The Economic Aspects of Medical Biotechnology 2 2 The biotechnology industry has grown rapidly in recent years, doubling in size between 2004 and 2014. Much attention is given to the potential of the biotechnology industry, from drugs and environmental products currently in the pipeline. These products have the potential to generate tremendous opportunities for society, by improving the quality of health care and producing a cleaner environment. Over the past few decades biotechnology sometimes described as the oldest profession in the world has evolved into a modern technology without which medical progress would be scarcely imaginable. Modern biotechnology plays a crucial role both in the elucidation of the molecular causes of disease and in the development of new diagnostic methods and better targeted drugs. Diagnosis and treatment are thus becoming increasingly intertwined. When a disease, rather than being diagnosed on the basis of more or less vague signs and symptoms, can be detected on the basis of molecular information, the possibility of successful treatment depends largely on what diagnostic techniques are available. Biotechnology is an important component of the worldwide economy, and could take on an increasingly significant role as the industry continues to develop. The economic impact of biotechnology as a distinct industry is currently difficult to evaluate because of the manner in which data is collected; however, it is possible to calculate the combined impact of the biotech and pharmaceutical industries 1,2. 1 1 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Iran Editorial 225.pdf Modabbernia A, Rezaei F, Salehi B, Jafarinia M, Ashrafi M, Tabrizi M, et al. Intranasal oxytocin as an adjunct to risperidone in patients with schizophrenia: an 8-week, randomized, double-blind placebo-controlled study. CNS Drugs 2013;27(1):57-65.##Hosseini SM, Farokhnia M, Rezaei F, Gougol A, Yekehtaz H, Iranpour N, et al. Intranasal desmopressin as an adjunct to risperidone for negative symptoms of schizophrenia: a randomized, double-blind, placebo-controlled, clinical trial. Eur Neuropsychopharmacol 2014;24(6):846-855.##

Differentiation of Definitive Endoderm from Human Induced Pluripotent Stem Cells on hMSCs Feeder in a Defined Medium 2 2 Background: The Definitive Endoderm (DE) differentiation using the undefined media and non-human feeders can cause contaminations in the generated cells for therapeutic applications. Therefore, generating safer and more appropriate DE cells is needed. This study compared five different methods to establish an appropriate method for inducing an efficient DE differentiation from Human Induced Pluripotent Stem Cells (hiPSCs) on an appropriate feeder in a more defined medium. Methods: Human Induced Pluripotent Stem Cells (hiPSCs) were cultured on inactivated feeders. Passaged hiPSCs, without feeder, were incubated for three days with Activin-A and different endodermal differentiation media including 1-FBS, 2-B27, 3-ITS and albumin fraction-V, 4-B27 and ITS and 5-like the third medium. The feeder cells in the first four methods were Mouse Embryonic Fibroblasts (MEFs) and in the fifth method were human adult bone marrow Mesenchymal Stem Cells (hMSCs). DE markers FOXA2, SOX17 and CXCR4 and also pluripotency marker OCT4 were evaluated using qRT-PCR, as well as FOXA2 by the immunocytochemistry. Results: QRT-PCR analysis showed that after three days, the expression levels of DE and pluripotency markers in the differentiated hiPSCs among all five groups did not have any significant differences. Similarly, the immunocytochemistry analysis demonstrated that the differentiated hiPSCs expressed FOXA2, with no significant differences. Conclusion: Despite this similarity in the results, the third differentiation medium has more defined and cost effective components. Furthermore, hMSC, a human feeder, is safer than MEF. Therefore, the fifth method is preferable among other DE differentiation methods and can serve as a fundamental method helping the development of regenerative medicine. 2 8 Zahra Jaafarpour Department of Biology, Science and Research Branch, Islamic Azad University Department of Biology, Science and Research Branch, Islamic Azad University Iran Masoud Soleimani Department of Hematology, Faculty of Medical Science, Tarbiat Modares University Department of Hematology, Faculty of Medical Science, Tarbiat Modares University Iran Saman Hosseinkhani Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University Iran Mohammad Hossein Karimi Transplant Research Center, Shiraz University of Medical Sciences Transplant Research Center, Shiraz University of Medical Sciences Iran Marjan Yaghmaei Department of Biology, Science and Research Branch, Islamic Azad University Department of Biology, Science and Research Branch, Islamic Azad University Iran Naser Mobarra Metabolic Disorders Research Center, Faculty of Medicine, Golestan University of Medical Sciences Metabolic Disorders Research Center, Faculty of Medicine, Golestan University of Medical Sciences Iran Bita Geramizadeh EndodermInduced pluripotent stem cellsMesenchymal stem cells 226.pdf Jayme DW, Smith SR. Media formulation options and manufacturing process controls to safeguard against introduction of animal origin contaminants in animal cell culture. Cytotechnology 2000;33(1-3):27-36.##Basma H, Soto-Gutiérrez A, Yannam GR, Liu L, Ito R, Yamamoto T, et al. Differentiation and transplantation of human embryonic stem cell-derived hepatocytes. Gastroenterology 2009;136(3):990-999.##Sekine K, Takebe T, Suzuki Y, Kamiya A, Nakauchi H, Taniguchi H. Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells. Transplant Proc 2012;44(4):1127-1129.##Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 2010;51(1):297-305.##Mobarra N, Soleimani M, Kouhkan F, Hesari Z, Lahmy R, Mossahebi-Mohammadi M, et al. Efficient differentiation of human induced pluripotent stem cell (hiPSC) derived hepatocyte-like cells on hMSCs feeder. Int J Hema-tol Oncol Stem Cell Res 2014;8(4):20-29.##Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131(5):861-872.##Havasi P, Nabioni M, Soleimani M, Bakhshandeh B, Parivar K. Mesenchymal stem cells as an appropriate feeder layer for prolonged in vitro culture of human induced pluripotent stem cells. Mol Biol Rep 2013;40(4):3023-3031.##Takahashi K, Narita M, Yokura M, Ichisaka T, Yamanaka S. Human induced pluripotent stem cells on autologous feeders. PLoS One 2009;4(12):e8067.##Unger C, Gao S, Cohen M, Jaconi M, Bergstrom R, Holm F, et al. Immortalized human skin fibroblast feeder cells support growth and maintenance of both human embryonic and induced pluripotent stem cells. Hum Reprod 2009;24(10):2567-2581.##Montes R, Ligero G, Sanchez L, Catalina P, de la Cueva T, Nieto A, et al. Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-beta and IGF-II. Cell Res 2009;19(6):698-709.##Agarwal S, Holton KL, Lanza R. Efficient differentiation of functional hepatocytes from human embryonic stem cells. Stem Cells 2008;26(5):1117-1127.##Takata A, Otsuka M, Kogiso T, Kojima K, Yoshikawa T, Tateishi R, et al. Direct differentiation of hepatic cells from human induced pluripotent stem cells using a limited number of cytokines. Hepatol Int 2011;5(4):890-898.##Mathew S, Jaramillo M, Zhang X, Zhang LA, Soto-Gutiérrez A, Banerjee I. Analysis of alternative signaling pathways of endoderm induction of human embryonic stem cells identifies context specific differences. BMC Syst Biol 2012;6:154.##McLean AB, D'Amour KA, Jones KL, Krishnamoorthy M, Kulik MJ, Reynolds DM, et al. Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem Cells 2007;25(1):29-38.##Chen L, Daley GQ. Molecular basis of pluripotency. Hum Mol Genet 2008;17(R1):R23-27.##Adams GN, LaRusch GA, Stavrou E, Zhou Y, Nieman MT, Jacobs GH, et al. Murine prolylcarboxypeptidase depletion induces vascular dysfunction with hypertension and faster arterial thrombosis. Blood 2011;117(14):3929-3937.##Bakhshandeh B, Soleimani M, Hafizi M, Ghaemi N. A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells. Cytotechnology 2012;64(5):523-540.##Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res 2009;19(11):1233-1242.##Duan Y, Ma X, Zou W, Wang C, Bahbahan IS, Ahuja TP, et al. Differentiation and characterization of metabolically functioning hepatocytes from human embryonic stem cells. Stem Cells 2010;28(4):674-686.##Cai J, Zhao Y, Liu Y, Ye F, Song Z, Qin H, et al. Directed differentiation of human embryonic stem cells into functional hepatic cells. Hepatology 2007;45(5):1229-1239.##Prasajak P, Leeanansaksiri W. Developing a new two-step protocol to generate functional hepatocytes from wharton's jelly-derived mesenchymal stem cells under hypoxic condition. Stem Cells Int 2013;2013:762196.##Ghodsizadeh A, Taei A, Totonchi M, Seifinejad A, Gourabi H, Pournasr B, et al. Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells. Stem Cell Rev 2010;6(4):622-632.##Katsuda T, Sakai Y, Ochiya T. Induced pluripotent stem cell-derived hepatocytes as an alternative to human adult hepatocytes. J Stem Cells 2012;7(1):1-17.##Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, et al. Development of definitive endoderm from embryonic stem cells in culture. Development 2004;131(7):1651-1662.##Ninomiya H, Mizuno K, Terada R, Miura T, Ohnuma K, Takahashi S, et al. Improved efficiency of definitive endoderm induction from human induced pluripotent stem cells in feeder and serum-free culture system. In Vitro Cell Dev Biol Anim 2015;51(1):1-8.##Brunner D, Frank J, Appl H, Schöffl H, Pfaller W, Gstraunthaler G. Serum-free cell culture: the serum-free media interactive online database. ALTEX 2010;27(1):53-62.##Thermo Fisher Scientific Company [Internet]. Available from: http://www.lifetechnologies.com/ir/en/home/technical-resources/media- formulation.250.html.##Thermo Fisher Scientific Company [Internet]. Available from: http://www.lifetechnologies.com/ir/en/home/technical-resources/media-formulation.334.html.##Usta SN, Scharer CD, Xu J, Frey TK, Nash RJ. Chemically defined serum-free and xeno-free media for multiple cell lineages. Ann Transl Med 2014;2(10):97.##

The Effect of Angiotensin on the Quality of In Vitro Produced (IVP) Sheep Embryos and Expression of Na+/K+/ATPase 2 2 Background: The presence of rennin-angiotensin components in mammalian ovaries and their involvement in ovarian physiology have been established. In the present study, effects of angiotensin II (Ang II) on sodium-potassium adenosine triphosphatase (Na+/K+/ATPase) expression and development of sheep embryos was evaluated. Methods: The abattoir-derived Cumulus Oocyte Complexes (COC) were randomly allocated into three experimental groups; group I) in vitro Maturation (IVM) of oocytes in the presence of Ang II followed by in vitro fertilization (IVF)/in vitro Culture (IVC) (IVM group), group II) IVM/IVF of oocytes followed by IVC wherein the embryos were exposed to Ang II on day 4 of IVC (D4 group), and  group III) IVM/IVF and IVC of oocytes without any angiotensin (Control). The blastocyst and hatching rates were recorded on days 6 to 8. Day 8 embryos were immunostained with primary and secondary antibodies against Na+/K+/ATPase α1 and β1 subunits. Results: Addition of Ang II during IVM and IVC significantly increased the hatching rate of blastocysts on day 8 compared to the control. The trophectoderm and total blastocyst cells’ numbers were significantly increased by addition of Ang II to the IVM and IVC media, though the expression of Na+/K+/ATPase α1 and β1 subunits were positively influenced by the addition of Ang II on day 4 (D4 group). Conclusion: In conclusion, it seems Ang II through positive effects on embryos, expressed as the greater hatching rate and blastocyst cell number, could increase the sheep embryo developmental rate. These improvements might be partly related to the greater expression of Na+/K+/ATPase α1 and β1 subunits when Ang II was added during IVC. 9 15 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 Ali Sarvari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Banafsheh Heidari Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Akhondi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Amir-Hassan Zarnani Reproductive Immunology Research Center, Avicenna Research Institute, ACECR Reproductive Immunology Research Center, Avicenna Research Institute, ACECR 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 Angiotensin IIDevelopmentEmbryoExpressionNa+/K+/ATPaseSheep 227.pdf Honorato-Sampaio K, Pereira VM, Santos RA, Reis AM. Evidence that angiotensin-(1-7) is an intermediate of gonadotrophin-induced oocyte maturation in the rat preovulatory follicle. Exp Physiol 2012;97(5):642-650.##Stefanello JR, Barreta MH, Porciuncula PM, Arruda JN, Oliveira JF, Oliveira MA, et al. Effect of angiotensin II with follicle cells and insulin-like growth factor-I or insulin on bovine oocyte maturation and embryo development. Theriogenology 2006;66(9):2068-2076.##Giometti IC, Bertagnolli AC, Ornes RC, da Costa LF, Carambula SF, Reis AM, et al. Angiotensin II reverses the inhibitory action produced by theca cells on bovine oocyte nuclear maturation. Theriogenology 2005;63(4):1014-1025.##Muscella A, Aloisi F, Marsigliante S, Levi G. Angiotensin II modulates the activity of Na+, K+-ATPase in cultured rat astrocytes via the AT1 receptor and protein kinase C-delta activation. J Neurochem 2000;74(3):1325-1331.##Min LJ, Mogi M, Iwai M, Horiuchi M. Signaling mechanisms of angiotensin II in regulating vascular senescence. Ageing Res Rev 2009;8(2):113-121.##Madan P, Rose K, Watson AJ. Na/K-ATPase beta1 subunit expression is required for blastocyst formation and normal assembly of trophectoderm tight junction-associated proteins. J Biol Chem 2007;282(16):12127-12134.##Blanco G, Mercer RW. Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 1998;275(5 Pt 2):F633-650.##Lim JM, Hansel W. Exogeneous substances affecting development of in vitro-derived bovine embryos before and after embryonic genome activation. Theriogenology 2000;53(5):1081-1091.##Tervit HR, Whittingham DG, Rowson LE. Successful culture in vitro of sheep and cattle ova. J Reprod Fertil 1972;30(3):493-497.##Naderi MM, Sarvari A, Saviz A, Naji T, Borjian Boroujeni S, Heidari B, et al. The effect of Aldosterone on Na+/K+/ATPase expression and development of embryos derived from vitrified-warmed sheep oocytes. Small Ruminant Research 2015;126:44-51.##Isenovic ER, Jacobs DB, Kedees MH, Sha Q, Milivojevic N, Kawakami K, et al. Angiotensin II regulation of the Na+ pump involves the phosphatidylinositol-3 kinase and p42/44 mitogen-activated protein kinase signaling pathways in vascular smooth muscle cells. Endocrinology 2004;145(3):1151-1160.##Gomes CP, Leão-Ferreira LR, Caruso-Neves C, Lopes AG. Adenosine reverses the stimulatory effect of angiotensin II on the renal Na+-ATPase activity through the A2 receptor. Regul Pept 2005;129(1-3):9-15.##Lara LS, De Carvalho T, Leão-Ferreira LR, Lopes AG, Caruso-Neves C. Modulation of the (Na(+)+K+)ATPase activity by Angiotensin-(1-7) in MDCK cells. Regul Pept 2005;129(1-3):221-226.##Violette MI, Madan P, Watson AJ. Na+/K+ -ATPase regulates tight junction formation and function during mouse preimplantation development. Dev Biol 2006;289(2):406-419.##Huang JC, Wun WS, Goldsby JS, Egan K, FitzGerald GA, Wu KK. Prostacyclin receptor signaling and early embryo development in the mouse. Hum Reprod 2007;22(11):2851-2856.##Liszewska E, Reinaud P, Billon-Denis E, Dubois O, Robin P, Charpigny G. Lysophosphatidic acid signaling during embryo development in sheep: involvement in prostaglandin synthesis. Endocrinology 2009;150(1):422-434.##Kim JS, Chae JI, Song BS, Lee KS, Choo YK, Chang KT, et al. Iloprost, a prostacyclin analogue, stimulates meiotic maturation and early embryonic development in pigs. Reprod Fertil Dev 2010;22(2):437-447.##Wang H, Wen Y, Mooney S, Behr B, Polan ML. Phospholipase A(2) and cyclooxygenase gene expression in human preimplantation embryos. J Clin Endocrinol Metab 2002;87(6):2629-2634.##Sen Roy S, Seshagiri PB. Expression and function of cyclooxygenase-2 is necessary for hamster blastocyst hatching. Mol Hum Reprod 2013;19(12):838-851.##Sugiyama T, Yoshimoto T, Sato R, Fukai N, Ozawa N, Shichiri M, et al. Endothelin-1 induces cyclooxygenase-2 expression and generation of reactive oxygen species in endothelial cells. J Cardiovasc Pharmacol 2004;44 Suppl 1:S332-335.##Sarvari A, Naderi MM, Sadeghi MR, Akhondi MM. A technique for facile and precise transfer of mouse embryos. Avicenna J Med Biotech 2012;5(1):62-65.##Uhm SJ, Gupta MK, Chung HJ, Kim JH, Park Ch, Lee HT. Relationship between developmental ability and cell number of day 2 porcine embryos produced by parthenogenesis or somatic cell nuclear transfer. Asian-Australas J Anim Sci 2009;22(4):483-491.##Gupta MK, Uhm SJ, Han DW, Lee HT. Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol Reprod Dev 2007;74(4):435-444.##

Comparison of Three Escherichia coli Strains in Recombinant Production of Reteplase 2 2 Background: Escherichia coli (E. coli) is the most extensively used host for the production of recombinant proteins. However, most of the eukaryotic proteins are typically obtained as insoluble, misfolded inclusion bodies that need solubilization and refolding. Reteplase as a highly disulfide-bonded recombinant protein is an example of difficult to express protein in E. coli. Methods: In this study, a codon optimized reteplase gene was synthetically prepared and cloned under the control of an IPTG inducible T7 promoter. The vector was simultaneously transformed and expressed in three different E. coli strains. The ability of strains for expression of this recombinant pharmaceutical was compared. Also, an attempt was made to increase the soluble production of reteplase in SHuffle T7 E. coli with alterations of expression condition like temperature, inducer concentration and oxygen supply. Results: High amounts of reteplase were expressed as inclusion bodies in all three strains. BL21 (DE3) showed the highest level of expression in inclusion bodies followed by Rosetta-gami (DE3) and Shuffle T7. Changes of expression conditions were insufficient for soluble expression of reteplase in SHuffle T7 as a genetically engineered host for production of disulfide bonded proteins. Conclusion: The oxidizing cytoplasm of Rosetta-gami and Shuffle T7 in addition to alterations of cultivation parameters could not result in soluble production of reteplase, although the inclusion bodies produced in these two strains might increase the rate of refolding procedure likely due to formation of folding intermediates. 16 22 Mehrnoosh Fathi-Roudsari National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh Iran Asal Akhavian-Tehrani National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh National Institute of Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh Iran Nader Maghsoudi Neurobiology Research Center, Shahid Beheshti University of Medical Sciences Neurobiology Research Center, Shahid Beheshti University of Medical Sciences Iran Escherichia coliRecombinant proteinsReteplaseTissue Plasminogen Activator 228.pdf Mandi N, Sundaram KR, Tandra SK, Bandyopadhyay S, Padmanabhan S. Asn and asn: critical residues for in vitro biological activity of reteplase. Adv Hematol 2010;2010:172484.##Geng Y, Wang S, Qi Q. Expression of active recombinant human tissue-type plasminogen activator by using in vivo polyhydroxybutyrate granule display. Appl Environ Microbiol 2010;76(21):7226-7230.##Topol EJ. A comparison of reteplase with alteplase for acute myocardial infarction. The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO III) Investigators. N Engl J Med 1997;337(16):1118-1123.##Qiu J, Swartz JR, Georgiou G. Expression of active human tissue-type plasminogen activator in Escherichia coli. Appl Environ Microbiol 1998;64(12):4891-4896.##Baruah DB, Dash RN, Chaudhari MR, Kadam SS. Plasminogen activators: a comparison. Vascul Pharmacol 2006;44(1):1-9.##Kunadian V, Gibson CM. Recombinant tissue-type plasminogen activators: "time matters". Drugs Today (Barc) 2011;47(7):559-570.##Nordt TK, Bode C. Thrombolysis: newer thrombolytic agents and their role in clinical medicine. Heart 2003;89(11):1358-1362.##Marisch K, Bayer K, Cserjan-Puschmann M, Luchner M, Striedner G. Evaluation of three industrial Escherichia coli strains in fed-batch cultivations during high-level SOD protein production. Microb Cell Fact 2013;12:58.##Long X, Gou Y, Luo M, Zhang S, Zhang, Bai L, et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli. BMC Biotechnol 2015;15:13.##Lee HJ, Im H. Soluble expression and purification of human tissue-type plasminogen activator protease domain. Bull Korean Chem Soc 2010;31(9):2607-2612.##Zhao Y, GE W, Kong Y, Zhang Ch. Cloning, expression, and renaturation studies of reteplase. J Microbiol Biotechnol 2003;13(6):989-992.##Aghaabdollahian S, Rabbani M, Ghaedi K, Sadeghi HM. Molecular cloning of Reteplase and its expression in E. coli using tac promoter. Adv Biomed Res 2014;3:190.##Khodabakhsh F, Dehghani Z, Zia MF, Rabbani M, Sadeghi HM. Cloning and Expression of Functional Reteplase in Escherichia coli TOP10. Avicenna J Med Biotechnol 2013;5(3):168-175.##Gao L, Zhang C, Li L, Liang L, Deng X, Wu W, et al. Construction, expression and refolding of a bifunctional fusion protein consisting of C-terminal 12-residue of hirudin-PA and reteplase. Protein J 2012;31(4):328-336.##Nausch H, Huckauf J, Koslowski R, Meyer U, Broer I, Mikschofsky H. Recombinant production of human interleukin 6 in Escherichia coli. PLoS One 2013;8(1):e54933.##Rabhi-Essafi I, Sadok A, Khalaf N, Fathallah DM. A strategy for high-level expression of soluble and functional human interferon alpha as a GST-fusion protein in E. coli. Protein Eng Des Sel 2007;20(5):201-209.##Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 2012;11:56.##Novagen, pET system manual, 10th edition, May, 2003. Available at: http://download.bioon.com.cn/upload/201011/25/141105fw9i8zjm1jfm19m8.attach.pdf.##Rosano GL, Ceccaelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 2014;5(172):1-17.##Kunadian V, Gibson CM. Thrombolytics and myocardial infarction. Cardiovasc Ther 2012;30(2):e81-88.##Collen D, Lijnen HR. The tissue-type plasminogen activator story. Arterioscler Thromb Vasc Biol 2009;29(8):1151-1155.##Collen D, Lijnen HR. Thrombolytic agents. Thromb Haemost 2005;93(4):627-630.##Wooster MB, Luzier AB. Reteplase: a new thrombolytic for the treatment of acute myocardial infarction. Ann Pharmacother 1999;33(3):318-324.##Ershadi S, Rashedi H, Fazeli A. A study on the mechanism of aggregation of therapeutic reteplase protein by using the monomer-loss model. J Appl Biotechnol Rep 2015;2(1):191-197.##Baneyx F, Mujacic M. 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Cloning and Optimization of Soluble Vascular Endothelial Growth Factor165 Expression in Escherichia coli 2 2 Background: Vascular Endothelial Growth Factor (VEGF) is a coordinate regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. There are several types of VEGF, including VEGF165. VEGFs stimulate endothelial cell growth, angiogenesis, and capillary permeability. Low induction temperature is a major factor for expression of the recombinant VEGF165 in soluble form. The purpose of this study was cloning and optimization of soluble vascular endothelial growth factor165 expression in Escherichia coli (E. coli). Methods: In this study, total RNA of HeLa cell [cervix epithelium] was extracted. The VEGF165 gene was amplified by reverse transcription polymerase chain reaction (RT-PCR), and then VEGF165 was subcloned into prokaryotic expression vectors pET-32a(+) and transformed into BL21 (DE3) E. coli strain. VEGF165 expression was optimized by fine adjustments such as induction time and incubation temperature. VEGF165 was analyzed by DNA sequencing prior to expression and the protein was further characterized by SDS-PAGE and immunoblotting using His•tag specific polyclonal antibody. Results: Our results demonstrated that VEGF165 was successfully cloned and expressed in pET-32a(+) vector. Optimization of the expression procedure showed that, induction by 1 mM IPTG at OD600=0.7 and overnight incubation at 22oC resulted in the highest expression levels of soluble VEGF165. Conclusion: In this study, the expression of VEGF165 in a high soluble level was successfully cloned and optimized. 23 28 Ali Salimi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mohammad Babashamsi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran ExpressionInclusion bodiesRecombinantVEGF165 protein 229.pdf Catena R, Muniz-Medina V, Moralejo B, Javierre B, Best CJ, Emmert-Buck MR, et al. Increased expression of VEGF121/VEGF165-189 ratio results in a significant enhancement of human prostate tumor angiogenesis. Int J Cancer 2007;120(10):2096-2109.##Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 1999;13(1):9-22.##Chang YS, Munn LL, Hillsley MV, Dull RO, Yuan J, Lakshminarayanan S, et al. Effect of vascular endothelial growth factor on cultured endothelial cell monolayer transport properties. Microvasc Res 2000;59(2):265-277.##Cooper ME, Vranes D, Youssef S, Stacker SA, Cox AJ, Rizkalla B, et al. Increased renal expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 in experimental diabetes. Diabetes 1999;48(11):2229-2239.##Kang W, Kim S, Lee S, Jeon E, Lee Y, Yun Y, et al. Characterization and optimization of vascular endothelial growth factor 165 (rhVEGF 165) expression in Escherichia coli. Protein Expr Purif 2013;87(2):55-60.##Harper SJ, Bates DO. VEGF-A splicing: the key to anti-angiogenic therapeutics? Nat Rev Cancer 2008;8(11):880-887.##Cébe Suarez S, Pieren M, Cariolato L, Arn S, Hoffmann U, Bogucki A, et al. A VEGF-A splice variant defective for heparan sulfate and neuropilin-1 binding shows attenuated signaling through VEGFR-2. Cell Mol Life Sci 2006;63(17):2067-2077.##Pizarro SA, Gunson J, Field MJ, Dinges R, Khoo S, Dalal M, et al. High-yield expression of human vascular endothelial growth factor VEGF(165) in Escherichia coli and purification for therapeutic applications. Protein Expr Purif 2010;72(2):184-193.##Molinkova D. Purification of Escherichia coli-expressed HIS-tagged Maedi-Visna p25 core antigen by Ni2+-chelate affinity chromatography. Vet Med (Praha) 2001;46(2):50-54.##Bitazar R, Taghi Naserpour F, Hajikhani B, Bagheri R, Salimi A. Cloning and expression of truncated chlamydial major outer membrane protein in E. coli: A miniature step Forward. J Vaccines Vaccin 2014;5: 1.##Novy R, Drott D, Yaeger K, Mierendorf R. Overcoming the codon bias of E. coli for enhanced protein expression. Innov 2001;12:1-3.##Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med 2005;9(4):777-794.##Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. 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Evaluation of the Anti-proliferative Effects of Ophiocoma erinaceus Methanol Extract Against Human Cervical Cancer Cells 2 2 Background: Marine organisms provide appreciable source of novel bioactive compounds with pharmacological potential. There is little information in correlation with anti-cancer activities of brittle star. In the present study, anti-neoplastic efficacy of Ophiocoma erinaceus methanol extract against human cervical cancer cells was investigated. Methods: The HeLa cells were cultured and exposed to brittle star methanol extract for 24 and 48 hr. The anti-proliferative properties were examined by MTT assay and the type of cell death induced was evaluated through morphological changes, flow cytometry, Annexin kit and caspase assay. To assess the anti-metastatic activity, wound healing assay was conducted and photographs were taken from the scratched areas. Further, to understand molecular mechanism of cell apoptosis, the expression of Bax was evaluated. Results: The morphological analysis and MTT assay exhibited that the brittle star methanol extract can exert dose dependent inhibitory effect on cells viability (IC50, 50 μg/ml). Flow cytometry and fluorescence microscopy demonstrated increment of sub-G1 peak, early and late apoptosis in HeLa treated cells. Wound healing migration assay showed that brittle star extract has anti-neoplastic efficacy by inhibiting cell migration. Caspase assay and RT-PCR analysis revealed that brittle star methanol extract induced caspase dependent apoptosis in HeLa cells through up-regulation of caspase-3 followed by up-regulation of Bax gene which is a hallmark of intrinsic pathway recruitment. Conclusion: These results represented further insights into the chemopreventive potential of brittle star as a valuable source of unknown therapeutic agents against human cervical cancer. 29 35 Javad Baharara Department of Biology, Research Center for Animal Developmental Applied Biology, Mashhad Branch, Islamic Azad University Department of Biology, Research Center for Animal Developmental Applied Biology, Mashhad Branch, Islamic Azad University Iran Elaheh Amini Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Iran Farideh Namvar Department of Biology, Research Center for Animal Developmental Applied Biology, Mashhad Branch, Islamic Azad UniversityInstitute of Tropical Forestry and Forest Products, University Putra Malaysia, 43400 UPM Serdang Department of Biology, Research Center for Animal Developmental Applied Biology, Mashhad Branch, Islamic Azad UniversityInstitute of Tropical Forestry and Forest Products, University Putra Malaysia, 43400 UPM Serdang IranMalaysia ApoptosisBrittle starUterine cervical neoplasms 230.pdf Saunders FR, Wallace HM. 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Computational Detection of piRNA in Human Using Support Vector Machine 2 2 Background: Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (ncRNAs), with a length of about 24-32 nucleotides, which have been discovered recently. These ncRNAs play an important role in germline development, transposon silencing, epigenetic regulation, protecting the genome from invasive transposable elements, and the pathophysiology of diseases such as cancer. piRNA identification is challenging due to the lack of conserved piRNA sequences and structural elements. Methods: To detect piRNAs, an appropriate feature set, including 8 diverse feature groups to encode each RNA was applied. In addition, a Support Vector Machine (SVM) classifier was used with optimized parameters for RNA classification. According to the obtained results, the classification performance using the optimized feature subsets was much higher than the one in previously published studies. Results: Our results revealed 98% accuracy, Mathew’ correlation coefficient of 98% and 99% specificity in discriminating piRNAs from the other RNAs. Also, the obtained results show that the proposed method outperforms its competitors. Conclusion: In this paper, a prediction method was proposed to identify piRNA in human. Also, 48 heterogeneous features (sequence and structural features) were used to encode RNAs. To assess the performance of the method, a benchmark dataset containing 515 piRNAs and 1206 types of other RNAs was constructed. Our method reached the accuracy of 99% on the benchmark dataset. Also, our analysis revealed that the structural features are the most contributing features in piRNA prediction. 36 41 Atefeh Seyeddokht Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad Iran Ali Asghar Aslaminejad Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad Iran Ali Masoudi-Nejad Laboratory of System Biology and Bioinformatics (LBB), Institute of Biochemistry and Biophysics, University of Tehran Laboratory of System Biology and Bioinformatics (LBB), Institute of Biochemistry and Biophysics, University of Tehran Iran Mohammadreza Nassiri Department of Animal Science, Research Institute of Biotechnology, Ferdowsi University of Mashhad Department of Animal Science, Research Institute of Biotechnology, Ferdowsi University of Mashhad Iran Javad Zahiri Bioinformatics and Computational Omics. LAB (BioCOOL), Faculty of Biological Sciences, Tarbiat Modares University (TMU) Bioinformatics and Computational Omics. LAB (BioCOOL), Faculty of Biological Sciences, Tarbiat Modares University (TMU) Iran Balal Sadeghi Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman Department of Food Hygiene and Public Health, Faculty of Veterinary Medicine, Shahid Bahonar University of Kerman Iran Piwi-interacting RNAs (piRNAs)RNASupport Vector Machines (SVM) 231.pdf Carninci P, Kasukawa T, Katayama S, Gough J, Frith MC, Maeda N, et al. The transcriptional landscape of the mammalian genome. Science 2005;309(5740):1559-1563.##Kadri S, Hinman V, Benos PV. HHMMiR: efficient de novo prediction of microRNAs using hierarchical hidden Markov models. BMC Bioinformatics 2009;10 Suppl 1:S35.##Kapranov P, Drenkow J, Cheng J, Long J, Helt G, Dike S, et al. 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Outer Membrane Protein C (ompC) Gene as the Target for Diagnosis of Salmonella Species Isolated from Human and Animal Sources 2 2 Background: The use of selective and differential plating media is a simple method for the isolation of Salmonella spp. Recently, there has been a general move toward molecular methods of Salmonella detection and typing. Methods: A total of 1200 different specimens collected from human and animal sources were involved in his study. 600 stool specimens from patients suffering from diarrhea and 600 specimens from gall bladder (bile) of cattle from Al-Diwaniya slaughter house, Iraq were used. Salmonella spp. were isolated and identified using bacterial culturing on selective media and colonies were tested by API 20Eand then serotyping through polyvalent antisera and conformation by Polymerase Chain Reaction (PCR). PCR was used to detect ompC gene encoding biosynthesis of outer membrane protein C of Salmonella genus. Results: The results revealed that the rate of Salmonella isolates was 0.5% (3/600) from human and 1% (6/600) from animals. The PCR technique revealed that 9 isolates of Salmonella spp. harbored ompC gene. The results of this study revealed that the PCR technique had a high specificity in detection of Salmonella spp., in comparison to culture and biochemical test, Mini API 20 E and serological tests. The present study found no significant differences between human and animal isolates. Conclusion: Detection of ompC gene is a good method for detection of Salmonella species isolated from clinical specimens. It has a high specificity in comparison with other tests, with its advantages of greater speed and effectiveness than conventional detection methods. 42 45 Alaa Abdel-Kadhim Jawad College of Veterinary Medicine, Al-Qadisiya University College of Veterinary Medicine, Al-Qadisiya University Iraq Alaa H. Al-Charrakh Department of Microbiology, College of Medicine, Babylon University Department of Microbiology, College of Medicine, Babylon University Iraq DetectionGenePolymerase chain reactionSalmonella 232.pdf Foley SL, Johnson TJ, Ricke SC, Nayak R, Danzeisen J. Salmonella pathogenicity and host adaptation in chicken-associated serovars. Microbiol Mol Biol Rev 2013;77 (4):582-607.##Fàbrega A, Vila J. Salmonella enterica serovar typhimurium skills to succeed in the host: virulence and regulation. Clin Microbiol Rev 2013;26(2):308-341.##Donnenberg MS. Pathogenic strategies of enteric bacteria. Nature 2000;406(6797):768-774.##Park SH, Ryu S, Kang DH. Development of an improved selective and differential medium for isolation of Salmonella spp. J Clin Microbiol 2012;50(10):3222-3226.##Pickup RW, Rhodes G, Hermon-Taylor J. Monitoring bacterial pathogens in the environment: advantages of a multilayered approach. Curr Opin Biotechnol 2003;14(3):319-325.##Mikoleit ML. Isolation of Salmonella and Shigella from Faecal Specimens. Atlanta, USA: WHO Global Foodborne Infections Network; 2010 Nov. 15 p.##Alvarez J, Sota M, Vivanco AB, Perales I, Cisterna R, Rementeria A, et al. Development of a multiplex PCR technique for detection and epidemiological typing of salmonella in human clinical samples. J Clin Microbiol 2004;42(4):1734-1738.##Kwang J, Littledike ET, Keen JE. Use of the polymerase chain reaction for Salmonella detection. Lett Appl Microbiol 1996;22(1):46-51.##Belle GV, Fisher LD, Heagerty PJ, Lumley T. Biostatistics a Methodology for the Health Sciences. 2nd ed. USA: A John Wiley & Sons, Inc., Publication; 2004. 888 p. ##Jawad AA. Detection of fim A, rfbj (B) and fimC Genes of Salmonella Isolates using singleplex and multiplex polymerase chain reaction [master’s thesis]. [Qadisiya, Iraq]: College of Medicine Al-Qadisiya University; 2010.##Al-Janabi JK. Microbiological study of some causative agents of diarrhea in children under five year of age in Al-Diwaniya city [dissertation]. [Qadisiya, Iraq]: College of Education Al-Qadisiya University; 2006. ##Al–Karawiy HAM. Isolation and identification of Salmonella typhimurium and detection of gene encoded type-1- fimbria by using polymerase chain reaction. [master’s thesis]. [Basra, Iraq]: College of Veterinary Medicine, University of Basrah; 2008.##van Dijk S, Bruins MJ, Ruijs GJ. Evaluation and implementation of a chromogenic agar medium for salmonella detection in stool in routine laboratory diagnostics. J Clin Microbiol 2009;47(2):456-458.##Nucera DM, Maddox CW, Hoien-Dalen P, Weigel RM. Comparison of API 20E and invA PCR for identification of Salmonella enterica isolates from swine production units. J Clin Microbiol 2006;44(9):3388-3390.##