US Editors and Reviewers can no Longer Handle Submissions by Authors Employed by the Government of Iran: Is it Fair and Logical? 2 2 We have recently become aware that the Office of Foreign Assets Control (OFAC) of the US Department of the Treasury has imposed new sanctions against Iran. Regulation 560.538 provides that US persons should not handle publishing services for written publications (whether journals or books) if any of the authors of the manuscript are employed by the Government of Iran. The Government of Iran is defined in Regulation 560.304. The regulation does not include authors at academic and research institutions or authors based in clinical settings such as hospitals, and should therefore only affect a very small number of submissions. Never-theless, some editors reject the manuscripts from Iran even from academic settings with abovementioned sanction. In practice the result of these sanctions will mean that: • Submissions where any author is based in Iran, and is not at an academic and research institution, cannot be handled by US-based editors, US Elsevier staff, US reviewers, or any US citizens based outside of the US. • If an Iranian author has dual affiliations (Eg. University and government), their submission cannot be handled by US-based editors, US Elsevier staff, US reviewers, or any US citizens based outside of the US. • Affiliations of Iranian authors should therefore be checked, and any manuscripts which fall under this OFAC regulation delegated to a non-US editor, before handling. • When assigning reviewers, affiliations of Iranian authors should also be checked, and any papers which fall under this OFAC regulation should only be sent to non-US reviewers. (As an editor you should do what is reasonable to determine the nationalities of a reviewer e.g. check their email address. This check does not extend to emailing reviewers directly to confirm their nationality or location). Unless there is specific knowledge that a non-US-based reviewer is a US citizen, editors can send such submissions to reviewers based outside the US. Iranian researchers and academicians are disappointed that some publishers have created the impression that work from Iran should be discriminated against. This attitude is contrary to the spirit and values of global science. Over the last two decades everyone has spoken regarding global village and this action is against that. As an editor from Iran I ask my counterparts over the world that do you think this behavior and action is fair and logical. 203 203 Shahin Akhondzadeh Tehran University of Medical Sciences Tehran University of Medical Sciences Iran Editorial 172.pdf ####

Cloning, Expression and Purification of Penicillin Binding Protein2a (PBP2a) from Methicillin Resistant Staphylococcus aureus : A Study on Immunoreactivity in Balb/C Mouse 2 2 Background: Staphylococcus aureus (S. aureus) is a major nosocomial pathogen and the infection with this organism in human is increasing due to the spread of antibiotic resistant strains. One of the resistance mechanisms of S. aureus comprises modification in binding proteins to penicillin. Vaccine strategy may be useful in controlling the infections induced by this organism. This study aimed at developing and producing the recombinant protein PBP2a as a vaccine candidate and evaluating the related humoral immune response in a murine model. Methods: A 242 bp fragment of mecA gene was amplified by PCR from S. aureus COL strain and then cloned into prokaryotic expression vector pET-24a. For expression of recombinant protein, pET24a-mec plasmid was transformed into competent E. coli BL21 (DE3) cells. Recombinant protein was over expressed with 1 mM isopropythio-β-D-galctoside (IPTG) and purified using Ni-NTA agarose. SDS-PAGE and western blotting were carried out to confirm protein expression. For immunization of experimental groups, Balb/c mice were injected subcutaneously with 20 µg of recombinant PBP2a three times with three weeks intervals. The sera of experimental groups were collected three weeks after the last immunization and then specific antibodies were evaluated by ELISA method. Results: Successful cloning of mecA was confirmed by colony-PCR, enzymatic digestion, and sequencing. SDS-PAGE and western blot analysis showed that recombinant protein with molecular weight of 13 kDa is over expressed. In addition, high titer of specific antibody against PBP2a in vaccinated mice was developed as compared with the control group and confirmed the immunogenicity of the vaccine candidate. Conclusion: Results suggest that PBP2a recombinant induced specific antibodies and can be used as Staphylococcal vaccine candidate after further studies. 204 211 Setareh Haghighat Department of Biology, Faculty of Basic Sciences, Science and Research branch, Islamic Azad University Department of Biology, Faculty of Basic Sciences, Science and Research branch, Islamic Azad University Iran Seyed Davar Siadat Department of Microbiology, Pasteur Institute of Iran Iran Seyed Mehdi Rezayat Sorkhabadi Department of Pharmacology, School of Advanced Sciences and Technology in Medicine, Tehran University of Medical Sciences Department of Pharmacology, School of Advanced Sciences and Technology in Medicine, Tehran University of Medical Sciences Iran Abbas Akhavan Sepahi Department of Microbiology, Faculty of Basic Sciences, North Tehran Branch, Islamic Azad University Department of Microbiology, Faculty of Basic Sciences, North Tehran Branch, Islamic Azad University Iran Mehdi Mahdavi Department of Virology, Pasteur Institute of Iran Iran Methicillin-resistant Staphylococcus aureusPBP2aRecombinant vaccine 126.pdf Kuklin NA, Clark DJ, Secore S, Cook J, Cope LD, McNeely T, et al. A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model. Infect Immun 2006;74(4):2215-2223.##Selvey LA, Whitby M, Johnson B. Nosocomial methicillin resistant Staphylococcus aureus bacteremia: is it any worse than nosocomical methicillin sensitive Staphylococcus aureus bacteremia. Infect Control Hosp Epidemiol 2000;21(10):645-648.##Cooper BS. Infection control in healthcare settings. parliamentary office of science and technology 2005;247:1-5.##Lowy FD. Staphylococcus aureus infection. N Engl J Med 1998;339(8):520-532.##Deleo RF, Otto M, Kreiswirth BN, Chambers HF. Community-associated methicillin-resistant Staphylococcus aureus. Lancet 2010;375(9725):1557-1568.##Ohwada A, Sekiya M, Hanaki H, Arai KK, Naga-oka I, Hori S, et al . DNA vaccination by mecA sequence evokes an antibacterial immune response against methicillin resistant Staphylococcus aureus. J Antimicrob Chemother 1999;44(6):767-774. ##Roth DM, Senna JP, Machado DC. Evaluation of the humoral immune response in BALB/c mice immunized with a naked DNA vaccine anti- methicillin-resistant Staphylococcus aureus. Genet Mol Res 2006;5(3):503-512.##Senna JP, Roth DM, Oliveira JS, Machado DC, Santos DS. Protective immune response against methicillin resistant Staphylococcus aureus in a murine model using a DNA vaccine approach. Vac-cine 2003;21(19-20):2661-2666.##Gofﬁn C, Ghuysen JM. Multimodular penicillin-binding proteins: an enigmatic family of orthologs and paralogs. Microbiol Mol Biol Rev 1998;62(4):1079-1093.##Katayama Y, Ito T, Hiramatsu K. A new class of genetic element, Staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 2000;44(6):1549-1555.##Pearson H. "Superbug" hurdles key drug barrier. Nature 2002;418(6897):469.##Chambers HF. Methicillin resistance in staphylococci: molecular and biochemical basis and clinical implications. Clin Microbiol Rev 1997;10(4):781-791.##Stranger-Jones YK, Bae T, Schneewind O. Vaccine assembly from surface proteins of Staphylococcus aureus. Proc Natl Acad Sci USA 2006;103(45):16942-16947.##Cohen ML. Staphylococcus aureus: biology, mechanism of virulence, epidemiology. J Pediatr 1986;108(5Pt 2):796-799.##Foster TJ. Potential for vaccination against infection caused by Staphylococcus aureus. Vaccine 1991;9(4):221-227.##Michie CA. Staphylococcal vaccines. Trends Immunol 2002;23(9):461-464.##Pourmand MR, Foster SJ. A novel bioinformatics approach for Staphylococcal vaccine development. Tehran Univ Med J 2006;19-27.##Weichhart T, Horky M, Söllner J, Gangl S, Henics T, Nagy E, et al. Functional selection of vaccine candidate peptides from Staphylococcus aureus whole-genome expression libraries in vitro. Infect Immun 2003;71(8):4633-4641.##Etz H, Minh DB, Henics T, Dryla A, Winkler B, Triska C, et al. Identification of in vivo expressed vaccine candidate antigens from Staphylococcus aureus. Proc Natl Acad Sci USA 2002;99(10):6573-6578.##Jones C. Revised structures for the capsular polysaccharides from Staphylococcus aureus Types 5 and 8, components of novel glycoconjugate vaccines. Carbohydrate Res 2005;340(6):1097-1106.##Josefsson E, Hartford O, O'Brien L, Patti JM, Foster T. Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis 2001;184(12):1572-1580.##Ito T, Hiramatsu K, Tomasz A, de Lencastre H, Perreten V, Holden MT, et al. Guidelines for reporting novel mecA gene homologues. Antimicrob Agents Chemother 2012;56(10):4997-4999.##Mamo W, Jonsson P, Flock JI, Lindberg M, Müller HP, Wadström T, et al. Vaccination against Staphylococcus aureus mastitis: Immunological response of mice vaccinated with fibronectin-binding protein to challenge with S. aureus. Vaccine 1994;12(11):988-992.##Lim D, Strynadka CJ. Structural basis for the ß-lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus. Nat Struct Biol 2002;9(11):870-876.##Hong N, Yunying W, Weixian CH. Clone and construction of prokaryotic expression plasmid of MRSA-PBP2a. Chinese J Microbiol 2011;05:007.##Goudarzi G, Sattari M, Roudkenar MH, Montajabi-Niyat M, Zavaran-Hosseini A, Mosavi-Hosseini K. Cloning, expression, purification, and characterization of recombinant flagellin isolated from Pseudomonas aueroginosa. Biotechnol Lett 2009;31(9):1353-1360.##Tsumoto K, Abe R, Ejima D, Arakawa T. Non denaturing solubilization of inclusion bodies. Curr Pharm Biotechnol 2010;11(3):309-312.##Casadevall A. Antibody-mediated immunity against intracellular pathogens: two-dimensional thinking comes full circle. Infect Immun 2003;71(8):4225-4228.##Abbas AK, Litchtman AH, Pillai SH. Cellular and molecular immunology. 6th ed. Philadelphia: Elsevier; 2007.##

Production and Characterization of a Murine Monoclonal Antibody Against Human Ferritin 2 2 Background: Ferritin is an iron storage protein, which plays a key role in iron metabolism. Measurement of ferritin level in serum is one of the most useful indicators of iron status and also a sensitive measurement of iron deficiency. Monoclonal antibodies may be useful as a tool in various aspects of ferritin investigations. In this paper, the production of a murine monoclonal antibody (mAb) against human ferritin was reported. Methods: Balb/c mice were immunized with purified human ferritin and splenocytes of hyper immunized mice were fused with Sp2/0 myeloma cells. After four times of cloning by limiting dilution, a positive hybridoma (clone: 2F9-C9) was selected by ELISA using human ferritin. Anti-ferritin mAb was purified from culture supernatants by affinity chromatography. Results: Determination of the antibody affinity for ferritin by ELISA revealed a relatively high affinity (2.34×109 M-1) and the isotype was determined to be IgG2a. The anti-ferritin mAb 2F9-C9 reacted with 79.4% of Hela cells in flow cytometry. The antibody detected a band of 20 kDa in K562 cells, murine and human liver lysates, purified ferritin in Western blot and also ferritin in human serum. Conclusion: This mAb can specifically recognize ferritin and may serve as a component of ferritin diagnostic kit if other requirements of the kit are met. 212 219 Ali Ahmad Bayat Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Omid Yeganeh Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Roya Ghods Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Amir-Hassan Zarnani Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Iran Reza Bahjati Ardekani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Ahmad Reza Mahmoudi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Jafar Mahmoudian Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Farzaneh Haghighat-Noutash Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran ELISAFerritinFlow cytometryMonoclonal antibodyWestern blotting 127.pdf Balla G, Jacob HS, Balla J, Rosenberg M, Nath K, Apple F, et al. Ferritin: a cytoprotective antioxidant strategem of endothelium. J Biol Chem 1992;267(25):18148-18153.##Goswami B, Tayal D, Mallika V. Ferritin: A multidimensional bio marker. The Internet Journal of Laboratory Medicine 2009;3(2):http://www.ispub.##Theil EC. Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu Rev Biochem 1987;56:289-315.##Andrews SC, Arosio P, Bottke W, Briat JF, von Darl M, Harrison PM, et al. Structure, function, and evolution of ferritins. J Inorg Biochem 1992;47(3-4):161-174.##Koorts AM, Viljoen M. Ferritin and ferritin isoforms I: Structure-function relationships, synthesis, degradation and secretion. Arch Physiol Biochem 2007;113(1):30-54.##Siimes MA, Addiego JE, Jr., Dallman PR. Ferritin in serum: diagnosis of iron deficiency and iron overload in infants and children. Blood 1974;43(4):581-590.##Shpyleva SI, Tryndyak VP, Kovalchuk O, Starlard-Davenport A, Chekhun VF, Beland FA, et al. Role of ferritin alterations in human breast cancer cells. Breast Cancer Res Treat 2011;126(1):63-71.##Alkhateeb AA, Han B, Connor JR. Ferritin stimulates breast cancer cells through an iron-independent mechanism and is localized within tumor-associated macrophages. Breast Cancer Res Treat 2013;137(3):733-744.##Ćujić D, Stefanoska I, Golubović S. Serum ferritin in healthy women and breast cancer patients. J Med Biochem 2010;30(1):33-37.##Kuvibidila SR, Gauthier T, Rayford W. Serum ferritin levels and transferrin saturation in men with prostate cancer. J Natl Med Assoc 2004;96(5):641-649.##Chou SF, Chen CY. Monoclonal and polyclonal antibodies against human ferritin, a nonspecific tumor marker. Hybridoma 2001;20(1):59-62.##Uesaka K. Production and clinical study of monoclonal antibodies against liver ferritin. Gan No Rinsho 1986;32(9):987-991.##Stevens RG, Graubard BI, Micozzi MS, Neriishi K, Blumberg BS. Moderate elevation of body iron level and increased risk of cancer occurrence and death. Int J Cancer 1994;56(3):364-369.##Lee CY, Leung WY, Tung JK. Applications of a monoclonal antibody to human ferritin in various immunoassays. Biotechnol Appl Biochem 1987;9(1):31-38.##Rath CE, Finch CA. Sternal marrow hemosiderin; a method for the determination of available iron stores in man. J Lab Clin Med 1948;33(1):81-86.##Lundin P, Lundquist A, Lundvall O. Evaluation of fine-needle aspiration biopsy smears in the diagnosis of liver iron overload. Acta Med Scand 1969;186(5):369-373.##Behjati Ardakani R, Ghods R, Bayat AA, Jeddi Tehrani M. Extraction and purification of ferritin from liver tissue. JBUM 2005;7(1).##Hadavi R, Zarnani AH, Ahmadvand N, Mahmoudi AR, Bayat AA, Mahmoudian J, et al. Production of monoclonal antibody against human nestin. Avicenna J Med Biotechnol 2010;2(2):69-77.##Loirat MJ, Gourbil A, Frioux Y, Muller JY, Blanchard D. A murine monoclonal antibody directed against the Gerbich 3 blood group antigen. Vox Sang 1992;62(1):45-48.##Kazemi T, Tahmasebi F, Bayat AA, Mohajer N, Khoshnoodi J, Jeddi-Tehrani M, et al. Characterization of novel murine monoclonal antibodies directed against the extracellular domain of human HER2 tyrosine kinase receptor. Hybridoma (Larchmt) 2011;30(4):347-353.##Mahmoudian J, Jeddi-Tehrani M, Bayat AA, Mahmoudi AR, Vojgani Y, Tavangar B, et al. A monoclonal antibody against leptin. Hybridoma (Larchmt) 2012;31(5):372-377.##Hajighasemi F, Shokri F. Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG. Avicenna J Med Biotechnol 2010;2(1):37-45.##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.##Cazzola M, Bergamaschi G, Tonon L, Arbustini E, Grasso M, Vercesi E, et al. Hereditary hyperferritinemia-cataract syndrome: relationship between phenotypes and specific mutations in the iron-responsive element of ferritin light-chain mRNA. Blood 1997;90(2):814-821.##Orino K, Eguchi K, Nakayama T, Yamamoto S, Watanabe K. Sequencing of cDNA clones that encode bovine ferritin H and L chains. Comp Biochem Physiol B Biochem Mol Biol 1997;118(3):667-673.##Boyd D, Jain SK, Crampton J, Barrett KJ, Drysdale J. Isolation and characterization of a cDNA clone for human ferritin heavy chain. Proc Natl Acad Sci USA 1984;81(15):4751-4755.##Mel'nikova IaI, Lunev VE, Preigerzon VA, Luneva NM, Koshkin SA, Rodionov MA, et al. Monoclonal antibodies to human spleen ferritin. II. Localization of epitopes and quantitative parameters of antigen binding. Biokhimiia 1993;58(5):759-771.##Nozawa S, Tsukazaki K, Narisawa S. Production of monoclonal antibodies to ferritin and development of the enzyme immunoassay system. Nihon Sanka Fujinka Gakkai Zasshi 1985;37(12):2775-2783.##

Production and Characterization of Recombinant Light Chain and Carboxyterminal Heavy Chain Fragments of Tetanus Toxin 2 2 Background: Light chain (LC) and heavy chain carboxyterminal subdomain (HCC) fragments are the most important parts of tetanus neurotoxin (TeNT) which play key roles in toxicity and binding of TeNT, respectively. In the present study, these two fragments were cloned and expressed in a prokaryotic system and their identity was confirmed using anti-TeNT specific polyclonal and monoclonal antibodies. Methods: LC and HCC gene segments were amplified from Clostridium tetani genomic DNA by PCR, cloned into pET28b(+) cloning vector and transformed in Escherichia coli (E. coli) BL21(DE3) expression host. Recombinant proteins were then purified through His-tag using Nickel-based chromatography and characterized by SDS-PAGE, Western blotting and ELISA techniques. Results: Recombinant light chain and HCC fragments were successfully cloned and expressed in (E. coli) BL21 (DE3). Optimization of the induction protocol resulted in production of high levels of HCC (~35% of total bacterial protein) and to lesser extends of LC (~5%). Reactivity of the His-tag purified proteins with specific polyclonal and monoclonal antibodies confirmed their renatured structure and identity. Conclusion: Our results indicate successful cloning and production of recombinant LC and HCC fragments of TeNT. These two recombinant proteins are potentially useful tools for screening and monitoring of anti-TeNT antibody response and vaccine production. 220 226 Mehdi Yousefi Department of Immunology, School of Public Health, Tehran University of Medical SciencesImmunology Research Center, Tabriz University of Medical SciencesDepartment of Immunology, School of Medicine, Tabriz University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical SciencesImmunology Research Center, Tabriz University of Medical SciencesDepartment of Immunology, School of Medicine, Tabriz University of Medical Sciences IranIranIran Roya Khosravi-Eghbal Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Azam Hemmati Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Fazel Shokri Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR IranIran Fragment CLight chainMonoclonal antibodyTetanus toxin 128.pdf Ataro PD, Mushatt D, Ahsan S. Tetanus: a review. South Med J 2011;104(8):613-617.##alli G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G. The journey of tetanus and botulinum neurotoxins in neurons. Trends Microbiol 2003;11(9):431-437.##Schiavo G, Rossetto O, Montecucco C. Clostridial neurotoxins as tools to investigate the molecular events of neurotransmitter release. Semin Cell Biol 1994;5(4):221-229.##Schiavo G, Papini E, Genna G, Montecucco C. An intact interchain disulfide bond is required for the neurotoxicity of tetanus toxin. Infect Immun 1990;58(12):4136-4141.##Jahn R, Niemann H. Molecular mechanisms of clostridial neurotoxins. Ann NY Acad ScI 1994;733(1):245-255.##Lacy DB, Tepp W, Cohen AC, DasGupta BR, Stevens RC. Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol 1998;5(10):898-902.##Swaminathan S, Eswaramoorthy S. Structural analysis of the catalytic and binding sites of Clostridium botulinum neurotoxin B. Nat Struct Biol 2000;7(8):693-699.##Schiavo G, Matteoli M, Montecucco C. Neurotoxins affecting neuroexocytosis. Physiol Rev 2000;80(2):717-766.##Montecucco C, Schiavo G. Structure and function of tetanus and botulinum neurotoxins. Q Rev Biophys 1995;28(04):423-472.##Grumelli C, Verderio C, Pozzi D, Rossetto O, Montecucco C, Matteoli M. Internalization and mechanism of action of clostridial toxins in neurons. Neurotoxicology 2005;26(5):761-767.##Galazka A, Gasse F. The present status of tetanus and tetanus vaccination. Curr Top Microbiol Immunol 1995;195:31.##Chen C, Fu Z, Kim JJ, Barbieri JT, Baldwin MR. Gangliosides as high affinity receptors for tetanus neurotoxin. J Biol Chem 2009;284(39):26569-26577.##Lang AB, Cryz SJ Jr, Schürch U, Ganss MT, Bruderer U. Immunotherapy with human monoclonal antibodies. Fragment A specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin. J Immunol 1993;151(1):466-472.##Yousefi M, Tahmasebi F, Younesi V, Razavi A, Khoshnoodi J, Bayat AA, et al. Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C. J Immunotoxicolo 2013;1-7.##Yoon TY, Shin YK. Progress in understanding the neuronal SNARE function and its regulation. Cell Mol Life Sci 2009;66(3):460-469.##Chaddock J, Marks PM. Clostridial neurotoxins: structure-function led design of new therapeutics. Cell Mol Life Sci 2006; 63(5):540-551.##Singh BR, Thirunavukkarasu N, Ghosal K, Ravichandran E, Kukreja R, Cai S, et al. Clostridial neurotoxins as a drug delivery vehicle targeting nervous system. Biochimie 2010;92(9):1252-1259.##Herreros J, Lalli G, Montecucco C, Schiavo G. Tetanus toxin fragment C binds to a protein present in neuronal cell lines and motoneurons. J Neurochem 2000;74(5):1941-1950.##Herreros J, Ng T, Schiavo G. Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons. Mol Biol Cell 2001;12(10):2947-2960.##Herreros J, Lalli G, Montecucco C, Schiavo G. Tetanus toxin fragment C binds to a protein present in neuronal cell lines and motoneurons. J Neurochem 2008;74(5):1941-1950.##Montecucco C, Rossetto O, Schiavo G. Presynaptic receptor arrays for clostridial neurotoxins. Trends Microbiol 2004;12(10):442-446.##Yeh FL, Dong M, Yao J, Tepp WH, Lin G, Johnson EA, et al. SV2 mediates entry of tetanus neurotoxin into central neurons. PLoS Pathogens 2010;6(11):e1001207.##Swaminathan S. Molecular structures and functional relationships in clostridial neurotoxins. FEBS J 2011;278(23):4467-4485.##

Expression and Purification of Recombinant ROP1 of Toxoplasma gondii in Bacteria 2 2 Background: Toxoplasmosis is a worldwide-distributed infection which is mostly asymptomatic but can cause serious health problems in congenitally-infected newborns and immunecompromised individuals. Research is undergoing both to improve Toxoplasma serological tests, which play the main role in laboratory diagnosis of the infection, and develop an effective vaccine to prevent the infection. Some studies showed usefulness of rhoptry protein 1 (ROP1) antigen of Toxoplasma gondii (T. gondii) in serodiagnosis of the infection and induction of protective immunity. The purpose of this study was to produce recombinant ROP1 and evaluate its antigenicity against human infected sera. Methods: DNA encoding ROP1, amino acids 171 to 574, was obtained from T. gondii RH strain by polymerase chain reaction amplification and cloned in prokaryotic expression plasmid pET-15b. rROP1 was expressed in Escherichia coli (E. coli) and purified in a single step by immobilized metal ion affinity chromatography. Results: DNA sequencing showed 99% similarity between the cloned sequence and the corresponding sequence in Gene bank. Results indicated the proper antigenicity of rROP1. Sera from Toxoplasma infected individuals specifically recognized rROP1 in Western blotting. Conclusion: rROP1 is antigenic toward human infected sera and can be used in studies for development of both a Toxoplasma serological test and a protective vaccine. 227 233 Reyhaneh Mohabati Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of IranDepartment of Biochemistry, Science and Research Branch, Islamic Azad University Department of Biochemistry, Science and Research Branch, Islamic Azad University IranIran Jalal Babaie Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran Iran Samira Amiri Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran Iran Mohammad Talebzadeh Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran Iran Pezhman Fard-Esfahani Department of Biochemistry, Pasteur Institute of Iran Iran Mojtaba Darbouy Department of Biochemistry, Science and Research Branch, Islamic Azad University Department of Biochemistry, Science and Research Branch, Islamic Azad University Iran Majid Golkar Molecular Parasitology Laboratory, Department of Parasitology, Pasteur Institute of Iran Iran Gene expressionPurificationROP1 proteinToxoplasma gondii 129.pdf Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30(12-13):1217-1258.##Dubey JP. The history of Toxoplasma gondii--the first 100 years. J Eukaryot Microbiol 2008;55(6):467-475.##Weiss LM, Dubey JP. Toxoplasmosis: A history of clinical observations. Int J Parasitol 2009;39(8):895-901.##Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004;363(9425):1965-1976.##Aubert D, Maine GT, Villena I, Hunt JC, Howard L, Sheu M, et al. Recombinant antigens to detect Toxoplasma gondii-specific immunoglobulin G and immunoglobulin M in human sera by enzyme immunoassay. J Clin Microbiol 2000;38(3):1144-50.##Jones JL, Kruszon-Moran D, Wilson M, McQuillan G, Navin T, McAuley JB. Toxoplasma gondii infection in the United States: seroprevalence and risk factors. Am J Epidemiol 2001;154(4):357-365.##Remington JS, Thulliez P, Montoya JG. Recent developments for diagnosis of toxoplasmosis. J Clin Microbiol 2004;42(3):941-945.##Ferrandiz J, Mercier C, Wallon M, Picot S, Cesbron-Delauw MF, Peyron F. Limited value of assays using detection of immunoglobulin G antibodies to the two recombinant dense granule antigens, GRA1 and GRA6 Nt of Toxoplasma gondii, for distinguishing between acute and chronic infections in pregnant women. Clin Diagn Lab Immunol 2004;11(6):1016-1021.##Montoya JG. Laboratory diagnosis of Toxoplasma gondii infection and toxoplasmosis. J Infect Dis 2002;185(Suppl 1):S73-S82.##Liesenfeld O, Press C, Montoya JG, Gill R, Isaac-Renton JL, Hedman K, et al. False-positive results in immunoglobulin M (IgM) toxoplasma antibody tests and importance of confirmatory testing: the Platelia Toxo IgM test. J Clin Microbiol 1997;35(1):174-178.##Wilson M, Remington JS, Clavet C, Varney G, Press C, Ware D. Evaluation of six commercial kits for detection of human immunoglobulin M antibodies to Toxoplasma gondii. The FDA Toxoplasmosis Ad Hoc Working Group. J Clin Microbiol 1997;35(12):3112-3115.##Gatkowska J, Hiszczynska-Sawicka E, Kur J, Holec L, Dlugonska H. Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model. Exp Parasitol 2006;114(3):220-227.##Beghetto E, Spadoni A, Bruno L, Buffolano W, Gargano N. Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol 2006;44(6):2133-2140.##Dlugonska H. Toxoplasma rhoptries: unique secretory organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis. J Biomed Biotechnol 2008;2008:632424.##Sam-Yellowe TY. Rhoptry organelles of the apicomplexa: Their role in host cell invasion and intracellular survival. Parasitol Today 1996;12(8):308-316.##Ossorio PN, Schwartzman JD, Boothroyd JC. A Toxoplasma gondii rhoptry protein associated with host cell penetration has unusual charge asymmetry. Mol Biochem Parasitol 1992;50(1):1-15.##Maine GT, Hunt JC, Brojanac S, Sheu MJT, Chovan LE, Tyner JD, et al., inventors; Abbott Laboratories, assignee. Antigen cocktails and uses thereof. United States patent 6329157-A. 2003.##Bradley PJ, Hsieh CL, Boothroyd JC. Unprocessed Toxoplasma ROP1 is effectively targeted and secreted into the nascent parasitophorous vacuole. Mol Biochem Parasitol 2002;125(1-2):189-193.##Reichmann G, Dlugonska H, Fischer HG. Characterization of TgROP9 (p36), a novel rhoptry protein of Toxoplasma gondii tachyzoites identified by T cell clone. Mol Biochem Parasitol 2002;119(1):43-54.##Saffer LD, Mercereau-Puijalon O, Dubremetz JF, Schwartzman JD. Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration. J Protozool 1992;39(4):526-530.##Holec-Gasior L, Drapala D, Lautenbach D, Kuri J. Toxoplasma gondii: usefulness of ROP1 recombinant antigen in an immunoglobulin G avidity assay for diagnosis of acute toxoplasmosis in humans. Pol J Microbiol 2010;59(4):307-310.##Pfrepper KI, Enders G, Gohl M, Krczal D, Hlobil H, Wassenberg D, et al. Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clin Diagn Lab Immunol 2005;12(8):977-982.##Wang Q, Jiang W, Chen YJ, Liu CY, Shi JL, Li XT. Prevalence of Toxoplasma gondii antibodies, circulating antigens and DNA in stray cats in Shanghai, China. Parasit Vectors 2012;5:190.##Li B, Oledzka G, McFarlane RG, Spellerberg MB, Smith SM, Gelder FB, et al. Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes. Parasite Immunol 2010;32(9-10):671-683.##Soldati D, Lassen A, Dubremetz JF, Boothroyd JC. Processing of Toxoplasma ROP1 protein in nascent rhoptries. Mol Biochem Parasitol 1998;96(1-2):37-48.##Yildir C, Oensan Z, Kirdar B. Optimization of starting time and period of induction and inducer concentration in the production of the restriction enzyme EcoRI from recombinant Escherichia coli 294. Turk J Chem 1998;22(3):221-226.##Holec-Gasior L, Kur J, Hiszczynska-Sawicka E. GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii-specific immunoglobulin G in humans with acute toxoplasmosis. Clin Vaccine Immunol 2009;16(4):510-514.##Chen G, Guo H, Lu F, Zheng H. Construction of a recombinant plasmid harbouring the rhoptry protein 1 gene of Toxoplasma gondii and preliminary observations on DNA immunity. Chin Med J (Engl) 2001;114(8):837-840.##

Micropatterning of ECM Proteins on Glass Substrates to Regulate Cell Attachment and Proliferation 2 2 Background: Micropatterning is becoming a powerful tool for studying cells in vitro. This method not only uses very small amount of material but also mimic the microenvironment structure present in living tissues better than flask culturing techniques. In previous studies using micropatterning of extracellular matrix proteins on glass surfaces, the rate of protein detachment from the surface was so high that the proteins and the cultivated cells detached after 3 three days of cell seeding. Methods: Here we optimized the glass surface modification method to fulfill the requirement of most in vitro studies. Results: in our study we showed that the optimum time for glass surface modification reaction is 1.5 hr, and the cells could be tracked in vitro for over 15 days after cell seeding which is enough for the most in vitro studies. As a model, we cultivated HEK 293T and HepG2 cells on the collagen micropatterns and showed that they have normal growth and morphology on these micropatterns. The HEK cells also transfected with pmaxGFP plasmid vector to show that the cells on collagen micropatterns could also used in transfection studies. Conclusion: Taking these together, this novel method is promising for efficient cell culture studies on micropatterened surfaces in the future. 234 240 Omid G. Alamdari Department of Biotechnology, College of Science, University of TehranDepartment of Nanotechnology and Tissue engineering, Stem Cell Technology Research Center Department of Biotechnology, College of Science, University of TehranDepartment of Nanotechnology and Tissue engineering, Stem Cell Technology Research Center IranIran Ehsan Seyedjafari Department of Biotechnology, College of Science, University of Tehran Department of Biotechnology, College of Science, University of Tehran Iran Masoud Soleimani Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University Iran Nasser Ghaemi Department of Biotechnology, College of Science, University of Tehran Department of Biotechnology, College of Science, University of Tehran Iran Cellular microenvironmentCollagen type I Tissue engineering 130.pdf Théry M. Micropatterning as a tool to decipher cell morphogenesis and functions. J Cell Sci 2010;123(Pt 24):4201-4213. ##Ruiz S, Chen C. Microcontact printing: A tool to pattern. Soft Matter 2007;3:168-177.##Théry M, Racine V, Pépin A, Piel M, Chen Y, Sibarita JB, et al. The extracellular matrix guides the orientation of the cell division axis. Nat Cell Biol 2005;7(10):947-953. ##Théry M, Racine V, Piel M, Pépin A, Dimitrov A, Chen Y, et al. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc Natl Acad Sci USA 2006;103(52):19771-19776.##Théry M, Bornens M. Cell shape and cell division. Curr Opin Cell Biol 2006;18(6):648-657.##Théry M, Piel M. Adhesive micropatterns for cells: a microcontact printing protocol. Cold Spring Harb Protoc 2009;2009(7):pdb.prot5255.##Li N, Tourovskaia A, Folch A. Biology on a chip: microfabrication for studying the behavior of cultured cells. Crit Rev Biomed Eng 2003;31(5-6):423-488.##Liu WW, Chen ZL, Jiang XY. Methods for cell micropatterning on two-dimensional surfaces and their applications in biology. Chinese J Anal Chem;2009;37(7):943-949.##Carter S. Haptotactic islands: a method of confining single cells to study individual cell reactions and clone formation. Exp Cell Res 1967;48(1):189-193.##Harris A. Behavior of cultured cells on substrata of variable adhesiveness. Exp Cell Res 1973;77(1):285-297.##Ricoult SG, Goldman JS, Stellwagen D, Juncker D, Kennedy TE. Generation of microisland cultures using microcontact printing to pattern protein substrates. J Neurosci Methods 2012;208(1):10-17. ##Lee JY, Jones C, Zern MA, Revzin A. Analysis of local tissue-specific gene expression in cellular micropatterns. Anal Chem 2006;78(24):8305-8312.##Xu W, Sims C, Allbritton N. Microcup arrays for the efficient isolation and cloning of cells. Anal Chem 2010;82(8):3161-3167.##Luchansky M, Bailey R. Rapid, multiparameter profiling of cellular secretion using silicon photonic microring resonator arrays. J Am Chem Soc 2011;133(50):20500-20506. ##Kuhn E, Naschberger E, Konrad A, Croner RS, Britzen-Laurent N, Jochmann R, et al. A novel chip-based parallel transfection assay to evaluate paracrine cell interactions. Lab Chip 2012;12(7):1363-1372.##Falconnet D, Csucs G, Grandin HM, Textor M. Surface engineering approaches to micropattern surfaces for cell-based assays. Biomaterials 2006;27(16):3044-3063.##Jones CN, Tuleuova N, Lee JY, Ramanculov E, Reddi a H, Zern MA, et al. Cultivating hepatocytes on printed arrays of HGF and BMP7 to characterize protective effects of these growth factors during in vitro alcohol injury. Biomaterials 2010;31(23):5936-5944.##Jones CN, Tuleuova N, Lee JY, Ramanculov E, Reddi a H, Zern MA, et al. Cultivating liver cells on printed arrays of hepatocyte growth factor. Biomaterials 2009;30(22):3733-3341. ##Lee JY, Tuleuova N, Jones CN, Ramanculov E, Zern MA, Revzin A. Directing hepatic differentiation of embryonic stem cells with protein microarray-based co-cultures. Integr Biol (Camb) 2009;1(7):460-468. ##Kim M, Lee JY, Shah SS, Tae G, Revzin A. On-cue detachment of hydrogels and cells from optically transparent electrodes. Chem Commun (Camb) 2009;(39):5865-5867. ##Kulkarni M, Greiser U, O’Brien T, Pandit A. Liposomal gene delivery mediated by tissue-engineered scaffolds. Trends Biotechnol 2010;28(1):28-36.##

Development of Polymer-coated Glass Slides as Optical Oligonucleotide Microarrays 2 2 Background: The microarray technology is in needed of cost-effective, low background noise and stable substrates for successful hybridization and analysis. Methods: In this research, we developed a three-dimentional stable and mechanically reliable microarray substrates by coating of two polymeric layers on standard microscope glass slides. For fabrication of these substrates, a thin film of oxidized agarose was prepared on the Poly-L-Lysine (PLL) coated glass slides. Unmodified oligonucleotide probes were spotted and immobilized on these double layered thin films by adsorption on the porous structure of the agarose film. Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; on the other side, they bound to amino groups of adsorbed tail of biomolecules. These linkages were fixed by UV irradiation at 254 nm using a CL-1000 UV. These prepared substrates were compared to only agarose-coated and PLL-coated slides. Results: Atomic Force Microscope (AFM) results demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. The nano-indentation tests demonstrated the prepared double coating was more reliable and flexible for mechanical robotic spotting. In addition, the repeated indentation on different substrates showed uniformity of coatings. The stability of novel coating was sufficient for hybridization process. The signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion: Finally, the agarose-PLL microarrays had the highest signal (2920) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes. 241 250 Atefeh Pourjahed Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Iran Mohammad Rabiee Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Iran Mohammadreza Tahriri Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology Iran AgaroseMicroarray analysisPLLSignal-to-noise ratio 131.pdf Dover JE, Hwang GM, Mullen EH, Prorok BC, Suh SJ. Recent advances in peptide-probe based biosensors for detection of infectious agents. J Microbiol Methods 2009;78(1):10-19.##Bagni G, Osella D, Sturchio E, Mascini M. Deoxyribonucleic acid (DNA) biosensors for environmental risk assessment and drug studies. Anal Chim Acta 2006;573-574:81-89.##Van Dorst B, Mehta J, Bekaert K, Rouah-Martin E, De Coen W, Dubruel P, et al. Recent advances in recognition elements of food and environmental biosensors: a review. Biosens Bioelectron 2010;26(4):1178- 1194.##Hook AL, Anderson DG, Langer R, Williams P, Davies MC, Alexander MR. High throughput methods applied in biomaterial development and discovery. Biomaterials 2010;31(2):187-198.##Zhou X, Wu L, Zhou J. Fabrication of DNA microarrays on nanoengineered polymeric ultrathin film prepared by self-assembly of polyelectrolyte multilayers. Langmuir 2004;20(20):8877-8885.##Beier M, Hoheisel JD. Versatile derivatisation of solid support media for covalent bonding on DNA-microchips. Nucleic Acids Res 1999;27(9):1970-1977.##Joos B, Kuster H, Cone R. Covalent attachment of hybridizable oligo¬nucleotides to glass supports. Anal Biochem 1997;247(1):96-101.##Dolan PL, Wu Y, Ista LK, Metzenberg RL, Nelson MA, Lopez GP. Robust and efficient synthetic method for forming DNA microarrays. Nucleic Acids Res 2001;29(21):E107-7.##Lindroos K, Liljedahl U, Raitio M, Syvänen AC. Minisequencing on oligonucleotide microarrays: comparison of immobilization chemistries. Nucleic Acids Res 2001;29(13):E69-9.##Steel AB, Levicky RL, Herne TM, Tarlov MJ. Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly. Biophys J 2000;79(2):975-981.##Sinitsyna ES, Walter JG, Vlakh EG, Stahl F, Kasper C, Tennikova TB. Macroporous methacrylate-based monoliths as platforms for DNA microarrays. Talanta 2012;93:139-146.##Ricciardi S, Castagna R, Severino SM, Ferrante I, Frascella F, Celasco E, et al. Surface functionalization by poly-acrylic acid plasma-polymerized films for microarray DNA diagnostics. Surf Coat Tech 2012;207:389-399.##Li YK, Chen YC, Jiang KJ, Wu JC, Chen-Yang YW. Three-dimensional arrayed amino aerogel biochips for molecular recognition of antigens. Biomaterials 2011;32(30):7347-7354.##Yong-Song Ch, Jung-Yeol L, Kyung-Sup L. Development of a DNA chip microarray using hydrophobic interaction between template and particle by using the random fluidic self-assembly method. Curr Appl Phys 2007;7(4):370-374.##Li YK, Yang DK, Chen YC, Su HJ, Wu JC, Chen-Yang YW. A novel three-dimensional aerogel biochip for molecular recognition of nucleotide acids. Acta Biomater 2010;6(4):1462-1470.##Kim SY, Yu J, Son SJ, Min J. Signal enhancement in a protein chip array using a 3D nanosurface. Ultramicroscopy 2010;110(6):659-665.##Buenger D, Topuz F, Groll PJ. Hydrogels in sensing applications. Prog Polym Sci 2012;37(12):1678-1719.##Mercey E, Obeïd P, Glaise D, Calvo-Muñoz ML, Guguen-Guillouzo C, Fouqué B. The application of 3D micropatterning of agarose substrate for cell culture and in situ comet assays. Biomaterials 2010;31(12):3156-3165.##Dufva M, Petronis S, Jensen LB, Krag C, Christensen CB. Characterization of an inexpensive, nontoxic, and highly sensitive microarray substrate. BioTech 2004;37(2):286-296.##Dufva M, Petersen J, Stoltenborg M, Birgens H, Christensen CB. Detection of mutations using microarrays of poly(C)10–poly(T)10 modified DNA probes immobilized on agarose films. Anal Biochem 2006;352(2):188-197.##Wei Y, Ning G, Hai-Qian Zh, Jian-Guao W, Yi-Hong W, Wesche K. Microarray preparation based on oxidation of agarose-gel and subsequent enzyme immunoassay. Sensor Actuat B-Chem 2004;98(1):83-91.##Koch CA, Li PC, Utkhede RS. Evaluation of thin films of agarose on glass for hybridization of DNA to identify plant pathogens with microarray technology. 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Mutation Analysis of SLC20A2 and SPP2 as Candidate Genes for Familial Idiopathic Basal Ganglia Calcification 2 2 Background: Familial Idiopathic Basal Ganglia Calcification (IBGC) is a rare neurodegenerative disorder which is usually transmitted as an autosomal dominant trait. IBGC is genetically heterogeneous and SLC20A2, on chromosome 8p21.1–8q11.23, is the first gene found in IBGC-affected patients with varied ancestry. On the other hand, several candidate genes for IBGC on chromosome 2q37, including the SPP2 gene, may play a role in inhibiting calcification. Methods: Totally, 22 members of a three generational Iranian family affected by IBGC, with an autosomal dominant pattern of inheritance were included in this study. DNA was extracted from the whole blood using standard salting out method. To find a mutation responsible for IBGC, we sequenced the coding region of SLC20A2 as well as promoter and coding region of SPP2 in the index subject of IBGC-affected family. Results: Pathogenic mutation was found neither in SLC20A2 nor in SPP2. Conclusion: Our results strengthen genetic heterogeneity of this condition. Additional mutation studies are necessary to find a gene or genes responsible for IBGC in this affected family. 251 256 Fereshteh Ashtari Neurology Department, Isfahan Neuroscience Research Centre, Isfahan University of Medical Sciences Neurology Department, Isfahan Neuroscience Research Centre, Isfahan University of Medical Sciences Iran Kioomars Saliminejad Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Ali Ahani Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Koorosh Kamali Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Zhamak Pahlevanzadeh Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, 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 Familial Idiopathic Basal Ganglia CalcificationMutationSLC20A2 proteinSPP2 protein 132.pdf Manyam BV. What is and what is not "Fahr’s disease". Parkinsonism Relat Disord 2005;11(2):73–80.##Oliveira JR, Spiteri E, Sobrido MJ, Hopfer S, Klepper J, Voit T, et al. Genetic heterogeneity in familial idiopathic basal ganglia calcification (“Fahr’s disease”). Neurology 2004;63(11):2165-2167.##Dai X, Gao Y, Xu Z, Cui X, Liu J, Li Y, et al. Identification of a novel genetic locus on chromosome 8p21.1-q11.23 for idiopathic basal ganglia calcification. Am J Med Genet B Neuropsychiatr Genet 2010;153B(7):1305-1310.##Lemos RR, Oliveira DF, Zatz M, Oliveira JR. Population and computational analysis of the MGEA6 P521A variation as a risk factor for familial idiopathic basal ganglia calcification (Fahr’s disease). J Mol Neurosci 2011(3);43:333-336.##Wang C, Li Y, Shi L, Ren J, Patti M, Wang T, et al. Mutations in SLC20A2 link familial idiopathic basal ganglia calcification with phosphate homeostasis. Nat Genet 2012;44(3):254-256.##Geschwind DH, Loginov M, Stern JM. Identification of a locus on chromosome 14q for idiopathic basal ganglia calcification (Fahr’s disease). Am J Hum Genet 1999;65(3):764-772.##Volpato CB, De Grandi A, Buffone E, Pichler I, Gebert U, Schifferle G, et al. Exclusion of linkage to chromosome 14q in a large South Tyrolean family with idiopathic basal ganglia calcification (IBGC). Am J Med Genet B Neuropsychiatr Genet 2008;147B(7):1319-1322.##Volpato CB, De Grandi A, Buffone E, Facheris M, Gebert U, Schifferle G, et al. 2q37 as a susceptibility locus for idiopathic basal ganglia calcification (IBGC) in a large South Tyrolean family. J Mol Neurosci 2009;39(3):346-353.##Price PA, Nguyen TMT, Williamson MK. Biochemical characterization of the serum fetuin-mineral complex. J Biol Chem 2003;278(24):22153-22160.##Hsu SC, Sears RL, Lemos RR, Quintáns B, Huang A, Spiteri E, et al. Mutations in SLC20A2 are a major cause of familial idiopathic basal ganglia calcification. Neurogenetics 2013;14(1):11-22.##Zhang Y, Guo X, Wu A. Association between a novel mutation in SLC20A2 and familial idiopathic basal ganglia calcification. PLoS One 2013;8(2):e57060.##Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16(3):1215.##Oliveira JR, Sobrido MJ, Spiteri E, Hopfer S, Meroni G, Petek E, et al. Analysis of candidate genes at the IBGC1 locus associated with idiopathic basal ganglia calcification (“Fahr’s disease”). J Mol Neurosci 2007;33(2):151-154.##Brodaty H, Mitchell P, Luscombe G, Kwok JJ, Badenhop RF, McKenzie R, et al. Familial idiopathic basal ganglia calcification (Fahr’s disease) without neurological, cognitive and psychiatric symptoms is not linked to the IBGC1 locus on chromosome 14q. Hum Genet 2002;110(1):8-14.##Kostić VS, Lukić-Ječmenica M, Novaković I, Dobričić V, Brajković L, Krajinović M, et al. Exclusion of linkage to chromosomes 14q, 2q37 and 8p21.1-q11.23 in a Serbian family with idiopathic basal ganglia calcification. J Neurol 2011;258(9):1637-1642.##Bennett CS, Khorram Khorshid HR, Kitchen JA, Arteta D, Dalgleish R. Characterization of the human secreted phosphoprotein 24 gene (SPP2) and comparison of the protein sequence in nine species. Matrix Biol 2004;22(8):641-651.##Saliminejad K, Ashtari F, Kamali K, Edalatkhah H, Khorram Khorshid HR. Analysis of the CTAGE5 P521A variation with the risk of familial idiopathic basal ganglia calcification in an Iranian population. J Mol Neurosci 2013;49(3):614-617.##

Retraction: Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos 2 2 <p>The author has requested that the original article entitled "Genotyping Analysis of Circulating Fetal Cells Reveals High Frequency of Vanishing Twin Following Transfer of Multiple Embryos" that was published in the April-June 2013 issue of <em>Avicenna Journal of Medical Biotechnology (AJMB)</em> be withdrawn because the results were published without the co-workers being aware of this publication. Therefore this paper is retracted, considering the fact that it is contrary to the scientific rules and unrespectful of the contribution of the other authors.</p> 257 257 Hussein Mouawia RetractRetraction 133.pdf ####