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Iranian science shows world's fastest growth: ranks 17th in science production in 2012 2 2 The Islamic Republic of Iran ranked 17th in terms of science production in the world in 2012, according to the latest statistics released by the Scopus database. According to the statistics, Iran produced 34,155 articles in 2012, which gained the country the world’s 17th rank in science production and fixed its top position in the region, above Turkey. Scientific progress over the past few years was the result of the country’s recent policies and programs to develop knowledge and facilitate researchers' access to the world’s top academic resources. Iran has the world's fastest-growing scientific output, measured by the number of peer-reviewed papers published in international journals. In addition, Iran ranked first in scientific growth in the world in 2011. In 2000, the Islamic Republic of Iran ranked 53rd in the world in terms of highly cited medical articles, but improved to the 23rd rank in 2011. According to the Institute for Scientific Information (ISI), Iranian researchers and scientists published a total of 60,979 scientific articles in major international journals from 1999 to 2008. It is being said that scientific growth in Iran has been fastest in the world, even more than China. Whatever the reason, it is commendable that the country has achieved this even at the worst possible sanctions from the most powerful and most influential country in the world. Iran with a science and technology yearly growth rate of 25% is doubling its total output every three years and at this rate will reach the level of Canadian annual output in 2017. "If Iran keeps moving with the present momentum, it can ascend to the 4th place from the current 17th (in the world ranking of scientific growth and scientific production) in the next 6 years. How about quality and impact? The USA still has a clear lead; taking as a measure of world impact the share of the most highly cited papers (taken as the top 1% in each discipline) puts the USA in the lead with 61%, while the UK outperforms its volume share with 13% of highly cited papers. Iran still underperforms on this measure but the gap is closing, and is likely to close further as citation counts are a lagging indicator – it takes some years for spending on science to translate, first into publication outputs, and only later into citations of those papers by other scientists. Under Iran's 'comprehensive plan for science', the country plans to be spending four per cent of GDP on research and development by 2030. Unfortunately, heavy sanctions against Iran and financial crisis over the last two years slowed down this scientific growth. Now Iranian scientific society hopes to next president to fasten this growth and again back to future. 139 139 Shahin Akhondzadeh Tehran University of Medical Sciences Tehran University of Medical Sciences Iran Editorial 171.pdf ####

The Role of Different Supplements in Expression Level of Monoclonal Antibody against Human CD20 2 2 Background: Recombinant monoclonal antibodies have been marketed in last three decades as the major therapeutic proteins against different cancers. However choosing a proper medium and supplements to reach the high expression is a challenging step. Despite of commercial serum free and chemically defined media, there are still numerous researches seeking the optimum media to gain higher expression titer. Selecting the best basal media followed by proper supplementation to increase the cell density and expression titer needs proper and accurate investigation. Methods: In this study, we have determined the expression titer of monoclonal antibody against human CD20 using soy extract, Essential Amino Acid, Non- Essential Amino Acid, Panexin NTS, Peptone, Yeast extract, Insulin-transferrin selenite, Human Serum Albumin, Bovine Serum Albumin, Lipid, and two commercially available supplements, Power and Xtreme feed. In each experiment, the expression level was compared with a well defined media, ProCHO5, RPMI 1640 and DMEM-F12. Results: It has been shown that supplementing the ProCHO5 basal medium with 10% power feed or combination of 5% PanexinNTS,1.5 g/L yeast and 1.5 g/L peptone results in the best production levels with 450 and 425 mg/L of anti CD20 mAb expression level, respectively. Conclusion: Panexin NTS, yeast and peptone cane be proper supplement for fed-batch cell culture instead of commercial Power feed supplement which is a cost effective way to increase expression level. And thereby ProCHO5 may be replaced with common media such as RPMI 1640 and DMEM-F12. 140 147 Fereidoun Mahboudi Biotechnology Research Center, Pasteur Institute of Iran Iran Mohammad Reza Abolhassan Department of Research and Development, Aryogen Biopharma Company Iran Armita Azarpanah Department of Research and Development, Aryogen Biopharma Company Iran Hamideh Aghajani-Lazarjani Department of Research and Development, Aryogen Biopharma Company Iran Mohammad Amin Sadeghi-Haskoo Department of Research and Development, Aryogen Biopharma Company Iran Shaian Maleknia Department of Research and Development, Aryogen Biopharma Company Iran Behrouz Vaziri Department of Research and Development, Aryogen Biopharma Company Iran CHO cellsGene expressionMonoclonal antibody 119.pdf Chee Furng Wong D, Tin Kam Wong K, Tang Goh L, Kiat Heng C, Gek Sim Yap M. Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. 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Predictions of Protein-Protein Interfaces within Membrane Protein Complexes 2 2 Background: Prediction of interaction sites within the membrane protein complexes using the sequence data is of a great importance, because it would find applications in modification of molecules transport through membrane, signaling pathways and drug targets of many diseases. Nevertheless, it has gained little attention from the protein structural bioinformatics community. Methods: In this study, a wide variety of prediction and classification tools were applied to distinguish the residues at the interfaces of membrane proteins from those not in the interfaces. Results: The tuned SVM model achieved the high accuracy of 86.95% and the AUC of 0.812 which outperforms the results of the only previous similar study. Nevertheless, prediction performances obtained using most employed models cannot be used in applied fields and needs more effort to improve. Conclusion: Considering the variety of the applied tools in this study, the present investigation could be a good starting point to develop more efficient tools to predict the membrane protein interaction site residues. 148 157 Ebrahim Barzegari Asadabadi Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University Iran Parviz Abdolmaleki Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University Iran Interaction sitesMembrane proteinsSupport vector machines 120.pdf Stagljar I, Fields S. Analysis of membrane protein interactions using yeast-based technologies. Trends Biochem Sci 2002;27(11):559-563.##Ge H, Walhout AJ, Vidal M. Integrating 'omic' information: a bridge between genomics and systems biology. 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Effect of Oral Supplementation of Biogenic Selenium Nanoparticles on White Blood Cell Profile of BALB/c Mice and Mice Exposed to X-ray Radiation 2 2 Background: Radiation therapy is an effective method used for treatment of many types of cancers. However, this method can cause unwanted side effects such as bone marrow suppression. In this study, the effect of oral administration of biogenic selenium nanoparticles (SeNPs) on total and differentiated white cells profile of BALB/c mice exposed to X-ray radiation was investigated and compared with non-irradiated mice. Methods: Sixty female BALB/c mice between six to eight weeks olds were divided into 4 test and control groups in two categories of normal and irradiated mice. In normal mice SeNPs administration was started from the day 0 and followed for a month. Irradiated mice were divided into three groups and were exposed to doses of 2, 4 and 8 Gy. After 72 hr of irradiation, the SeNPs treatment was started and continued for a month. Total and differentiated blood cells counts of both irradiated and non-irradiated groups were monitored during 30 days and the obtained results were compared. Also, the deposition of Se in different tissues and blood serum of normal mice was determined in normal mice after 30 days period of supplementation. Results: In normal mice an increase in the count of neutrophils was observed after 30 days of supplementation. In irradiated mice, SeNPs supplementation led to increase in both lymphocytes and neutrophils counts especially in mice exposed to 2 and 4 Gys radiation. Conclusion: Radiotherapy is categorized as an invasive method which can cause tissue damage and suppress the host immune defense. A restore of lymphocytes which was observed after SeNPs supplementation in irradiated mice can be highly interesting and provide cellular immunity against malignant diseases or other bacterial or fungal infections after radiotherapy. 158 167 Mohammad Hossein Yazdi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Maryam Masoudifar Department of Radiation-Oncology, Mahak Hospital Iran Bardia Varastehmoradi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Ehsan Mohammadi Imam Khomeini Hospital, Tehran University of Medical Sciences Imam Khomeini Hospital, Tehran University of Medical Sciences Iran Erfan Kheradmand Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Somayeh Homayouni Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Ahmad Reza Shahverdi Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences Iran Bone marrow suppressionRadiotherapySelenium nanoparticleSide effect 121.pdf Li Y, Tang JH, Huang XE, Li CG. 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Cloning and Expression of Functional Reteplase in Escherichia coli TOP10 2 2 <p>Background: Production of tissue Plasminogen Activator protein (t-PA) in prokaryotes systems has many problems such as the lack of active protein production, multiple purification steps, and renaturation process which has been shown to be costly and time-consuming. Methods: In this study, reteplase which is the nonglycosylated active domain of t-PA was used to transform TOP10 Escherichia coli (E. coli) bacteria to resolve some of the above mentioned problems. Reteplase cDNA was ligated into pBAD/gIII plasmid which allowed secretion of this protein into the periplasmic space and would allow the correct formation of disulfide bonds in protein structure. The presence of reteplase cDNA in pBAD/gIII plasmid was confirmed by restriction digestion and sequencing. After induction of the expression of this protein by adding 0.0002% L-Arabinose to the medium, the proteins in periplasmic space as well as the inclusion bodies formed inside the cell were extracted. Subsequently, these proteins were purified and detected by Western blot method. Results: Our results showed that the amount of reteplase extracted from periplasmic space was much lower than the extracted inclusion bodies and large quantities of the recombinant protein were present as inclusion bodies. Therefore, it was more efficient to use inclusion body extraction method for protein isolation and purification. Conclusion: We produced active reteplase after its expression in E. coli TOP10 and isolation of inclusion bodies produced the best results for purification and extraction of this protein.</p> 168 175 Fatemeh Khodabakhsh Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Iran Zohreh Dehghani Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Iran Mohammad Farid Zia Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Iran Mohammad Rabbani Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Iran Hamid Mir Mohammad Sadeghi Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Sciences Research Centre, School of Pharmacy, Isfahan University of Medical Sciences Iran Arabinose<i>Escherichia coli</i>Gene expressionReteplase 122.pdf Lim HK, Jung KH, Park DH, Chung SI. Production characteristic of interferon-a using an L-arabinose promoter system in a high-cell-density culture. Appl Microbiol Biotechnol 2000;53(2):201-208.##Baruah DB, Dash RN, Chaudhari MR, Kadam SS. Plasminogen activators: A comparison. Vascul Pharmacol 2006;44(1):1-9.##Clark EDB. Protein refolding for industrial processes. Curr Opin Biotechnol. 2001;12:202-207.##Youchun Z, Wang G, Yang K, Changkai Z. Cloning, expression, and renaturation studies of reteplase. J Microbiol Biotechnl 2003;13:989- 992.##Sambrook J, Russell DW. Molecular cloning. A laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 2001. ##Llevadot J, Giugliano RP, Antman EM. Bolus fibrinolytic therapy in acute myocardial infarction. JAMA 2001;286(4):442-449.##Majidzadeh KA, Mahboudi F, Hemayatkar M, Davami F, Barkhordary F, Adeli A, et al. Human tissue plasminogen activator expression in Escherichia coli using cytoplasmic and periplasmic cumulative power. 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Overexpression and Purification of C-terminal Fragment of the Passenger Domain of Hap Protein from Nontypeable Haemophilus influenzae in a Highly Optimized Escherichia coli Expression System 2 2 Background: Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory tract disease and initiates infection by colonization in nasopharynx. The Haemophilus influenzae (H. influenzae) Hap adhesin is an auto transporter protein that promotes initial interaction with human epithelial cells. Hap protein contains a 110 kDa internal passenger domain called “HapS” and a 45 kDa C-terminal translocator domain called “Hapβ”. Hap adhesive activity has been recently reported to be connected to its Cell Binding Domain (CBD) which resides within the 311 C-terminal residues of the internal passenger domain of the protein. Furthermore, immunization with this CBD protein has been shown to prevent bacterial nasopharynx colonization in animal models. Methods: To provide enough amounts of pure HapS protein for vaccine studies, we sought to develop a highly optimized system to overexpress and purify the protein in large quantities. To this end, pET24a-cbd plasmid harboring cbd sequence from NTHi ATCC49766 was constructed and its expression was optimized by testing various expression parameters such as growth media, induction temperature, IPTG inducer concentration, induction stage and duration. SDS-PAGE and Western-blotting were used for protein analysis and confirmation and eventually the expressed protein was easily purified via immobilized metal affinity chromatography (IMAC) using Ni-NTA columns. Results: The highest expression level of target protein was achieved when CBD expressing E. coli BL21 (DE3) cells were grown at 37C in 2xTY medium with 1.0 mM IPTG at mid-log phase (OD600 nm equal to 0.6) for 5 hrs. Amino acid sequence alignment of expressed CBD protein with 3 previously published CBD amino acid sequences were more than %97 identical and antigenicity plot analysis further revealed 9 antigenic domains which appeared to be well conserved among different analyzed CBD sequences. Conclusion: Due to the presence of high similarity among CBD from NTHi ATCC49766 and other NTHi strains, CBD protein expressed here sounds to be theoretically ideal as a universal candidate for being used in vaccine studies against NTHi strains of various geographical areas. Further investigations to corroborate the potency of this protein as a vaccine candidate are under process. 176 185 Akram Tabatabaee Department of Biology, Science and Research Branch, Islamic Azad University Department of Biology, Science and Research Branch, Islamic Azad University Iran Seyed Davar Siadat Department of Bacteriology, Pasteur Institute of Iran Iran Seyed Fazllolah Mousavi Department of Bacteriology, Pasteur Institute of Iran Iran Mohammad Reza Aghasadeghi Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Arash Memarnejadian Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Mohammad Hassan Pouriayevali Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Neda Yavari Department of Microbiology, Karaj Islamic Azad University Department of Microbiology, Karaj Islamic Azad University Iran DensitometryNontypeable Haemophilus influenzaeOptimization 123.pdf Murphy TF. Current and future prospects for a vaccine for nontypeable Haemophilus influenzae. J Current Infect Dis 2009;11(3):177-182.##Murphy TF, Faden H, Bakaletz LO, Kyd JM, Campos J, Virji M, et al. Nontypeable Haemophilus influenzae as a pathogen in children. Pediatric Infect Dis J 2009;28(1):43-8.##Murphy TF, Apicella MA. Nontypeable Haemophilus influenzae: a review of clinical aspects, surface antigens, and the human immune response to infection. Rev Infect Dis 1987:9(1):1-15.##Cutter D, Mason KW, Howell AP, Fink DL, Green BA, St Geme JW 3rd. Immunization with Haemophilus influenzae hap adhesin protects against nasopharyngeal colonization in experimental mice. J Infect Dis 2002;186(8):1115-1121.##Murphy TF, Sethi S. Bacterial infection in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992;146(4):1067-1083.##St Geme JW 3rd. Insights into the mechanism of respiratory tract colonization by nontypable Haemophilus influenzae. Pediatr Infect Dis J 1997;16(10):931-935.##Murphy TF, Bernstein JM, Dryja DM, Campagnari AA, Apicella MA. Outer membrane protein and lipooligosaccharide analysis of paired nasopharyngeal and middle ear isolates in otitis media due to nontypeable Haemophilus influenzae: pathogenic and epidemiologic observations. J Infect Dis 1987;156(5):723-731.##Rao VK, Krasan GP, Hendrixson DR, Dawid S, St Geme JW 3rd. Molecular determinants of the pathogenesis of disease due to non-typeable Haemophilus influenzae. FEMS Microbiol Rev 1999;23(2):99-129.##St Geme JW 3rd. Pathogens due to nontypeable Haemophilus influenzae. In: Herbert MA, Hood DW, Moxon ER. Haemophilus influenza protocols. Michigan: Humana Press; 2003, 7-48.##Hendrixson DR, de la Morena ML, Stathopoulos C, St Geme JW 3rd. Structural determinants of processing and secretion of the Haemophilus influenzae hap protein. Mol Microbiol 1997;26(3):505-518.##Kenjale R, Meng G, Fink DL, Juehne T, Erickson HP, Waksman G, et al. Structural determinants of autoproteolysis of the Haemophilus influenzae hap autotransporter. Infect Immun 2009;77(11):4704-4713.##St Geme JW 3rd, de la Morena ML, Falkow S. A Haemophilus influenzae IgA protease-like protein promotes intimate interaction with human epithelial cells. Mol Microbiol 1994;14(2):217-233.##Kurono Y, Suzuki M, Yamamoto GM, Fujihashi K, McGhee JR, Kiyono H. Effects of intranasal immunization on protective immunity against otitis media. Int J Pediatr Otorhinolaryngol 1999;49(Suppl 1):S227-S229.##Fink DL, Buscher AZ, Green BA, Fernsten P, St Geme JW 3rd. The Haemophilus influenzae Hap autotransporter mediates microcolony formation and adherence to epithelial cells and extracellular matrix via binding regions in the C-terminal end of the passenger domain. Cell Microbiol 2003;5(3):175-186.##Liu DF, Mason KW, Mastri M, Pazirandeh M, Cutter D, Fink DL, et al. The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate. Infect Immun 2004;72(12):6961-6968.##Coleman HN, Daines DA, Jarisch J, Smith AL. Chemically defined media for growth of Haemophilus influenza strains. J Clin Microbiol 2003;41(9):4408-4410.##Sambrook J, Russell DW. In vitro amplification of DNA by the polymerase chain reaction. In: Janssen K. Molecular cloning. New York: Cold Spring Harbor Laboratory; 2001, 213-226.##Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 1979:76(9):4350-4354.##Kolaskar AS, Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 1990;276(1-2):172-174.##Fink DL, Green B, St Geme JW 3rd. The Haemophilus influenzae hap autotransporter binds to fibronectin, laminin, and collagen IV. Infect Immun 2002;70(9):4902-4907.##Hendrixson, DR, St Geme JW 3rd. The Haemophilus influenzae hap serine protease promotes adherence and microcolony formation, potentiated by a soluble host protein. Mol Cell 1998;2(6):841-850.##Performance standards for antimicrobial disk susceptibility tests; Approved Standard-Ninth Edition. CLSI Document M2A9, 26 (1), Wayne, PA, USA. ##Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 2009;18(5):936-948.##

Production of Cloned Mice by Nuclear Transfer of Cumulus Cells 2 2 Background: Over the past several years, mammals have been successfully cloned by either the splitting of an early stage embryo or nuclear transfer of adult somatic cells (NT) into oocytes. Although it has been 15 years since the generation of the first cloned mammals from somatic cells by NT, the success rate for producing live offspring by this technique is low regardless of the cell type and animal species used. However, these techniques have the potential to be important tools for future research in basic biology. In the present study, we described our experiences in producing successfully cloned mouse using NT method and piezo-actuated micromanipulator. Methods: B6D2F1 mice, 8-12 weeks old, were superovulated with injections of 5 IU of pregnant mare serum gonadotropin and 5 IU of human chorionic gonadotropin administered 48 hr apart. Enucleation and donor nuclei cumulus cell injection were performed with a piezo-actuated micromanipulator after which activation and trichostatin A treatment were used for reconstructed oocytes. Two-cell stage cloned embryos that developed in the mWM medium were transferred into the oviducts of pseudopregnant NMRI mice. Results: Of 367 oocytes collected, 131 (69%) developed into 2-cell stage embryos. Of these, 5 (1%) live pups were successfully delivered. We used NMRI foster mother to raise the pups by lactation. One adult cloned mouse was mated, after which she delivered and raised normal offspring. Conclusion: For mouse cloning, the present study also successfully tested the capability of somatic cell nuclear transfer SCNT using a piezo unit. 186 192 Soleiman Kurd Department of Biological Sciences and Biotechnology, School of Science, University of Kurdistan Department of Biological Sciences and Biotechnology, School of Science, University of Kurdistan Iran Mohammad Ali Zarei Department of Biological Sciences and Biotechnology, School of Science, University of Kurdistan Department of Biological Sciences and Biotechnology, School of Science, University of Kurdistan Iran Fardin Fathi Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Iran Tayyeb Ghadimi Department of Surgery, School of Medicine, Kurdistan University of Medical Sciences Department of Surgery, School of Medicine, Kurdistan University of Medical Sciences Iran Mohammad Saeed Hakhamaneshi Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Iran Ali Jalili Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Cellular and Molecular Research Center, Kurdistan University of Medical Sciences Iran MouseNuclear transfer techniquesTrichostatin A 124.pdf Yanagimachi R. Cloning: experience from the mouse and other animals. Mol Cell Endocrinol 2002;187(1-2):241-248.##Panno J. Animal cloning: the science of nuclear transfer. 1st ed. New York: Facts on File; 2005.##Pells S. Nuclear reprogramming: Methods and protocols.1st ed. Humana Press; 2005. ##Willadsen S.M. Nuclear transplantation in sheep embyos. Nature 1986;320(6057):63-65.##Wilmut I Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature 1997;385 (6619):810-813.##Wakayama T, Pery AC, Zuccotti M, Johnson KR, Yanagimachi R. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998;394(6691):369-374.##Kato Y, Tani T, Sotomaru Y, Kurokawa K, Kato J, Doguchi H, et al. Eight cattle cloned from somatic cells of a single adult. Science 1998;282(5396):2095-2098.##Baguisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, et al. Production of goats by somatic cell nuclear transfer. Nat Biotechnol 1999;17(5):456-461.##Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, et al. Dogs cloned from adult somatic cells. Nature 2005;436(7051):641.##Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, et al. A cat cloned by nuclear transplantation. Nature 2002;415(6874):859.##Chesne P, Adenot PG, Viglietta C, Baratte M, Boulanger L, Renard JP. Cloned rabbits produced by nuclear transfer from adult somatic cells. Nat Biotechnol 2002;20(4):366-369.##Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, et al. Pregnancy: a cloned horse born to its dam twin. Nature 2003;424(6949):635.##Zhou Q, Renard JP, Le Friec G, Brochard V, Beaujean N, Cherifi Y, et al. Generation of fertile cloned rats by regulating oocyte activation. Science 2003;302(5648):1179.##Li Z, Sun X, Chen J, Liu X, Wisely MS, Zhou Q, et al. Cloned ferrets produced by somatic cell nuclear transfer. Dev Biol 2007;293(2):439-448.##Ju B, Huang H, Lee KY, Lin S. Cloning zebrafish by nuclear transfer. Methods Cell Biol 2004;77:403-411.##Kazemi Ashtiani S, Nasr-Esfahani MH, Hosseini SM, Moulavi F, Hajian M, Frouzanfar M, et al. Royana: successful experience in cloning the sheep. Yakhteh Med J 2008;10(3):193-200.##Wakayama T, Yanagimachi R. Cloning of male mice from adult tail-tip cells. Nat Genet 1999; 22(2):127-128.##Ogura A, Inoue K, Ogonuki N, Noguchi A, Takano K, Nagano R, et al. Production of male cloned mice from fresh, cultured and cryopreserved immature Sertoli cells. Biol Reprod 2000;62(6):1579-1584.##Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 2001;58(4):376-383.##Ono Y, Shimozawa N, Ito M, Kono T. Cloned mice from fetal fibroblast cells arrested at metaphase by a serial nuclear transfer. Biol Reprod 2001;64(1):44-50.##Hu M, Wei H, Zhang J, Bai Y, Gao F, Li L, et al. Efficient production of chimeric mice from embryonic stem cells injected into 4-cell to 8-cell and blastocyst embryos. J Anim Sci Biotechnol 2013;4(1):12.##Wolf E, Zakhartchenko V, Brem G. Nuclear transfer in mammals: recent developments and future perspectives. J Biotechnol 1998;65(2-3):99-110.##Yang X, Smith LS, Tian CX, Lewin AH, Renard PJ, Wakayama T. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nat Genet 2007;39(3):295-302.##Mombaerts P. Therapeutic cloning in the mouse. PNAS 2003;100:11924-11925.##Kishigami S, Wakayama S, Thuan NV, Ohta H, Mizutani E, Hikichi T, et al. Production of cloned mice by somatic cell nuclear transfer. Nat Protoc 2006;1(1):125-138.##Kuhn R, Wurst W. Gene knockout protocols. 2nd ed. Humana Press; 2009.##Kishigami S, Mizutani E, Ohta H, Hikichi T, Thuan NV, Wakayama S, et al. Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer. Biochem Biophys Res Commun 2006;340(1):183-189.##Lyon MF, Rastan S, Brown SDM. Genetic variations and strains of the laboratory mouse. 3rd ed. New York: Oxford University Press; 1996.##Silver LM. Mouse genetics: concepts and applications. 1st ed. New York: Oxford University Press; 1995.##Hogan B, Costantini F, Lacy E. Manipulating the mouse embryo: A laboratory manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press; 1986.##Wassarman PM, DePamphilis ML. Guide to techniques in mouse development. Methods in enzymology. 2nd ed. Orlando, FL: Academic Press; 1993.##

A Novel Approach for High Level Expression of Soluble Recombinant Human Parathyroid Hormone (rhPTH 1-34) in Escherichia coli 2 2 Background: Parathyroid hormone (PTH) secreted by parathyroid glands regulates the metabolism of calcium and phosphorus in bone and kidney. Thereby, it can stimulate bone formation, and is a promising agent in the treatment of osteoporosis. Mature form of PTH consists of 84 amino acids; however, the first 34 residues of PTH cover the majority of hormonal action. Methods: In this study, the fusion form of highly soluble rhPTH was expressed at high level in Escherichia coli (E. coli). His6-thioredoxin as an extension for rhPTH improves the solubility of inclusion body. His6-thioredoxin-hPTH (1-34) was ligated into pET32a expression vector. The insertion of 5 amino acids (Asp-Asp-Asp-Asp-Lys) in the N-terminal of PTH made this protein to be digestable specifically by enterokinase enzyme. The fusion form of rhPTH was harvested and purified by immobilized affinity chromatography followed by digestion with enterokinase. Digested rhPTH was purified by applying on size exclusion and ion exchange chromatography to get the highest purity. Results: The mass spectroscopy analysis shows rhPTH molecular weight was 4117.5 Da. The purity was measured by HPLC column which showed more than 97%. Bioassay analysis of rhPTH was performed on rat sarcoma cell UMR-106 in parallel with commercially available rhPTH, Forteo. The result was measured through immunofluorescence detection kit. The data showed that the potency of rhPTH was comparable with commercially available medicine. Conclusion: Thioredoxin was applied as a fusion partner for production of highly soluble rhPTH. This specific fusion partner increased protein solubility and decreased protease reactivity. Purification process was optimized for high recovery and for purity more than 99%. As its biological activity is comparable with marketed drug, this protein is qualified for biopharmaceutical usage. 193 201 Haleh Hamedifar Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Firoozeh Salamat Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Mohammad Saffarion Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Mohammad Ghiasi Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Alireza Hosseini Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Hadi Lahiji Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Zomorrod Nouri Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Hamed Arfae Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Fereidoun Mahboudi Research and Development Department, CinnaGen, Simindasht Industrial Area Iran Enterokinase<i>Escherichia coli</i>Fusion proteinGene expressionParathyroid hormone 125.pdf Jin L, Briggs SL, Chandrasekhar S, Chirgadze NY, Clawson DK, Schevitz RW, et al. Crystal structure of human parathyroid hormone 1-34 at 0.9-A resolution. J Biol Chem 2000;275(35):27238-27244.##Murray TM, Cedergren L, Nilsson J, Nygren PA, Hammarberg B, Nilsson B, Enfors SO, Uhlén M. Parathyroid hormone secretion and action: evidence for discrete receptors for the carboxyl-terminal region and related biological actions of carboxyl- terminal ligands. Endocr Rev 2005;26(1):78-113.##Schluter KD, Weber M, Piper HM. Parathyroid hormone induces protein kinase C but not adenylate cyclase in adult cardiomyocytes and regulates cyclic AMP levels via protein kinase C-dependent phosphodiesterase activity. Biochem J 1995;310(Pt 2):439-444.##Sone T, Fukunaga M,Ono S, Nishiyama T. A small dose of human parathyroid hormone(1-34) increased bone mass in the lumbar vertebrae in patients with senile osteoporosis. Miner Electrolyte Metab 1995;21(1-3):232-235.##Brommage R, Hotchkiss CE, Lees CJ, Stancill MW, Hock JM, Jerome CP. Daily treatment with human recombinant parathyroid hormone-(1-34), LY333334, for 1 year increases bone mass in ovariectomized monkeys. J Clin Endocrinol Metab 1999;84(10):3757-3763.##Hock JM, Gera I, Fonseca J, Raisz LG. Human parathyroid hormone-(1-34) increases bone mass in ovariectomized and orchidectomized rats. Endocrinology 1988;122(6):2899-2904.##Neer RM, Arnaud CD, Zanchetta JR, Prince R, Gaich GA, Reginster JY, Het al. Effect of parathyroid hormone (1-34) on fractures and bone mineral density in postmenopausal women with osteoporosis. N Engl J Med 2001;344(19):1434-1441.##Bonn D. Parathyroid hormone for osteoporosis. Lancet 1996;347(8993):50.##Vad R, Nafstad E, Dahl LA, Gabrielsen OS. Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone. J Biotechnol 2005;116(3):251-260.##Liu Q, Lin J, Liu M, Tao X, Wei D, Ma X, et al. Large scale preparation of recombinant human parathyroid hormone 1-84 from Escherichia coli. Protein Expr Purif 2007;54(2):212-219.##Murby M, Cedergren L, Nilsson J, Nygren PA, Hammarberg B, Nilsson B, et al. Stabilization of recombinant proteins from proteolytic degradation in Escherichia coli using a dual affinity fusion strategy. Biotechnol Appl Biochem 1991;14(3):336-346.##Suzuki Y, Yabuta M, Ohsuye K. High-level production of recombinant human parathyroid hormone 1-34. Appl Environ Microb 1998;64(2):526-529.##Kostenuik PJ, Ferrari S, Pierroz D, Bouxsein M, Morony S, Warmington KS, et al. Infrequent delivery of a long-acting PTH-Fc fusion protein has potent anabolic effects on cortical and cancellous bone. J Bone Miner Res 2007;22(10):1534-1547.##Chunxiao W, Jingjing L, Yire X, Min D, Zhaohui W, Gaofu Q, et al. Study on preparation and activity of a novel recombinant human parathyroid hormone(1-34) analog with N-terminal Pro-Pro extension. Regul Pept 2007;141(1-3):35-43.##Chunxiao W, Yu Z, Wentao L, Jingjing L, Jiahui Y, Qingmei C. 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