. J Med Virol 2007;79(8):1187-1193.##Karnowski A, Achatz-Straussberger G, Klockenbusch C, Achatz G, Lamers MC. Inefficient processing of mRNA for the membrane form of IgE is a genetic mechanism to limit recruitment of IgE-secreting cells. Eur J Immunol 2006;36(7):1917-1925. ##Teloni R, von Hunolstein C, Mariotti S, Donati S, Orefici G, Nisini R. Antibody classes & subclasses induced by mucosal immunization of mice with Streptococcus pyogenes M6 protein & oligodeoxynucleotides containing CpG motifs. Indian J Med Res 2004;119 Suppl:126-130. ##Altannavch Ts, Roubalova K, Broz J, Hruba D, Andel M. Serological markers of Chlamydia pneumoniae, cytomegalovirus and Helicobacter pylori infection in diabetic and non-diabetic patients with unstable angina pectoris. Cent Eur J Public Health 2003;11(2):102-106.##Rigopoulou EI, Davies ET, Pares A, Zachou K, Liaskos C, Bogdanos DP, et al. Prevalence and clinical significance of isotype specific antinuclear antibodies in primary biliary cirrhosis. Gut 2005;54(4):528-532.##Abd El-Aal AA, El-Arousy MH, El-Gendy AM, Tunkul Ael-K, Ismail SA, El-Badry AA. Early post-treatment immunoglobulin profile in human schistosomiasis. J Egypt Soc Parasitol 2005;35(1):167-180.##Singer RE, Moss K, Beck JD, Offenbacher S. Association of systemic oxidative stress with suppressed serum IgG to commensal oral biofilm and modulation by periodontal infection. Antioxid Redox Signal 2009;11(12):2973-2983.##Al-Ghamdi HS, Anil S. Serum antibody levels in smoker and non-smoker saudi subjects with chronic periodontitis. J Periodontol 2007;78(6):1043-1050. ##Khajuria A, Gupta A, Singh S, Malik F, Singh J, Suri KA, et al. RLJ-NE-299A: a new plant based vaccine adjuvant. Vaccine 2007;25(14):2706-2715.##Morais CG, Soares IS, Carvalho LH, Fontes CJ, Krettli AU, Braga EM. IgG isotype to C-terminal 19 kDa of Plasmodium vivax merozoite surface protein 1 among subjects with different levels of exposure to malaria in Brazil. Parasitol Res 2005;95(6):420-426. ##Chevrier MC, Chateauneuf I, Guerin M, Lemieux R. Sensitive detection of human IgG in ELISA using a monoclonal anti-IgG-peroxidase conjugate. Hybrid Hybridomics 2004;23(6):362-367.##Hajighasemi F, Shokri F. Generation and characterization of mouse hybridomas secreting monoclonal antibodies specific for human IgG3. Avicenna J Med Biotech 2009;1(1)19-26.##Edelman GM, Gally JA. Somatic recombination of duplicated genes. An hypothesis on the origin of antibody diversity. Proc Natl Acad Sci USA 1967;57(2):353-358.##Hajighasemi F, Khoshnoodi J, Shokri F. Development of two murine monoclonal antibodies recognizing human nG1m(a)-like isoallotypic markers. Hybridoma (Larchmt) 2008;27(6):473-479.##Loirat MJ, Gourbil A, Frioux Y, Muller JY, Blan-chard D. A murine monoclonal antibody directed against the Gerbich 3 blood group antigen. Vox Sang 1992;62(1):45-48.##Hajighasemi F, Gharagozlou S, Ghods R, Khoshnoodi J, Shokri F. Private idiotypes located on light and heavy chains of human myeloma proteins characterized by monoclonal antibodies. Hybridoma (Larchmt) 2006;25(6):329-335.##Hajighasemi F, Saboor-Yaraghi AA, Shokri F. Measurement of affinity constant of anti-human IgG monoclonal antibodies by an Elisa-based method. Iranian J Immunol 2004;1(3):154-161.##Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 1987;100(1-2):173-179.##Hadji-Ghasemi F, Gharagozlou S, Ghods R, Roohi A, Khoshnoodi J, Shokri F. Generation and characterization of a mouse monoclonal antibody with specificity similar to staphylococcal protein A (SPA). Hybrid Hybridomics 2003;22(1):33-39.##Reimer CB, Phillips DJ, Aloisio CH, Moore DD, Galland GG, Wells TW, et al. Evaluation of thirty-one mouse monoclonal antibodies to human IgG epitopes. Hybridoma 1984;3(3):263-275.##Jefferis R, Reimer CB, Skvaril F, de Lange G, Ling NR, Lowe J, et al. Evaluation of monoclonal antibodies having specificity for human IgG sub-classes: Results of an IUIS/WHO collaborative study. Immunol lett 1985;10(3-4):223-252. ##Girkontaite I, Leckiene M, Mauricas M. Immunochemical study of human immunoglobulin G Fc region. Cancer Biother Radiopharm 1996;11(1):87-96.##Jefferis R, Low J, Ling NR, Porter P, Senior S. Immunogenic and antigenic epitopes of immunoglobulins. I. Cross-reactivity of murine monoclonal antibodies to human IgG with the immunoglobulins of certain animal species. Immunology 1982;45(1):71-77. ##Phillips DJ, Wells TW, Reimer CB. Estimation of association constants of 42 monoclonal antibodies to human IgG epitopes using a fluorescent sequential- saturation assay. Immunol Lett 1987;17(2):159-168.##Carvalho FR, Silva DA, Cunha-Júnior JP, Souza MA, Oliveira TC, Béla SR, et al. Reverse enzyme-linked immunosorbent assay using monoclonal antibodies against SAG1-related sequence, SAG2A, and p97 antigens from Toxoplasma gondii to detect specific immunoglobulin G (IgG), IgM, and IgA antibodies in human sera. Clin Vaccine Immunol 2008;15(8):1265-1271. ##Hamilton RG, Morrison SL. Epitope mapping of human immunoglobulin-specific murine monoclonal antibodies with domain- switched, deleted and point-mutated chimeric antibodies. J Immunol Meth 1993;158(1):107-122. ##

Periplasmic Expression of a Novel Human Bone Morphogenetic Protein-7 Mutant in Escherichia coli 2 2 Background: Bone Morphogenetic Proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily, and play an important role in bone metabolism. Recombinant forms of BMP-2 and BMP-7 are the only BMPs used clinically. In this study the mature part of human bone morphogenetic protein-7 (BMP-7) was engineered through substitution of the BMP-7 N-terminal sequence by heparin-binding site of BMP-2. This targeted substitution was made to enhance the binding affinity of the novel protein to the extracellular matrix components such as heparin and heparan sulfate proteoglycans (HSPGs). Methods: The engineered protein was expressed in Escherichia coli (E.coli). The PelB signal sequence was used to translocate soluble pro¬teins into the periplasmic space of E.coli. The protein was purified from periplasmic extract using Ni-NTA chromatography and the SDS-PAGE and western blot analysis confirmed the successful expression of the novel protein. Results: The novel hBMP-7 mutant was produced as approximately 16 kDa monomer. It was found that the heparin binding of this protein was approximately 50% more than that of the wild-type at a protein concentration of 500 ng/ml. Conclusion: The findings showed that the periplasmic expression may be suitable to produce complex proteins like BMPs. 178 185 Leila Nematollahi Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Vahid Khalaj Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Seyedeh Maliheh Babazadeh Department of Microbiology, Islamic Azad University of Pharmaceutical Sciences Department of Microbiology, Islamic Azad University of Pharmaceutical Sciences Iran Azam Rahimpour Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Hoda Jahandar Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Fatemeh Davami Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Fereidoun Mahboudi Medical Biotechnology Department, Biotechnology Research Center, Pasteur Institute of Iran Iran Bone morphogenetic protein-7 (BMP-7)<i>Escherichia coli</i>Periplasmic expression 97.pdf Bustos-Valenzuela J, Halcsik E, Bassi E, Demasi M, Granjeiro J, Sogayar M. Expression, purification, bioactivity, and partial characterization of a recombinant human bone morphogenetic protein-7 produced in human 293T cells. Mol Biotechnol 2010;46(2):118-126.##Kim CK, Oh SD, Rhee JI, Lee EM, Yoon TR. Expression and purification of recombinant human bone morphogenetic protein-7 (rhBMP-7) in Bacillus subtilis. Biotechnol Bioprocess Eng 2010;15(5):830-836.##Lee DH, Baek HS, Lee MH, Park JC. Production of bone morphogenetic protein-7 using pET expression system. Curr Appl Phys 2005;5(5):422-425.##Chen L, Hung YD, Zhang YD. Expression and sec-retion of human bone morphogenetic protein-7 in pichia pastoris. Chin J Biotech 2006;22(6):907-914.##Dinopoulos H, Giannoudis PV. (iv) The use of bone morphogenetic proteins (BMPs) in long-bone non-unions. Curr Orthopaed 2007;21(4):268-279.##Alaoui-Ismaili MH, Falb D. Design of second generation therapeutic recombinant bone morphogenetic proteins. Cytokine Growth Factor Rev 2009;20(5-6):501-507.##Han D, Liu W, Ao Q, Wang G. Optimal delivery systems for bone morphogenetic proteins in orthopedic applications should model initial tissue repair structures by using a heparin-incorporated fibrin-fibronectin matrix. Med Hypotheses 2008;71(3):374-378.##Kübler N, Würzler K, Reuther J, Sieber E, Kirchner T, Seebald W. Effect of different factors on the bone forming properties of recombinant BMPs. Mund Kiefer Gesichtschir 2000;4(8):465-469.##Takada T, Katagiri T, Ifuku M, Morimura N, Kobayashi M, Hasegawa K, et al. Sulfated polysaccharides enhance the biological activities of bone morphogenetic proteins. J Biol Chem 2003;278(44):43229-43235.##Irie A, Takami M, Kubo H, Sekino-Suzuki N, Kasahara K, Sanai Y. Heparin enhances osteoclastic bone resorption by inhibiting osteoprotegerin ac-tivity. Bone 2007;41(2):165-174.##Johnson Z, Proudfoot A, Handel T. Interaction of chemokines and glycosaminoglycans: a new twist in the regulation of chemokine function with opportunities for therapeutic intervention. Cytokine Growth Factor Rev 2005;16(6):625-636.##Würzler K, Emmert J, Eichelsbacher F, Kübler N, Sebald W, Reuther J. Evaluation of osteoinductive potency of genetically modified BMP-2 variations. Mund Kiefer Gesichtschir 2004;8(2):83-92.##Depprich R, Handschel J, Sebald W, Kübler N, Würzler K. Comparison of the osteogenic activity of bone morphogenetic protein (BMP) mutants. Mund Kiefer Gesichtschir 2005;9(6):363-368.##Robbens J, De Coen W, Fiers W, Remaut E. Improved periplasmic production of biologically active murine interleukin-2 in Escherichia coli through a single amino acid change at the cleavage site. Process Biochem 2006;41(6):1343-1346.##Soares CRJ, Gomide FIC, Ueda EKM, Bartolini P. Periplasmic expression of human growth hormone via plasmid vectors containing the λPL promoter: use of HPLC for product quantification. Protein Eng 2003;16(12):1131-1138.##Balderas Hernández VE, Paz Maldonado LMT, Medina Rivero E, Barba de la Rosa AP, JiménezBremont JF, Ordoñez Acevedo LG, et al. Periplasmic expression and recovery of human inter-feron gamma in Escherichia coli. Protein Expr Purif 2008;59(1):169-174.##Ambrus A, Torocsik B, Adam-Vizi V. Periplasmic cold expression and one-step purification of human dihydrolipoamide dehydrogenase. Protein Expr Purif 2009;63(1):50-57.##Rastgar Jazii F, Karkhane A, Yakhchali B, Fatemi S, Deezagi A. A simplified purification procedure for recombinant human granulocyte macrophage-colony stimulating factor from periplasmic space of Escherichia coli. J Chromatogr B Analyt Technol Biomed Life Sci 2007;856(1-2):214-221.##Nossal NG, Heppel LA. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J Biol Chem 1966;241(13):3055-3062.##Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227(5259):680-685.##Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193(1):265-275.##Mahoney DJ, Whittle JD, Milner CM, Clark SJ, Mulloy B, Buttle DJ, et al. A method for the non-covalent immobilization of heparin to surfaces. Anal Biochem 2004;330(1):123-129.##Fisher MC, Li Y, Seghatoleslami MR, Dealy CN, Kosher RA. Heparan sulfate proteoglycans including syndecan-3 modulate BMP activity during limb cartilage differentiation. Matrix Biol 2006;25(1):27-39.##Granjeiro J, Oliveira R, Bustos-Valenzuela J, Sogayar M, Taga R. Bone morphogenetic proteins: from structure to clinical use. Braz J Med Biol Res 2005;38(10):1463-1473.##Carlisle E, Fischgrund JS. Bone morphogenetic proteins for spinal fusion. Spine J 2005;5(6 Suppl):S240-S249.##Ruppert R, Hoffmann E, Sebald W. Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity. Eur J Biochem 1996;237(1):295-302.##Sampath TK, Maliakal J, Hauschka P, Jones W, Sasak H, Tucker R, et al. Recombinant human osteogenic protein-1 (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimu-lates osteoblast proliferation and differentiation in vitro. J Biol Chem 1992;267(28):20352-20362.##Lee DH, Suh H, Han DW, Park BJ, Lee JW, Park JC. The effects of recombinant human BMP-7, prepared from a COS-7 expression system, on the proliferation and differentiation of rat newborn calvarial osteoblasts. Yonsei Med J 2003;44(4):593-601.##29. Swencki-Underwood B, Mills JK, Vennarini J, Boakye K, Luo J, Pomerantz S, et al. Expression and characterization of a human BMP-7 variant with improved biochemical properties. Protein Expr Purif 2008;57(2):312-319.##Vallejo LF, Brokelmann M, Marten S, Trappe S, Cabrera-Crespo J, Hoffmann A, et al. Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. J Biotechnol 2002;94(2):185-194.##Kirker-Head CA. Potential applications and delivery strategies for bone morphogenetic proteins. Adv Drug Deliv Rev 2000;43(1):65-92.##Kiany J, Zomorodipour A, Raji MA, Sanati MH. Construction of recombinant plasmids for periplasmic expression of human growth hormone in Escherichia coli under T7 and lac promoters. J Sci IR Iran 2003;14(4):311-316.##

Cloning and Expression of Leishmania infantum LPG3 Gene by the Lizard Leishmania Expression System 2 2 Background: Various prokaryotic and eukaryotic expression systems have been developed for the production of recombinant proteins. In the present study, we used a new protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host, for the expression of LPG3 gene from Leishmania infantum (L.infantum). Methods: The LPG3 gene was cloned in the expression cassette for integration into the small subunit of the ribosomal RNA locus of Lizard Leishmania genome by electroporation. Expression of the recombinant LPG3 protein was confirmed by western blotting and immunofluorescence staining. Results: Western blotting confirmed the expression and production of rLPG3 protein. Immunofluoresence analysis also revealed the staining throughout the cytoplasm of transfected parasites, indicating that the protein has been expressed. Conclusion: These results demonstrate that Leishmania cells can be suggested an expression system for the production of recombinant LPG3 (rLPG3) to further research in vaccine designing against leishmaniasis. 186 192 Leila Pirdel Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Iran Ahmad Zavaran Hosseini Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Iran Bahram Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Iran Manoochehr Rasouli Department of Immunology, Clinical Microbiology Research Centre, Shiraz University of Medical Sciences Department of Immunology, Clinical Microbiology Research Centre, Shiraz University of Medical Sciences Iran Mojgan Bandehpour Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Iran Sara Soudi Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University Iran Leishmania infantumLeishmaniaRecombinant proteinsVaccines 98.pdf Basile G, Peticca M. Recombinant protein expression in Leishmania tarentolae. Mol Biotechnol 2009;43(3):273-278.##Nagill R, Kuar S. Vaccine candidates for leishmaniasis: A review. Int Immunopharm 2011;11(10):1464-1488.##Goyal N, Duncan R, Selvapandiyan A, Debrabant A, Baig MS, Nakhasi HL. Cloning and characterization of angiotension converting enzyme related dipeptidylcarboxypeptidase from Leishmania dono-vani. Mol Biochem Parasitol 2006;145(2):147-157.##Raymond F, Biosvert S, Roy G, Ritt JF, Legare D, Isnard A, et al. Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species. Nucleic Acids Res 2011;40(3):1-17.##Ilg T, Demar M, Harbecke D. Phosphoglycan repeat-deficient Leishmania mexicana parasites remain infectious to macrophages and mice. J Biol Chem 2001;276(7):4988-4997.##Beverley SM, Turco SJ. Lipophosoglycan (LPG) and the identification of virulence genes in the protozoan parasite Leishmania. Trends Microbiol 1998;6(1):35-40.##Larreta R, Soto M, Alonso C, Requena JM. Leishmania infantum: Gene cloning of the GRP94 homologue, its expression as recombinant protein, and analysis of antigenicity. Exp Parasitol 2000;96(2):108-115.##Larreta R, Guzman F, Patarryo ME, Alonso C, Requena JM. Antigenic properties of the Leishmania infantum GRP94 and mapping of linear B-cell epitopes. Immunol Lett 2002;80(3):199-205.##Zhang WW, Charest H, Matlashewski G. The expression of biologically active human p53 in Leishmania cells: A novel eukaryotic system to produce recombinant proteins. Nucleic Acids Res 1995;23(20):4073-4080.##Clayton CE, Hausler T, Blattner J. Protein trafficking in kinetoplastid protozoa. Microbiol Review 1995;59(3):325-344.##Hemayatkar M, Mahboudi F, Majidzade K, Davami F, Vaziri B, Barkhordari F, et al. Increased expression of recombinant human tissue plasminogen activator in Leishmania tarentolae. Biotechnol J 2010;5(11):1198-1206.##Tobin JF, Reiner SL, Hatam F, Zheng S, Leptek CL, Writh DF, et al. Transfected leishmania expressing biologically active IFN-gamma. J Immunol 1993;150(11):5059-5069.##Phan HP, Sugino M, Niimi T. The production of recombinant human laminin-332 in a Leishmania tarentolae expression system. Protein Expr Purif 2009;68(1):79-84. ##Basak A, Shervani NJ, Mbikay M, Kolajova M. Recombinant proprotein convertase 4 (PC4) from Leishmania tarentolae expression system: purification, biochemical study and inhibitor design. Protein Expr Purif 2008;60(2):117-126.##Mirzaahmadi S, Asaadi Tehrani G, Bandehpour M, Davoudi N, Tahmasbi L, Mirzahoseini H, et al. Expression of recombinant human coagulation factor VII by the Lizard Leishmania expression system. J Biomed Biotechol 2011. 2011:873874. Epub 2011 Sep 8.##Kazemi B, Tahvildari GH, Feshareki SR, Javadian E. Isolation a lizard leishmania promastigote from its natural host in Iran. J Biol Sciences 2004;4:620-623.##Mißlitz A, Mottram JC, Overath P, Aebischer T. Targeted integration into a rRNA locus results in uniform and high level expression of transgenes in Leishmania amastigotes. Mol Biochem Parasitol 2000;107(2):251-261.##Tomoaki N. Recombinant protein production in the eukaryotic protozoa parasite Leishmania tarentolae: A review. In: Lorence A (eds). Recombinant gene expression: reviews and protocols. Berlin: Springer Science; 2012, 307-315.##Soleimani M, Mahboudi F, Davoudi N, Amanzadeh A, Azizi M, Adeli A, et al. Expression of human tissue plasminogen activator in the trypanosomatid protozoan Leishmania tarentolae. Biotechnol Appl Biochem 2007;48(Pt 1):55-61. ##Fernandez-Robledo JA, Vasta GR. Production of recombinant proteins from protozoan parasites. Trend Parasitol 2010;26(5):244-254.##Breitling R, Klingner S, Callewaert N, Pietrucha R, Anett Geyer A, Ehrlich G, et al. Non-pathogenic trypanosomatid protozoa as a platform for protein research and production. Protein Expr Purif 2002;25(2):209-218.##

Genotyping of Polymorphic Microsatellite Markers Linked to RB1 Locus in Iranian Population 2 2 Background: Retinoblastoma is the most common intraocular tumor in childhood and mutation in the RB1 gene will trigger the tumorigenesis. So far, a wide range of the mutations along the length of RB1 gene have been reported. However, some could not be detected by common detection methods. In such condition, linkage analysis using microsatellite markers is suggested to trace unknown RB1 mutations in the affected family. The aim of the present study was to evaluate the heterozygosity rates and genotyping of three microsatellite markers near or inside of the RB1 gene. Methods: Totally, 120 unrelated healthy people from Fardis, Karaj, Iran were recruited and from each participant genomic DNA was extracted from 5 ml of peripheral blood. Three microsatellite markers D13S153, D13S156 and D13S128 located within or adjacent to the RB1 gene were selected for linkage analysis. The reliability of microsatellite markers and linkage analysis were investigated in 10 members of 2 families with familial retinoblastoma. Results: Our results showed that heterozygosity rates for the three markers D13S153, D13S156 and D13S128 were 74, 70 and 78%, respectively. On the other hand, 2 and 36 out of 120 people were homozygote and heterozygous for all loci, respectively. Conclusion: Given the heterozygosity rates, it may be concluded that all microsatellite markers D13S153, D13S156 and D13S128 are informative and have a high rate of heterozygosity and sensitivity. Therefore, tracing the unknown RB1 mutated alleles using linkage analysis in Iranian family with familial retinoblastoma could be recommended by means of these three microsatellite markers. 193 199 Saman Mohamad Zahery Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture, and Research (ACECR)Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture, and Research (ACECR)Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) IranIran Kioomars Saliminejad Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Iran Hamid Reza Khorram Khorshid Genetic Research Center, University of Social Welfare and Rehabilitation Science Genetic Research Center, University of Social Welfare and Rehabilitation Science Iran Ali Ahani Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Iran Genetic markersGenotyping techniquesIranRetinoblastoma 99.pdf Gallie B. Canadian guidelines for retinoblastoma care. Can J Ophthalmol 2009;44(6):639-642.##Richter S, Vandezande K, Chen N, Zhang K, Sutherland J, Anderson J, et al. Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. Am J Hum Genet 2003;72(2):253-269.##Noorani HZ, Khan HN, Gallie BL, Detsky AS. Cost comparison of molecular versus conventional screening of relatives at risk for retinoblastoma. Am J Hum Genet 1996;59(2):301-307.##Mallipatna AC, Sutherland JE, Gallie BL, Chan H, Heon E. Management and outcome of unilateral retinoblastoma. J AAPOS 2009;13(6):546-550.##Gallie BL, Zhao J, Vandezande K, White A, Chan HS. Global issues and opportunities for optimized retinoblastoma care. Pediatr Blood Cancer 2007;49(7 Suppl):1083-1090.##O'Doherty M, Lanigan B, Breathnach F, O'Meara A, Gallie B, Chan H, et al. A retrospective review of visual outcome and complications in the treatment of retinoblastoma. Ir Med J 2005;98(1):17-20.##Dommering CJ, van den Heuvel MR, Moll AC, Imhof SM, Meijers-Heijboer H, Henneman L. Reproductive decision-making: a qualitative study among couples at increased risk of having a child with retinoblastoma. Clin Genet 2010;78(4):334-341.##Mitter D, Rushlow D, Nowak I, Ansperger-Rescher B, Gallie BL, Lohmann DR. Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma. Familial Cancer 2009;8(1):55-58.##Zielinski B, Gratias S, Toedt G, Mendrzyk F, Stange DE, Radlwimmer B, et al. Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization. Genes Chromosomes Cancer 2005;43(3):294-301.##Schuler A, Weber S, Neuhauser M, Jurklies C, Lehnert T, Heimann H, et al. Age at diagnosis of isolated unilateral retinoblastoma does not distinguish patients with and without a constitutional RB1 gene mutation but is influenced by a parent-of-origin effect. Eur J Cancer 2005;41(5):735-740.##Albrecht P, Ansperger-Rescher B, Schuler A, Zeschnigk M, Gallie B, Lohmann DR. Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression. Hum Mutat 2005;26(5):437-445.##Leone PE, Vega ME, Jervis P, Pestana A, Alonso J, Pazy-Mino C. Two new mutations and three novel polymorphisms in the RB1 gene in Ecuadorian patients. J Hum Genet 2003;48(12):639-641.##Zeschnigk M, Lohmann D, Horsthemke B. A PCR test for the detection of hypermethylated alleles at the retinoblastoma locus. J Med Genet 1999;36(10):793-794.##Dimaras H, Rushlow D, Halliday W, Doyle JJ, Babyn P, Abella EM, et al. Using RB1 mutations to assess minimal residual disease in metastatic retinoblastoma. Transl Res 2010;156(2):91-97.##Klutz M, Horsthemke B, Lohmann DR. RB1 gene mutations in peripheral blood DNA of patients with isolated unilateral retinoblastoma. Am J Hum Genet 1999;64(2):667-668.##Yilmaz S, Horsthemke B, Lohmann DR. Twelve novel RB1 gene mutations in patients with hereditary retinoblastoma. Mutations in brief no. 206. Online. Hum Mutat 1998;12(6):434.##Lohmann DR, Gerick M, Brandt B, Oelschlager U, Lorenz B, Passarge E, et al. Constitutional RB1-gene mutations in patients with isolated unilateral retinoblastoma. Am J Hum Genet 1997;61(2):282-294.##Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. The spectrum of RB1 germ-line mutations in hereditary retinoblastoma. Am J Hum Genet 1996;58(5):940-949.##Szijan I, Lohmann DR, Parma DL, Brandt B, Horsthemke B. Identification of RB1 germline mutations in Argentinean families with sporadic bilateral retinoblastoma. J Med Genet 1995;32(6):475-479.##Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. Spectrum of small length germline mutations in the RB1 gene. Hum Mol Genet 1994;3(12):2187-2193.##Ishak SR, Hanafi H, Alagaratnam JV, Zilfalil BA, Tajudin LS. RB pocket domain B mutation frequency in Malaysia. Ophthalmic Genet 2010;31(3):159-161.##Ahani A, Behnam B, Khorshid HR, Akbari MT. RB1 gene mutations in Iranian patients with retinoblastoma: report of four novel mutations. Cancer Genet 2011;204(6):316-322.##Rushlow D, Piovesan B, Zhang K, Prigoda-Lee NL, Marchong MN, Clark RD, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat 2009;30(5):842-851.##Emre S, Sungur A, Hazar V, Bilgic S, Buyukpamukcu M, Gunalp I. A linkage analysis in two families with bilateral retinoblastoma. Turk J Pediatr 1996;38(4):413-417.##Haines JL, Ozelius L, St George-Hyslop P, Wexler NS, Gusella JF, Conneally PM. Partial linkage map of chromosome 13q in the region of the Wilson disease and retinoblastoma genes. Genet Epidemiol 1988;5(6):375-380.##Alonso J, Garcia-Miguel P, Abelairas J, Mendiola M, Pestana A. A microsatellite fluorescent method for linkage analysis in familial retinoblastoma and deletion detection at the RB1 locus in retinoblastoma and osteosarcoma. Diagn Mol Pathol 2001;10(1):9-14.##Wilimas JA, Wilson MW, Haik BG, Barnoya M, Fu L, Castellanos M, et al. Development of retinoblastoma programs in Central America. Pediatr Blood Cancer 2009;53(1):42-46.##Onadim Z, Cowell JK. Application of PCR amplification of DNA from paraffin embedded tissue sections to linkage analysis in familial retinoblastoma. J Med Genet 1991;28(5):312-316.##Lahiri DK, Bye S, Nurnberger JI, Jr., Hodes ME, Crisp M. A non-organic and non-enzymatic extraction method gives higher yields of genomic DNA from whole-blood samples than do nine other methods tested. J Biochem Biophys Methods 1992;25(4):193-205.##Rabbani B, Khanahmad H, Bagheri R, Mahdieh N, Zeinali S. Characterization of minor bands of STR amplification reaction of FVIII gene by PCR cloning. Clin Chim Acta 2008;394(1-2):114-115.##Rabbani B, Rezaeian A, Khanahmad H, Bagheri R, Kamali E, Zeinali S. Analysing two dinucleotide repeats of FVIII gene in Iranian population. Haemophilia 2007;13(6):740-744.##Chunder N, Basu D, Roy A, Roychoudhury S, Panda CK. Prediction of retinoblastoma and osteosarcoma: linkage analysis of families by using polymorphic markers around RB1 locus. J BUON 2003;8(4):365-369.##Lee CC, Wu MC, Wu JY, Li TC, Tsai FJ, Tsai CH. Carrier detection of Duchenne/Becker muscular dystrophy by using fluorescent linkage analysis in Taiwan. Acta Paediatr Taiwan 2000;41(2):69-74.##Schwartz LS, Tarleton J, Popovich B, Seltzer WK, Hoffman EP. Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy. Am J Hum Genet 1992;51(4):721-729.##O'Leary CA, Duffy D, Biros I, Corley S, Seddon JM. Linkage analysis excludes the involvement of the canine PKD2 homologue in bull terrier polycystic kidney disease. Anim Genet 2006;37(5):527-528.##Onoe T, Konoshita T, Miyagi K, Yamada K, Mutoh H, Koni I. An efficient linkage analysis strategy for autosomal dominant polycystic kidney disease. Clin Nephrol 2003;59(6):406-414.##Emre S, Sungur A, Bilgic S, Buyukpamukcu M, Gunalp I, Ozguc M. Loss of heterozygosity in the VNTR region of intron 1 of P53 in two retinoblastoma cases. Pediatr Hematol Oncol 1996;13(3):253-256.##Kato MV, Ishizaki K, Ejima Y, Kaneko A, Tanooka H, Sasaki MS. Loss of heterozygosity on chromosome 13 and its association with delayed growth of retinoblastoma. Int J Cancer 1993;54(6):922-926.##Choy KW, Pang CP, Yu CB, Wong HL, Ng JS, Fan DS, et al. Loss of heterozygosity and mutations are the major mechanisms of RB1 gene inactivation in Chinese with sporadic retinoblastoma. Hum Mutat 2002;20(5):408.##

Medical Biotechnology Trends and Achievements in Iran 2 2 A healthcare system has been the most important priority for all governments worldwide. Biotechnology products have affected the promotion of health care over the last thirty years. During the last several decades, Iran has achieved significant success in extending healthcare to the rural areas and in reducing the rates of infant mortality and increasing population growth. Biomedical technology as a converging technology is considered a helpful tool to fulfill the Iranian healthcare missions. The number of biotechnology products has reached 148 in 2012. The total sales have increased to 98 billion USD without considering vaccines and plasma derived proteins in 2012. Iran is one of the leading countries in the Middle East and North Africa in the area of Medical biotechnology. The number of biotechnology medicines launched in Iran is 13 products until 2012. More than 15 products are in pipelines now. Manufacturers are expecting to receive the market release for more than 8 products by the end of 2012. Considering this information, Iran will lead the biotechnology products especially in area of biosimilars in Asia after India in next three years. The present review will discuss leading policy, decision makers’ role, human resource developing system and industry development in medical biotechnology. 200 205 Fereidoun Mahboudi Biotechnology Research Center, Pasteur Institute of Iran Iran Haleh Hamedifar CinnaGen Co. Iran Hamideh Aghajani Aryogen Co. Iran BiopharmaceuticsHuman resourcesIranMedical biotechnology 100.pdf Fári MG, Kralovánszky UP. The founding father of biotechnology: Károly (Karl) Ereky. Int J Hortic Sci 2006;12(1):9-12.##Young FE. Biotechnology: the view from the FDA. Health Matrix 1986;4(3):10-15.##http://www.fao.org/biotech/fao-statement-on-biotechnology/en/ [Online].##Rader RA. Paucity of biopharma approvals raises alarm, lower numbers, novelty, and economic impact indicate problems. GEN 2008.##Rader RA. FDA biopharmaceutical product ap-provals and trends. Biotechnology Information Institute, www.biopharma.com/approvals_2011 .html.##Sasson A. Medical biotechnology: achievements, prospects and preception. Tokyo, New York, Paris: United Nations University press; 2006.##Samadikuchaksaraei A, Mousavizadeh K. Hightech biomedical research: lessons from Iran's experience. BioMed Eng OnLine 2008;7(17):1-6.##

Amplification of GC-rich Putative Mouse PeP Promoter using Betaine and DMSO in Ammonium Sulfate Polymerase Chain Reaction Buffer 2 2 Background: Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. Methods: Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl2 was used. Results: The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. Conclusion: Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions. 206 209 Tahere Seifi Department of Biology, School of Sciences, University of Isfahan Department of Biology, School of Sciences, University of Isfahan Iran Kamran Ghaedi Department of Biology, School of Sciences, University of IsfahanDepartment of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Biology, School of Sciences, University of IsfahanDepartment of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR IranIran Ahmad Salamian Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Somayeh Tanhaei Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran Forouzan Safari Department of Biology, School of Sciences, University of Isfahan Department of Biology, School of Sciences, University of Isfahan Iran Zohreh Hojati Department of Biology, School of Sciences, University of Isfahan Department of Biology, School of Sciences, University of Isfahan Iran Manuchehr Tavassoli Department of Biology, School of Sciences, University of Isfahan Department of Biology, School of Sciences, University of Isfahan Iran Hossein Baharvand Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECRDepartment of Developmental Biology, University of Science and Culture, ACECR Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECRDepartment of Developmental Biology, University of Science and Culture, ACECR IranIran Mohammad Hossein Nasr-Esfahani Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Department of Cell and Molecular Biology, Cell Science Research Center, Royan Institute for Animal Biotechnology, ACECR Iran AmplificationGC-rich regionPeroxsiomal proteinPromoter 101.pdf Sahdev S, Saini S, Tiwari P, Sexena S, Saini KS. Amplification of GC-rich genes by following a combination strategy of primer design, enhancers and modified PCR cycle conditions. Mol Cell Probs 2007;21(4):303-307. ##Hube F, Reverdiau P, Lochmann S, Gruel Y. Improved PCR method for amplification of GC-rich DNA sequences. Mol Biotech 2005;31(1):81-84. ##Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun 2006;347(3):747-751.##Dutton CM, Paynton C, Sommer SS. General method for amplifying regions of high GC content. Nucleic Acids Res 1993;21(12):2953-2954.##Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I. Betaine, dimethyl sulfoxide, and 7-Deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn 2006;8(5):544-550.##Wei M, Deng J, Feng K, Yu B, Chen Y. Universal method facilitating the amplification of extremely GC-rich DNA fragments from genomic DNA. Anal Chem 2010;82(14):6303-6307. ##Henke W, Herdel K, Jung K, Schnorr D, Loenong SA. Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic acid Res 1997;25(19):3957-3958.##Jensen MA, Fukushima M, Davis RW. DMSO and betaine greatly improve amplification of GC-rich constructs in de novo synthesis. PLoS One 2010;5(6):e11024.##Zhang Z, Yang X, Meng l, Liu F, Shen C, Yang W. Enhanced amplification of GC-rich DNA with two organic reagents. Bio-techniques 2009;47(3):775-779.##Ferrer-Martinez A, Ruiz-Luzanp P, Chien KR. Mouse PEP: A novel peroxisomal protein linked to myoblast differentiation and development. Dev Dyn 2002;224(2):154-167. ##Bostrom P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, et al. A PGC-α-dependent myokine that derives brown-fat-like development of white fat and thermogenesis. Nature 2012;148 (7382):463-468.##Ostadsharif M, Ghaedi K, Nasr Esfahani MH, Mojbafan M, Tanhaie S, Karbalaie K, et al. Retinoic acid increased peroxisomal protein gene expression during neural differentiation. Differentiation 2011;81(2):127-132.##Sobocki T, Jayman F, Sobocka MB, Marmur JD, Banerjee P. Isolation, Sequencing, and Functional Analysis of the TATA-Less murine ATPase II promoter and structural analysis of the ATPase II gene. Biochem Biophys Acta 2007;1769(1):61-75. ##Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acid Res 2003;31(13):3406-3415. ##http://www.med.yale.edu/genetics/ward/tavi/p08.html.##http://www.protocol-online.org/biology-forums/posts/4854.html. ##http://wiki.answers.com/Q/why is a variable in PCR.##Brito M, Malta-Vacas J, Carmong B, Aires C, Costa P, Martins AP, et al. Polyglycine expansions in eRF3/GS PT1 are associated with gastric cancer susceptibility. Carcinogenesis 2005;26(12):2046-2049.##Malta-Vacas J, Chauvin C, Gonçalves L, Nazaré A, Carvalho C, Monteiro C, et al. eRF3a/GSPT1 12-GGC allele increases the susceptibility for breast cancer development. Oncol Rep 2009;21(6):1551-1558.##Malta-Vacas J, Ferreira P, Monteiro C, Brito M. Differential expression of GSPT1 GGCn alleles in cancer. Cancer Genet Cytogenet 2009;195(2):132-142.##Wijesekera LC, Leigh PN. Amyotrophic lateral sclerosis. Orphanet J Rare Dis 2009;4:3.##Fischbeck KH, Lieberman A, Bailey CK, Abel A, Merry DE. Androgen receptor mutation in Kennedy's disease. Philos Trans R Soc Lond B Biol Sci 1999;354(1386):1075-1078.##Chen CJ, Fischbeck KH. Clinical aspects and the genetic and molecular biology of Kennedy's disease. In: Wells RD, Warren ST, Sarmiento M. Genetic instabilities and hereditary neurological diseases. Boston: Academic Press; 2006, 211-222. ##Kennedy WR, Alter M, Sung JH. Progressive proximal spinal and bulbar muscular atrophy of late onset. A sex-linked recessive trait. Neurology 1968;18(7):671-680.##La Spada AR, Wilson EM, Lubahn DB, Harding AE, Fischbeck KH. Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature 1991;352(6330):77-79.##

Host-Microbiota Interaction is MyD88-Independent in the Intestinal Tract under Physiologic Condition 2 2 The role of microbiota in health and disease is the subject of rigorous investigation. Several studies have demonstrated that microbiota and the pattern-recognition receptors contribute to intestinal tumourigenesis; the exact mechanism of which is still obscure. MyD88 is the downstream effector of all Toll-like receptors (TLRs) except TLR3. However, the alternative MyD88-independent pathway is functional downstream of not only TLR3, but also TLR1/2, 2/6, 4, and 5. TLR4 stimulation with intraperitoneal lipopolysaccharide exerts distinct functional effect on the intestinal motility via MyD88-dependent and-independent pathways. 210 211 Shirin Moossavi Digestive Disease Research Center, Tehran University of Medical Sciences Digestive Disease Research Center, Tehran University of Medical Sciences Iran Nima Rezaei Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical SciencesMolecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical SciencesDepartment of Infection and Immunity, School of Medicine and Biomedical Sciences, University of Sheffield, Research Center for Immunodeficiencies, Pediatrics Center of Excellence, Children’s Medical Center, Tehran University of Medical SciencesMolecular Immunology Research Center, Department of Immunology, School of Medicine, Tehran University of Medical SciencesDepartment of Infection and Immunity, School of Medicine and Biomedical Sciences, University of Sheffield, IranIranUK Host-microbiota interactionIntestinal tractToll-like receptors 102.pdf Larsson E, Tremaroli V, Lee YS, Koren O, Nookaew I, Fricker A, et al. Analysis of gut microbial regulation of host gene expression along the length of the gut and regulation of gut microbial ecology through MyD88. Gut 2012;61(8):1124-1131.##Gao Q, Qi L, Wu T, Wang J. Clostridium butyricum activates TLR2-mediated MyD88-independent signaling pathway in HT-29 cells. Mol Cell Biochem 2012;361(1-2):31-37.##Biswas SK, Bist P, Dhillon MK, Kajiji T, Del Fresno C, Yamamoto M, et al. Role for MyD88-independent, TRIF pathway in lipid A/TLR4-induced endotoxin tolerance. J Immunol 2007;179(6):4083-4092.##Choi YJ, Im E, Chung HK, Pothoulakis C, Rhee SH. TRIF mediates Toll-like receptor 5-induced signaling in intestinal epithelial cells. J Biol Chem 2010;285(48):37570-37578.##Zughaier SM, Zimmer SM, Datta A, Carlson RW, Stephens DS. Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins. Infec Immun 2005;73(5):2940-2950.##Buchholz BM, Billiar TR, Bauer AJ. Dominant role of the MyD88-dependent signaling pathway in mediating early endotoxin-induced murine ileus. Am J Physiol Gastrointest Liver Physiol 2010;299(2):G531-G538.##Gavin AL, Hoebe K, Duong B, Ota T, Martin C, Beutler B, et al. Adjuvant-enhanced antibody responses occur without toll-like receptor signaling. Science 2006;314(5807):1936-1938.##