An Overview of 3D Bioprinting as a Novel Strategy in the Field of Tissue Engineering 2 2 Tissue engineering and regenerative medicine have typically matured from benchtop ideas to commercially applicable products in the clinic 1. However, despite of typical advances in tissue engineering field, some limitations such as no reproducibility, no control of structure geometry including pore size and pore distribution and no integrity of cell distribution and migration in the construct have impelled the scientists into bioprinting technology. The most advantage of 3D bioprinting sounds to be precise fabrication of 3D deposition with controlled geometric structure and cells distribution 2. Over the past decade, lots of researches in bioprinting of different tissues and organs has been carried out using different bioprinting modalities particularly inkjet based printing for skin tissue engineering and extrusion based printing for 3D depositions like bone, cartilage, heart, liver and heart valve. The key factor in extrusion-based bioprinting is bioink preparation, cell encapsulation in the bioink and bioprinting procedure. Indeed, preparation of bioink with appropriate gelation rate, suitable mechanical strength and elasticity which preserve cell viability and proliferation is the most challenge of bioprinting technology. So far, different strategies such as dual bioink cross-linkers, multi-step polymerization and using of core-shell nozzle have been reported to improve viability, quality and functionality of the printed product 3. However, some issues including creation of constructs supporting in vivo vascularization, scaling up tissue constructs and in situ bioprinting have been remained to resolve. A few bioprinting products have been commercialized especially in orthopedic and skin tissue engineering fields and given the fast development of this industry over the past years; it supposed that the bioprinting products will eventually take a big proportion of the medical market to help patients suffering from a wide range of diseases in the future. 201 201 Somaieh Kazemnejad Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Editorial 285.pdf Mobini S, Khanmohammadi M, Heidari-Vala H, Samadikuchaksaraei A, Moshiri A, Kazemnejad S. Tissue Engineering and Regenerative Medicine in Iran: Current State of Research and Future Outlook. Mol Biotechnol 2015;57(7):589-605. ##Derakhshanfar S, Mbeleck R, Xu K, Zhang X, Zhong W, Xing M. 3D bioprinting for biomedical devices and tissue engineering: A review of recent trends and advances. Bioact Mater 2018;3(2):144-156.##Zhang YS, Arneri A, Bersini S, Shin SR, Zhu K, Goli-Malekabadi Z, et al. Bioprinting 3D microfibrous scaffolds for engineering endothelialized myocardium and heart-on-a-chip. Biomaterials 2016;110:45-59. ##

Inclusion Body Expression and Refolding of Recombinant Bone Morphogenetic Protein-2 2 2 Background: Bone Morphogenetic Protein-2 (BMP-2) is a cysteine rich growth factor expressed in homodimeric form and has a pivotal role in osteochondral development and fracture healing. Recent studies have benefited more from recombinant BMP-2 in osteochondral tissue engineering. Cost-effective and easy production at large scale makes Escherichia coli (E. coli) the first choice for recombinant protein expression programs. However, inclusion body aggregation and refolding process limits production and purification of recombinant BMP-2 in bacterial systems. Methods: BMP-2 encoded gene was optimized for expression in bacterial expression system and synthesized with proper restriction sites. The optimized sequence was then cloned in a pET28a expression vector and expressed in OrigamiTM E. coli strain. The aggregated and monomeric BMP-2 was refolded and purified comparing two oxidoreductase systems and refolding methods as well as different purification techniques. The biological activity of recombinant protein was investigated by increasing alkaline phosphatase activity (ALK) of ATDC-5 cell line. Results: No difference was observed between oxidoreductase systems in improving the efficiency of protein refolding. However, comparisons between two refolding methods showed that pooling monomeric BMP-2 that was refolded under mild condition with equal volume of it refolded under severe oxidoreductase condition resulted in production of more active dimeric protein. Conclusion: A new method for production of biologically active dimeric form of BMP-2 in E. coli expression system was established in this study. 202 207 Davood Nasrabadi Department of Medical Biotechnology, Semnan University of Medical SciencesDepartment of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECRStudent Research Committee, Semnan University of Medical Sciences Department of Medical Biotechnology, Semnan University of Medical SciencesDepartment of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECRStudent Research Committee, Semnan University of Medical Sciences IranIranIran Siamak Rezaeiani Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Iran Ali Sayadmanesh Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Department of Molecular Systems Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Iran Mohammadreza Baghaban Eslaminejad Department of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Department of Stem Cell and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR Iran Aliakbar Shabani Department of Medical Biotechnology, Semnan University of Medical Sciences Department of Medical Biotechnology, Semnan University of Medical Sciences Iran Bone morphogenetic protein -2CloningEscherichia coliInclusion bodiesProtein refolding 326.pdf Wang RN, Green J, Wang Z, Deng Y, Qiao M, Peabody M, et al. Bone morphogenetic protein (BMP) signaling in development and human diseases. Genes Dis 2014;1(1):87-105.##Wu M, Chen G, Li YP. TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease. Bone Res 2016;4:16009.##Carreira AC, Zambuzzi WF, Rossi MC, Astorino Filho R, Sogayar MC, Granjeiro JM. Bone morphogenetic proteins: promising molecules for bone healing, bioengineering, and regenerative medicine. Vitam Horm 2015;99:293-322.##Kuo MM, Nguyen PH, Jeon YH, Kim S, Yoon SM, Choe S. MB109 as bioactive human bone morphogenetic protein-9 refolded and purified from E. coli inclusion bodies. Microb Cell Fact 2014;13(1):29.##Vallejo LF, Brokelmann M, Marten S, Trappe S, Cabrera-Crespo J, Hoffmann A, et al. Renaturation and purification of bone morphogenetic protein-2 produced as inclusion bodies in high-cell-density cultures of recombinant Escherichia coli. J Biotechnol 2002;94(2):185-194.##Vallejo LF, Rinas U. Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-beta family. FEBS J 2013;280(1):83-92.##Chen J, Liu Y, Li X, Wang Y, Ding H, Ma G, et al. Cooperative effects of urea and L-arginine on protein refolding. Protein Expr Purif 2009;66(1):82-90.##Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog 2004;20(5):1301-1308.##Samuel D, Kumar TK, Ganesh G, Jayaraman G, Yang PW, Chang MM, et al. Proline inhibits aggregation during protein refolding. Protein Sci 2000;9(2):344-352.##Han B, Hall FL, Nimni ME. Refolding of a recombinant collagen-targeted TGF-beta2 fusion protein expressed in Escherichia coli. Protein Expr Purif 1997;11(2):169-178.##Kim YV, Gasparian ME, Bocharov EV, Chertkova RV, Tkach EN, Dolgikh DA, et al. New strategy for high-level expression and purification of biologically active monomeric TGF-beta1/C77S in Escherichia coli. Mol Biotechnol 2015;57(2):160-171.##Vallejo LF, Rinas U. Optimized procedure for renaturation of recombinant human bone morphogenetic protein-2 at high protein concentration. Biotechnol Bioeng 2004;85(6):601-609.##Sharapova NE, Kotnova AP, Galushkina ZM, Lavrova NV, Poletaeva NN, Tukhvatulin AE, et al. [Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo]. Mol Biol (Mosk) 2010;44(6):1036-1044. Russian.##Zhang Y, Ma Y, Yang M, Min S, Yao J, Zhu L. Expression, purification, and refolding of a recombinant human bone morphogenetic protein 2 in vitro. Protein Expr Purif 2011;75(2):155-160.##Nakamura K, Shirai T, Morishita S, Uchida S, Saeki-Miura K, Makishima F. p38 mitogen-activated protein kinase functionally contributes to chondrogenesis induced by growth/differentiation factor-5 in ATDC5 cells. Exp Cell Res 1999;250(2):351-363.##Long S, Truong L, Bennett K, Phillips A, Wong-Staal F, Ma H. Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli. Protein Expr Purif 2006;46(2):374-378.##

Improvement Efficacy of Influenza Nanovaccine in Combination with Hemokinin-1 Molecular Adjuvant 2 2 Background: H9N2 avian influenza viruses have the potential to become the next human pandemic threat and next generation vaccine technologies are needed. Current studies introduce nanoparticles as a proper vaccine delivery vehicle for induction of protective immunity. In this study, the efficacy of chitosan nanoparticle-based H9N2 influenza vaccine with and without hemokinin-1 (HK-1) as a molecular adjuvant to induce protective immunity against the virus was examined. Methods: The H9N2 antigen was prepared in MDCK cells and inactivated with formalin. The inactivated antigen alone and in combination with HK-1 was encapsulated into chitosan nanoparticles. Groups of BALB/c mice received chitosan nanoparticle-based H9N2 antigen alone or in combination with HK-1 in a prime/boost platform via eye drop method. To evaluate the efficacy of the adjuvanted-nanovaccine candidate, systemic antibody responses were compared among the groups of animals. Results: Serological analysis indicated that mice receiving the HK-1/H9N2 nanoparticles formulation induced higher antibody titers that were sustained until the end of experiment. However, in the immunized mice, influenza specific antibody titers were comparable to that in the animals which were immunized either with inactivated antigen alone or the H9N2 nanoparticles without HK-1 adjuvant. Conclusion: The data demonstrate the synergy between HK-1 as an adjuvant and chitosan nanoparticles as a delivery antigen/adjuvant carrier in the improvement of influenza immune responses. 208 213 Atefeh Dehghan Department of Microbiology, Karaj Branch, Islamic Azad University Department of Microbiology, Karaj Branch, Islamic Azad University Iran Shahla Shahsavandi Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization Iran Leila Jabalameli Department of Microbiology, Karaj Branch, Islamic Azad University Department of Microbiology, Karaj Branch, Islamic Azad University Iran ChitosanImmunizationInfluenza vaccinesInfluenza virusNanoparticles 327.pdf Baigent SJ, McCauley JW. Influenza type A in humans, mammals and birds: determinants of virus virulence, host-range and interspecies transmission. Bioessays 2003;25(7):657-671.##Palese P, García-Sastre A. Influenza vaccines: present and future. J Clin Invest 2002;110(1):9-13.##Cox RJ, Brokstad KA, Ogra P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol 2004;59(1):1-15.##Bi Y, Lu L, Li J, Yin Y, Zhang Y, Gao H, et al. Novel genetic reassortants in H9N2 influenza A viruses and their diverse pathogenicity to mice. Virol J 2011;8:505.##Lee YT, Kim KH, Ko EJ, Lee YN, Kim MC, Kwon YM, et al. New vaccines against influenza virus. Clin Exp Vaccine Res 2014;3(1):12-28.##Ben-Yedidia T, Arnon R. Epitope-based vaccine against influenza. Expert Rev Vaccines 2007;6(6):39-48.##Hashem AM. Prospects of HA-based universal influenza vaccine. Biomed Res Int 2015;2015:414637.##Qiu M, Fang F, Chen Y, Wang H, Chen Q, Chang H, et al. Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin-or neuraminidase-expressing DNA in BALB/c mice. Biochem Biophy Res Commun 2006;343(4):24-31.##Luo J, Zheng D, Zhang W, Fang F, Wang H, Sun Y, et al. Induction of cross-protection against influenza A virus by DNA prime-intranasal protein boost strategy based on nucleoprotein. Virol J 2012;9:286.##Fox CB, Kramer RM, Barnes VL, Dowling QM, Vedvick TS. Working together: interactions between vaccine antigens and adjutants. Ther Adv Vaccines 2013;1(1):7-20.##Even-Or O, Samira S, Ellis R, Kedar E, Barenholz Y. Adjuvanted influenza vaccines. Expert Rev Vaccines 2013;12(9):1095-1108.##Lambrecht BN, Kool M, Willart MA, Hammad H. Mechanism of action of clinically approved adjuvants. Curr Opin Immunol 2009;21(1):23-29.##Wang W, Singh M. Selection of adjuvants for enhanced vaccine potency. World J Vaccines 2011;1(2):33-78.##Liniger M, Summerfield A, Ruggli N. MDA5 can be exploited as efficacious genetic adjuvant for DNA vaccination against lethal H5N1 influenza virus infection in chickens. PLoS One 2012;7(12):e49952.##Crowe JE Jr, Sannella EC, Pfeiffer S, Zorn GL, Azimzadeh A, Newman R, et al. CD154 regulates primate humoral immunity to influenza. Am J Transplant 2003;3(6):680-688.##Sadghi K, Shahsavandi Sh, Ebrahimi MM, Mahravani H, Fazel, H. Hemokinin-1 molecular adjuvant: an approach to enhance the efficacy of influenza vaccine. Arak Med Uni J 2014;17(11):62-69.##Soleimani S, Shahsavandi S, Madadgar O. Improvement influenza HA2 DNA vaccine cellular and humoral immune responses with Mx bio adjuvant. Biologicals 2017;46:6-10.##Rose MA, Zielen S, Baumann U. Mucosal immunity and nasal influenza vaccination. Expert Rev Vaccines 2012;11(5):595-607.##Tumpey TM, Renshaw M, Clements JD, Katz JM. Mucosal delivery of inactivated influenza vaccine induces B-cell-dependent heterosubtypic cross-protection against lethal influenza A H5N1 virus infection. J Virol 2001;75(11):5141-5150.##Kanekiyo M, Wei CJ, Yassine HM, Mc Tamney PM, Boyington JC, Whittle JR, et al. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013;499(7456):102-106.##Ghendon Y, Markushin S, Krivtsov G, Akopova I. Chitosan as an adjuvant for parenterally administered inactivated influenza vaccines. Arch Virol 2008;153(5):831-837.##Ghendon Y, Markushin S, Vasiliev Y, Akopova I, Koptiaeva I, Krivtsov G, et al. Evaluation of properties of chitosan as an adjuvant for inactivated influenza vaccines administered parenterally. J Med Virol 2009;81(3):494-506.##Shahsavandi S, Ebrahimi MM, Sadghi K, Mahravani H. Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses. Virol Sin 2015;30(3):200-207.##Avian influenza. In: Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, fifth ed. Office International des Epizooties, Paris, France; 2008; Chapter 2.7.12.##Lu X, Renshaw M, Tumpey TM, Kelly GD, Primmer JH, Katz JM. Immunity to influenza A H9N2 viruses induced by infection and vaccination. J Virol 2001;75(10):4896-4901.##Sawaengsak C, Mori Y, Yamanishi K, Mitrevej A, Sinchaipanid N. Chitosan nanoparticle encapsulated hemagglutinin-split influenza virus mucosal vaccine. AAPS PharmSciTech 2014;15(2):317-325.##Khalili I, Ghadimipour R, SadighEteghad S, FathiNajafi M, Ebrahimi MM, Godsian N, et al. Evaluation of immune response against inactivated avian influenza (H9N2) vaccine, by using chitosan nanoparticles. Jundishapur J Microbiol 2015;8(12):e27035.##Amidi M, Romeijn SG, Verhoef JC, Junginger HE, Bungener L, Huckriede A, et al. N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza subunit antigen for intranasal vaccination: Biological properties and immunogenicity in a mouse model. Vaccine 2007;25(1):144-153.##Liu Q, Zheng X, Zhang C, Shao X, Zhang X, Zhang Q, et al. Conjugating influenza a (H1N1) antigen to n-trimethylaminoethylmethacrylate chitosan nanoparticles improves the immunogenicity of the antigen after nasal administration. J Med Virol 2015;87(11):1807-1815.##Tao W, Gill HS. M2e-immobilized gold nanoparticles as influenza A vaccine: Role of soluble M2e and longevity of protection. Vaccine 2015;33(20):2307-2315.##Waithman J, Mintern, JD. Dendritic cells and influenza A virus infection. Virulence 2012;3(7):603-608.##Zhang Y, Lu L, Furlonger C, Wu GE, Paige CJ. Hemokinin is a hematopoietic-specific tachykinin that regulates B lymphopoiesis. Nat Immunol 2000;1(5):392-397.##Wang W, Li Q, Zhang J, Wu H, Yin Y, Ge Q, et al. Hemokinin-1 activates the MAPK pathway and enhances B cell proliferation and antibody production. J Immunol 2010;184(7):3590-3597.##Borges O, Borchard G, de Sousa A, Junginger HE, Cordeiro-da-Silva A. Induction of lymphocytes activated marker CD69 following exposure to chitosan and alginate biopolymers. Int J Pharm 2007;337(1-2):254-264.##Schoenberger SP. CD69 guides CD4+T cells to the seat of memory. Proc Natl Acad Sci USA 2012;109(22):8358-8359.##Bernkop-Schnürch A, Dünnhaup S. Chitosan-based drug delivery systems. Eur J Pharm Biopharm 2012;81(3):463-469.##AbdElgadir A, Uddin MS, Ferdosh S, Adam A, Chowdhury AJK, Sarker MZI. Impact of chitosan composites and chitosan nanoparticle composites on various drug delivery systems: A review. J Food Drug Anal 2015;23(4):619-629.##

Effects of Dietary Polyunsaturated Fatty Acids on DNA Methylation and the Expression of DNMT3b and PPARα Genes in Rats 2 2 Background: Previous studies have suggested a protective role for Polyunsaturated Fatty Acids (PUFA) against cancer, cardiovascular, and other diseases. To provide new insights into the in vivo effects of PUFA on gene expression, the effects of dietary PUFA on DNMT3b and PPARα gene expression and global DNA methylation were investigated in selected rat tissues.  Methods: Thirty sprague-dawley rats were allotted into 3 dietary groups of ten animals each, received experimental diets containing PUFAs every day by gavages for 12 weeks as follows: control group fed a normal diet and water; n-3 PUFAs group received 300 mg/kg/day n-3 PUFAs supplementation; mixed-PUFAs group received 300 mg/kg/day of a mixture of n-3, -6, -9 PUFAs supplementations. The expressions of DNMT3b and PPARα genes were quantitated using real-time RT-PCR. The genome-wide 5-methylcytosine contents in rat tissues were determined by ELISA method.  Results: The average expression of the DNMT3b mRNA was 50% lower in the colon and liver of rats fed the n-3- or mixed-PUFAs supplemented diet than control group (p=0.00). However, PPARα expression was significantly upregulated both in the colon and liver of PUFAs-supplemented rats (p<0.001). No significant difference was observed in the blood, colon, and liver DNA methylation levels between PUFAs-supplemented and control animals. Conclusion: The results indicate that dietary PUFAs could modulate the expressions of PPARα and DNMT3b genes in various rat tissues. The findings of this study provide additional insights into the in vivo mechanism of PUFA-mediated regulation of gene expression and could provide an opportunity to develop personalized diets for related disease control. 214 219 Ehsan Maktoobian Baharanchi Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Iran Mostafa Moradi Sarabi Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Iran Fakhraddin Naghibalhossaini Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical SciencesAutoimmune Research Center, Faculty of Medicine, Shiraz University of Medical Sciences Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical SciencesAutoimmune Research Center, Faculty of Medicine, Shiraz University of Medical Sciences IranIran DNA methylationGene expressionDietary supplement regulationFatty acids omega-3 325.pdf Mennitti LV, Oliveira JL, Morais CA, Estadella D, Oyama LM, Oller do Nascimento CM, et al. Type of fatty acids in maternal diets during pregnancy and/or lactation and metabolic consequences of the offspring. J Nutr Biochem 2015;26(2):99-111.##Burdge GC, Lillycrop KA. Nutrition, epigenetics, and developmental plasticity: implications for understanding human disease. Annu Rev Nutr 2010;30:315-339.##Cockbain AJ, Toogood GJ, Hull MA. Omega-3 polyunsaturated fatty acids for the treatment and prevention of colorectal cancer. Gut 2012;61(1):135-149.##Saravanan P, Davidson NC, Schmidt EB, Calder PC. Cardiovascular effects of marine omega-3 fatty acids. Lancet 2010;376(9740):540-550.##Fetterman JW, Zdanowicz MM. Therapeutic potential of n-3 polyunsaturated fatty acids in disease. Am J Health Syst Pharm 2009;66(13):1169-1179.##Song M, Nishihara R, Wu K, Qian ZR, Kim SA, Sukawa Y, et al. Marine ω-3 polyunsaturated fatty acids and risk of colorectal cancer according to microsatellite instability. J Natl Cancer Inst 2015;107(4), pii: djv007.##West NJ, Clark SK, Phillips RK, Hutchinson JM, Leicester RJ, Belluzzi A, et al. Eicosapentaenoic acid reduces rectal polyp number and size in familial adenomatous polyposis. Gut 2010;59(7):918-925.##Mund RC, Pizato N, Bonatto S, Nunes EA, Vicenzi T, Tanhoffer R, et al. Decreased tumor growth in Walker 256 tumor-bearing rats chronically supplemented with fish oil involves COX-2 and PGE2 reduction associated with apoptosis and increased peroxidation. Prostaglandins Leukot Essent Fatty Acids 2007;76(2): 113-120.##Tsuzuki T, Igarashi M, Miyazawa T. Conjugated eicosapentaenoic acid (EPA) inhibits transplanted tumor growth via membrane lipid peroxidation in nude mice. J Nutr 2004;134(5):1162-1166.##Azrad M, Turgeon C, Demark-Wahnefried W. Current evidence linking polyunsaturated fatty acids with cancer risk and progression. Front Oncol 2013;3:224.##Xu HE, Lambert MH, Montana VG, Parks DJ, Blanchard SG, Brown PJ, et al. Molecular recognition of fatty acids by peroxisome proliferator activated receptors. Mol Cell 1999;3(3):397-403.##Sarraf P, Mueller E, Jones D, King FJ, DeAngelo DJ, Partridge JB, et al. Differentiation and reversal of malignant changes in colon cancer through PPAR gamma. Nat Med 1998;4(9):1046-1052.##Bishop-Bailey D, Hla T. Endothelial cell apoptosis induced by the peroxisome proliferator-activated receptor (PPAR) ligand 15-deoxy-D2, 14-prostaglandin J2. J Biol Chem 1999;274(24):17042-17048.##Larsson SC, Kumlin M, Ingelman-Sundberg M, Wolk A. Dietary long-chain n-3 fatty acids for the prevention of cancer: a review of potential mechanisms. Am J Clin Nutr 2004;79(6):935-945.##Pancione M, Sabatino L, Fucci A, Carafa V, Nebbioso A, Forte N, et al. Epigenetic silencing of peroxisome proliferator-activated receptor γ is a biomarker for colorectal cancer progression and adverse patients’ outcome. PLoS One 2010;5(12):e14229.##Capaccio D, Ciccodicola A, Sabatino L, Casamassimi A, Pancione M, Fucci A, et al. A novel germline mutation in Peroxisome Proliferator-Activated Receptor γ gene associated with large intestine polyp formation and dyslipidemia. Biochim Biophys Acta 2010;1802(6):572-581.##Pancione M, Forte N, Sabatino L, Tomaselli E, Parente D, Febbraro A, et al. Reduced beta-catenin and peroxisome proliferator-activated receptor-gamma expression levels are associated with colorectal cancer metastatic progression: correlation with tumor-associated macrophages, cyclooxygenase 2, and patient outcome. Hum Pathol 2009;40(5):714-725.##Bae JM, Kim JH, Cho NY, Kim TY, Kang GH. Prognostic implication of the CpG island methylator phenotype in colorectal cancers depends on tumour location. Br J Cancer 2013;109(4):1004-1012.##Linhart HG, Lin H, Yamada Y, Moran E, Steine EJ, Gokhale S, et al. Dnmt3b promotes tumorigenesis in vivo by gene-specific de novo methylation and transcriptional silencing. Genes Dev 2007;21(23):3110-3122.##Nosho K, Shima K, Irahara N, Kure S, Baba Y, Kirkner GJ, et al. DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin Cancer Res 2009;15(11):3663-3671.##Ibrahim AE, Arends MJ, Silva AL, Wyllie AH, Greger L, Ito Y, et al. Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut 2011;60(4):499-508.##Kulkarni A, Dangat K, Kale A, Sable P, Chavan-Gautam P, Joshi S. Effects of altered maternal folic acid, vitamin B12 and docosahexaenoic acid on placental global DNA methylation patterns in Wistar rats. PLoS One 2011;6(3):e17706.##Lee HS, Barraza-Villarreal A, Hernandez-Vargas H, Sly PD, Biessy C, Ramakrishnan U, et al. Modulation of DNA methylation states and infant immune system by dietary supplementation with ω-3 PUFA during pregnancy in an intervention study. Am J Clin Nutr 2013;98(2):480-487.##Amaral CL, Crisma AR, Masi LN, Martins AR, Hirabara SM, Curi R. DNA methylation changes induced by a high-fat diet and fish oil supplementation in the skeletal muscle of mice. J Nutrigenet Nutrigenomics 2014;7(4-6):314-326.##Jacobsen SC, Brøns C, Bork-Jensen J, Ribel-Madsen R, Yang B, Lara E, et al. Effects of short-term high-fat overfeeding on genome-wide DNA methylation in the skeletal muscle of healthy young men. Diabetologia 2012;55(12):3341-3349.##Hoile SP, Irvine NA, Kelsall CJ, Sibbons C, Feunteun A, Collister A, et al. Maternal fat intake in rats alters 20:4𝑛-6 and 22:6𝑛-3 status and the epigenetic regulation of Fads2 in offspring liver. J Nutr Biochem 2013;24(7):1213-1220.##Niculescu MD, Lupu DS, Craciunescu CN. Perinatal manipulation of 𝛼-linolenic acid intake induces epigenetic changes in maternal and offspring livers. FASEB J 2013;27(1):350-358.##Sarabi MM, Naghibalhossaini F. Association of DNA methyltransferases expression with global and gene-specific DNA methylation in colorectal cancer cells. Cell Biochem Funct 2015;33(7):427-433.##Pakdel A, Naghibalhossaini F, Makarram P, Jaberipour M, Hosseini A. Regulation of carcinoembryonic antigen release from colorectal cancer cells. Mol Biol Rep 2012;39(4):3695-3704.##Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25(4):402-408.##Kanai Y, Hirohashi S. Alterations of DNA methylation associated with abnormalities of DNA methyltransferases in human cancers during transition from a precancerous to a malignant state. Carcinogenesis 2007;28(12):2434-2442.##Dodge JE, Okano M, Dick F, Tsujimoto N, Chen T, Wang S, et al. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem 2005;280(18):17986-17991.##Brøns C, Jensen CB, Storgaard H, Hiscock NJ, White A, Appel JS, et al. Impact of short-term high-fat feeding on glucose and insulin metabolism in young healthy men. J Physiol 2009;587(Pt 10):2387-2397.##Bestor TH. The DNA methyltransferases of mammals. Hum Mol Genet 2000;9(16):2395-2402.##Fan C, Liu X, Shen W, Deckelbaum RJ, Qi K. The regulation of leptin, leptin receptor and pro-opiomelanocortin expression by 𝑁-3 PUFAs in diet-induced obese mice is not related to the methylation of their promoters. Nutr Metab (Lond) 2011;8(1):31.##Bordoni A, Di Nunzio M, Danesi F, Biagi PL. Polyunsaturated fatty acids: from diet to binding to ppars and other nuclear receptors. Genes Nutr 2006;1(2):95-106.##Poulsen LI, Siersbæk M, Mandrup S. PPARs fatty acid sensors controlling metabolism. Semin Cell Dev Biol 2012;23(6):631-639.##Kurokawa T, Shimomura Y, Bajotto G, Kotake K, Arikawa T, Ito N, et al. Peroxisome proliferator-activated receptor α (PPARα) mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue. World J Surg Onco 2011;9:167.##Peyrou M, Ramadori P, Bourgoin L, Foti M. PPARs in liver diseases and cancer: epigenetic regulation by microRNAs. PPAR Res 2012;2012:757803.##Tapia G, Valenzuela R, Espinosa A, Romanque P, Dossi C, Gonzalez-MañánD, et al. N-3 long-chain PUFA supplementation prevents high fat diet induced mouse liver steatosis and inflammation in relation to PPAR-α upregulation and NF-κB DNA binding abrogation. Mol Nutr Food Res 2014;58(6):1333-1341.##Mejía-Barradas CM, Del-Río-Navarro BE, Domínguez-López A, Campos-Rodríguez R, Martínez-GodínezMd, Rojas-Hernández S, et al. The consumption of n-3 polyunsaturated fatty acids differentially modulates gene expression of peroxisome proliferator-activated receptor alpha and gamma and hypoxia-inducible factor 1 alpha in subcutaneous adipose tissue of obese adolescents. Endocrine 2014;45(1):98-105.##

Effects of Anethum graveolens L. on In Vitro Matured Mouse Oocytes and Granulosa Cells 2 2 Background: According to previous studies, Anethum graveolens L. (dill) aqueous extracts decreased the fertility of female rats. Therefore, the present study aimed to examine the effects of this herb on cultured granulosa cells and immature oocytes.  Methods: The cells were obtained from 27-29 day immature superovulated mice. The oocytes were cultured in a petri dish consisting of 30 μl drops of MEM-α and granulosa cells in a 24-well plate consisting of DMEM/F12 and different concentrations of 0, 10, 50, 100, 500, 1000, 10000 μg/ml of dill seed aqueous extract (DSAE) in 37oC and 5% CO2. Then, the in vitro maturation of oocytes, including Germinal Vesicle (GV), Germinal Vesicle Breakdown (GVBD), and meiosis ІІ (MІІ) and oocyte bioviability were determined. Granulosa cells were then extracted and their bioviability, apoptosis, chromatin condensation, and lipid synthesis were examined. Estrogen and progesterone concentrations and Alkaline Phosphatase (ALP) activity were measured by RIA and spectrophotometry respectively from the supernatant of granulosa cell culture. Results: The results revealed that concentration of 10000 μg/ml of DSAE were toxic and damaged granulosa cell growth and oocytes maturation. Lower concentrations were the same in the control group and did not have any side effects on cell growth. The number of lipid droplets, estrogen and progesterone concentrations, and ALP activity increased with higher doses of DSAE compared to those in the control culture. Additionally, apoptosis and chromatin condensation increased in higher concentrations of DSAE-(500 and 1000 μg/ml) treated cells. This herb extract decreased the oocytes maturation in dose-dependent manner.  Conclusion: It was concluded that DSAE increased granulosa cells activity but damaged oocytes maturation, therefore it might be introduced as infertility agent. 220 226 Malihezaman Monsefi Department of Biology, Faculty of Sciences, Shiraz University Department of Biology, Faculty of Sciences, Shiraz University Iran Bahareh Khalifeh Department of Biology, Faculty of Sciences, Shiraz University Department of Biology, Faculty of Sciences, Shiraz University Iran Samaneh Nikeghbal Department of Biology, Faculty of Sciences, Shiraz University Department of Biology, Faculty of Sciences, Shiraz University Iran Anethum graveolens L.CultureGranulosa cellsOocytes 328.pdf Weiss RF. Weiss’s herbal medicine. 2nd ed. New York: Stuttgart Thieme; 2001. 362 p.##Jana S, Shekhawat GS. Phytochemical analysis and antibacterial screening of in vivo and in vitro extracts of Indian medicinal herb: Anethum graveolens. Res J Med Plant 2001;14(1):206-212.##Monsefi M, Ghasemi M, Bahaoddini. The effects of Anethum graveolens L. on female reproductive system of rats. Daru 2006;14(3):131-135.##Monsefi M, Ghasemi M, Bahaoddini A. The effect of Anethum graveolens L. on female reproductive system. Phytother Res 2006;20(10):865-868.##Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem 2014;96(9):1402-1413.##Singh S, Anand A, Srivastava P. Regulation and properties of glucose-6-phosphate dehydrogenase. Int J Plant Physiol Biochem 2012;4(1):1-19.##Odet F, Duan C, Willis WD, Goulding EH, Kung A, Eddy EM, et al. Expression of the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility. Biol Reprod 2008;79(1):26-34.##Malihezaman M, Mojaba M, Elham H, Farnaz G, Ramin M. Anti-fertility effects of different fractions of Anethum graveolens L. extracts on female rats. Afr J Tradit Complement Altern Med 2012;9(3):336-341.##Handa SS, Khanuja SPS, Longo G, Rakesh DD. Extraction technologies for medicinal and aromatic plants. 1 st ed. Italy/Trieste: United Nations Industrial Development Organization and the International Centre for Science and High Technology; 2008. 266 p.##Azwanida NN. A review on the extraction methods use in medicinal plants, principle, strength and limitation. Med Aromats Plants 2015;4(3):1-6.##Bucci M, Murphy CR. Hormonal control of enzyme activity during the plasma membrane transformation of uterine epithelial cells. Cell Biol Int 2001;25(9):859-871.##Niknafs B, Afshar F, Dezfulian AR. The effects of different luteal support hormones on endometrial alkaline phosphatase activity and endometrial thickness in superovulated mice. Int J Reprod BioMed 2010;8(1):18-23.##Gómez-Coronado DJ, Ibañez E, Rupérez FJ, Barbas C. Tocopherol measurement in edible products of vegetable origin. J Chromatogr 2004;1054(1-2): 227-233.##Jana S, Shekhawat GS. Plant growth regulators, adenine sulfate and carbohydrates regulate organogenesis and in vitro flowering of Anethum graveolens. Acta Physiol Plant 2011;33(2):305-311.##Hall JM, Couse JF, Korach KS. The Multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem 2001;276(40):36869-36872.##Ososki AL, Kennelly EJ. Phytoestrogens: a review of the present state of research. Phytother Res 2003;17(8):845-869.##Nynca A, Jablonska O, Slomczynska M, Petroff BK, Ciereszko RE. Effects of phytoestrogen daidzein and estradiol on steroidogenesis and expression of estrogen receptors in porcine luteinized granulosa cells from large follicles. J Physiol Pharmacol 2009;60(2):95-105.##Wober J, Weisswange I, Vollmer G. Stimulation of alkaline phosphatase activity in Ishikawa cells induced by various phytoestrogens and synthetic estrogens. J Steroid Biochem Mol Biol 2002;83(1-5):227-233.##Yoshida N, Mizuno K. Effect of physiological levels of phytoestrogens on mouse oocyte maturation in vitro. Cytotechnology 2012;64(3):241-247.##Hošková K, Křivohlávková L, Kadlečková L, Rajmon R, Drábek O, Jílek F. Biochanin a and daidzein influence meiotic maturation of pig oocytes in a different manner. Sci Agric Bohemica 2014;45(3):155-161. ##Kang JT, Kwon DK, Park SJ, Kim SJ, Moon JH, Koo OJ, et al. Quercetin improves the in vitro development of porcine oocytes by decreasing reactive oxygen species levels. J Vet Sci 2013;14(1):15-20.##Orlovschi D, Miclea I, Zahan M, Miclea V, Pernes AJ. Quercetin efficacy on in vitro maturation of porcine oocytes. Anim Sci Biotechnol 2014;47(1):113-115.##Liu Y, He XQ, Huang X, Ding L, Xu L, Shen YT, et al. Resveratrol protects mouse oocytes from methylglyoxal-induced oxidative damage. PLoS One 2013;8(10):e77960.##

HER-3 Knocking Down Induces G2/M Arrest in Gastric Cancer Cells 2 2 Background: The Human Epidermal growth factor Receptor-3 (HER-3) is a member of ErbB receptor family and has deficient kinase activity. HER-3 should heterodimerize with other members of ErbB receptor family, especially with HER-2, to transduce downstream signaling pathways. HER-3 co-expresses with other ErbB receptors in different cancers and overexpresses while the oncogenic signaling pathways such as Jak/Stat, MAPK, and PI3K/Akt are activated and promoted. Here, the expression level of HER-3 was evaluated in Iranian gastric adenocarcinoma's patients and the effects of HER-3 knocking down was investigated on cell cycle and cell viability of human gastric adenocarcinoma cell line of MKN45. Methods: In this study, 38 paraffin-embedded surgical adenocarcinoma specimens and their marginal non-tumor tissue samples were collected. Total RNAs were extracted and cDNAs were synthesized. Finally, the expression level of HER-3 was evaluated by real time PCR approach. Moreover, the human adenocarcinoma cell line of MKN45 was transfected with siRNA against HER-3 and the effects of its down-regulation were evaluated using MTT assay and cell-cycle analysis. Results: The data obtained from this study revealed HER-3 is significantly overexpressed in gastric tumors rather than non-tumor marginal tissues. Also, it was found that the expression level of HER-3 is elevated with tumor depth of invasion. Moreover, HER-3 knocking down promotes cell accumulation in G2/M phase of cell cycle and decreases cell viability in MKN45 cells which suggests a potential role for HER-3 in gastric adenocarcinoma tumorigenesis. Conclusion: Taken together, these results emphasize the importance of HER-3 receptor in diagnosis and prognosis of gastric adenocarcinoma. 227 232 Ehsan Mokhtari Department of Biology, Faculty of Biological Sciences, Islamic Azad University, East Tehran Branch Department of Biology, Faculty of Biological Sciences, Islamic Azad University, East Tehran Branch Iran Hesamodin Mokhtari International Campus, Shahid Sadoughi University of Medical Sciences International Campus, Shahid Sadoughi University of Medical Sciences Iran Elham Moslemi Department of Biology, Faculty of Biological Sciences, Islamic Azad University, East Tehran Branch Department of Biology, Faculty of Biological Sciences, Islamic Azad University, East Tehran Branch Iran Epidermal growth factorGastric adenocarcinomaHER-3Iran 329.pdf Center MM, Jemal A, Ward E. International trends in colorectal cancer incidence rates. Cancer Epidemiol Biomarkers Prev 2009;18(6):1688-1694.##Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010;127(12):2893-2917.##Hu B, El Hajj N, Sittler S, Lammert N, Barnes R, Meloni-Ehrig A. Gastric cancer: classification, histology and application of molecular pathology. J Gastrointest Oncol 2012;3(3):251-261.##Xia W, Lau YK, Zhang HZ, Xiao FY, Johnston DA, Liu AR, et al. Combination of EGFR, HER-2/neu, and HER-3 is a stronger predictor for the outcome of oral squamous cell carcinoma than any individual family members. Clin Cancer Res 1999;5(12):4164-4174.##Hubbard SR, Till JH. Protein tyrosine kinase structure and function. Annu Rev Biochem 2000;69:373-398.##Klapper LN, Kirschbaum MH, Seta M, Yarden Y. Biochemical and clinical implications of the ErbB/HER signaling network of growth factors. Adv Cancer Res 1999;77:25-79.##Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene 2000;19(56):6550-6565.##Atalay G, Cardoso F, Awada A, Piccart MJ. Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer. Ann Oncol 2003;14(9):1346-1363.##Lemmon MA, Schlessinger J. Regulation of signal transduction and signal diversity by receptor oligomerization. Trends Biochem Sci 1994;19(11):459-463.##Holbro T, Civenni G, Hynes NE. The ErbB receptors and their role in cancer progression. Expe Cell Res 2003;284(1):99-110.##Amin DN, Sergina N, Ahuja D, McMahon M, Blair JA, Wang D, et al. Resiliency and vulnerability in the HER2-HER3 tumorigenic driver. Sci Transl Med 2010;2(16):16ra7.##Jaiswal BS, Kljavin NM, Stawiski EW, Chan E, Parikh C, Durinck S, et al. Oncogenic ERBB3 mutations in human cancers. Cancer Cell 2013;23(5):603-617.##Sithanandam G, Anderson LM. The ERBB3 receptor in cancer and cancer gene therapy. Cancer Gene Ther 2008;15(7):413-448.##Ma J, Lyu H, Huang J, Liu B. Targeting of erbB3 receptor to overcome resistance in cancer treatment. Mol Cancer 2014;13:105.##Citri A, Skaria KB, Yarden Y. The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp Cell Res 2003;284(1):54-65.##McDonagh CF, Huhalov A, Harms BD, Adams S, Paragas V, Oyama S, et al. Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3. Mol Cancer Ther 2012;11(3):582-593.##Lee Y, Ma J, Lyu H, Huang J, Kim A, Liu B. Role of erbB3 receptors in cancer therapeutic resistance. Acta Biochim Biophys Sin (Shanghal) 2014;46(3):190-198.##Carcas LP. Gastric cancer review. J Carcinog 2014;13:14.##Cervantes A, Roda D, Tarazona N, Roselló S, Pérez-Fidalgo JA. Current questions for the treatment of advanced gastric cancer. Cancer Treat Rev 2013;39(1):60-67.##Stern DF. ERBB3/HER3 and ERBB2/HER2 duet in mammary development and breast cancer. J Mammary Gland Biol Neoplasia 2008;13(2):215-223.##Hsieh AC, Moasser MM. Targeting HER proteins in cancer therapy and the role of the non-target HER3. Br J Cancer 2007;97(4):453-457.##Koutras AK, Fountzilas G, Kalogeras KT, Starakis I, Iconomou G, Kalofonos HP. The upgraded role of HER3 and HER4 receptors in breast cancer. Crit Rev Oncol Hematol 2010;74(2):73-78.##Green AR, Barros FF, Abdel-Fatah TM, Moseley P, Nolan CC, Durham AC, et al. HER2/HER3 heterodimers and p21 expression are capable of predicting adjuvant trastuzumab response in HER2+ breast cancer. Breast Cancer Res Treat 2014;145(1):33-44.##Seruga B, Tannock IF. Chemotherapy-based treatment for castration-resistant prostate cancer. J Clin Oncol 2011;29(27):3686-3694.##Jathal MK, Chen L, Mudryj M, Ghosh PM. Targeting ErbB3: the new RTK (id) on the prostate cancer block. Immunol Endocr Metab Agents Med Chem 2011;11(2):131-149.##Wang Y, Yang H, Duan G. HER3 over-expression and overall survival in gastrointestinal cancers. Oncotarget 2015;6(40):42868-42878.##Jeong EG, Soung YH, Lee JW, Lee SH, Nam SW, Lee JY, et al. ERBB3 kinase domain mutations are rare in lung, breast and colon carcinomas. Int J Cancer 2006119(12):2986-2987.##Wang K, Kan J, Yuen ST, Shi ST, Chu KM, Law S, et al. Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer. Nat Genet 2011;43(12):1219-1223.##Cao GD, Chen K, Xiong MM, Chen B. HER3, but not HER4, plays an essential role in the cinicopathology and prognosis of gastric cancer: a meta-analysis. PLoS One 2016;11(8):e0161219.##Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004;9(6):667-676.##Zhang Y, Gonzalez RM, Zangar RC. Protein secretion in human mammary epithelial cells following HER1 receptor activation: influence of HER2 and HER3 expression. BMC Cancer 2011;11:69.##Wu X, Chen Y, Li G, Xia L, Gu R, Wen X, et al. Her3 is associated with poor survival of gastric adenocarcinoma: Her3 promotes proliferation, survival and migration of human gastric cancer mediated by PI3K/AKT signaling pathway. Med Oncol 2014;31(4):903.##

Exploring Potential Biomarkers Underlying Pathogenesis of Alzheimer’s Disease by Differential Co-expression Analysis 2 2 Background: Alzheimer's Disease (AD) is the most common form of dementia in the elderly. Due to the facts that biological causes of AD are complex in addition to increasing rates of AD worldwide, a deeper understanding of AD etiology is required for AD treatment and diagnosis.  Methods: To identify molecular pathological alterations in AD brains, GSE36980 series containing microarray data samples from temporal cortex, frontal cortex and hippocampus were downloaded from Gene Expression Omnibus (GEO) database and valid gene symbols were subjected to building a gene co-expression network by a bioinformatics tool known as differential regulation from differential co-expression (DCGL) software package. Then, a network-driven integrative analysis was performed to find significant genes and underlying biological terms.  Results: A total of 17088 unique genes were parsed into three independent differential co-expression networks. As a result, a small number of differentially co-regulated genes mostly in frontal and hippocampus lobs were detected as potential biomarkers related to AD brains. Ultimately differentially co-regulated genes were enriched in biological terms including response to lipid and fatty acid and pathways mainly signaling pathway such as G-protein signaling pathway and glutamate receptor groups II and III. By conducting co-expression analysis, our study identified multiple genes that may play an important role in the pathogenesis of AD.  Conclusion: The study aimed to provide a systematic understanding of the potential relationships among these genes and it is hoped that it could aid in AD biomarker discovery. 233 241 Fereshteh Izadi Department of Genetics, Evolution and Environment, Darwin Building, University College London (UCL) Department of Genetics, Evolution and Environment, Darwin Building, University College London (UCL) UK Mohammad Hasan Soheilifar Research Center for Molecular Medicine, Hamedan University of Medical Sciences Research Center for Molecular Medicine, Hamedan University of Medical Sciences Iran Alzheimer’s diseaseComputational biologyDementia 330.pdf Ferri CP, Prince M, Brayne C, Brodaty H, Fratiglioni L, Ganguli M, et al. Global prevalence of dementia: a Delphi consensus study. Lancet 2005;366(9503):2112-2117.##Alzheimer's Association. 2013 Alzheimer’s disease facts and figures. Alzheimer’s Dement 2013;9(2):208-245.##Dubois B, Feldman HH, Jacova C, Hampel H, Molinuevo JL, Blennow K, et al. Advancing research diagnostic criteria for Alzheimer’s disease: the IWG-2 criteria. Lancet Neurol 2014;13(6):614-629. ##Alzheimer A. Ueber eine eigenartige erkrankung der hirnrinde. Z Psychiatr 1907;64:146-148.##Alzheimer A, Stelzmann RA, Schnitzlen HN, Murthagh FR. An english translation of alzheimer’s 1907 paper, “Uber eine eigenartige erkankung der hirnrinde”. Clin Anat 1995;8(6):429-431.##Braak E, Braak H. Alzheimer’s disease: transiently developing dendritic changes in pyramidal cells of sector CA1 of the Ammon's horn. Acta Neuropathol 1997;93(4):323-325.##Glass CK, Saijo K, Winner B, Marchetto MC, Gage FH. Mechanisms underlying inflammation in neurodegeneration. Cell 2010;140(6):918-934.##Varadarajan S, Yatin S, Aksenova M, Butterfield DA. Review: Alzheimer’s amyloid beta-peptide-associated free radical oxidative stress and neurotoxicity. J Struct Biol 2000;130(2-3):184-208.##Guglielmotto M, Giliberto L, Tamagno E, Tabaton M. Oxidative stress mediates the pathogenic effect of different Alzheimer’s disease risk factors. Front Aging Neurosci 2010;2:3.##Gaiteri C, Ding Y, French B, Tseng GC, Sibille E. Beyond modules and hubs: the potential of gene co-expression networks for investigating molecular mechanisms of complex brain disorders. Genes Brain Behav 2014;13(1):13-24.##Stoner R, Chow ML, Boyle MP, Sunkin SM, Mouton PR, Roy S, et al. Patches of disorganization in the neocortex of children with autism N Engl J Med 2003;370(13):1209-1219.##Jinawath N, Bunbanjerdsuk S, Chayanupatkul M, Ngamphaiboon N, Asavapanumas N, Svasti J, et al. Bridging the gap between clinicians and systems biologists: from network biology to translational biomedical research. J Transl Med 2016;14(1):324.##Yang J, Yu H, Liu BH, Zhao Z, Liu L, Ma LX, et al. DCGL v2.0: an R package for unveiling differential regulation from differential co-expression. PLoS One 2013;8(11):e79729.##Liu BH, Yu H, Tu K, Li C, Li YX, Li YY. DCGL: an R package for identifying differentially co-expressed genes and links from gene expression microarray data. Bioinformatics 2010;26(20):2637-2638.## Assenov Y, Ramirez F, Schelhorn SE, Lengauer T, Albrecht M. Computing topological parameters of biological networks. Bioinformatics 2008;24(2):282-284.##Maere S, Heymans K, Kuiper M. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 2005;21(16):3448-3449.##Gatta V, D'Aurora M, Granzotto A, Stuppia L, Sensi SL. Early and sustained altered expression of aging-related genes in young 3xTg-AD mice. Cell Death Dis 2014;5:e1054.##Mendoza-Naranjo A, Gonzalez-Billault C, Maccioni RB. Abeta1-42 stimulates actin polymerization in hippocampal neurons through Rac1 and Cdc42 Rho GTPases. J Cell Sci 2007;120(Pt 2):279-288.##Williams C, Mehrian Shai R, Wu Y, Hsu YH, Sitzer T, Spann B, et al. Transcriptome analysis of synaptoneurosomes identifies neuroplasticity genes overexpressed in incipient Alzheimer's disease. PLoS One 2009;4(3):e4936.##Potts RC, Zhang P, Wurster AL, Precht P, Mughal MR, Wood WH 3rd, et al. CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes. PLoS One 2011;6(9):e24515.##Borger E, Herrmann A, Mann DA, Spires-Jones T, Gunn-Moore F. The calcium-binding protein EFhd2 modulates synapse formation in vitro and is linked to human dementia. J Neuropathol Exp Neurol 2014;73(12):1166-1182.##Vega IE. EFhd2, a protein linked to Alzheimer’s disease and other neurological disorders. Front Neurosci 2016;10:150.##Cumming RC, Dargusch R, Fischer WH, Schubert D. Increase in expression levels and resistance to sulfhydryl oxidation of peroxiredoxin isoforms in amyloid beta-resistant nerve cells. J Biol Chem 2007;282(42):30523-30534.##Braunewell KH. The visinin-like proteins VILIP-1 and VILIP-3 in Alzheimer’s disease-old wine in new bottles. Front Mol Neurosci 2012;5:20.##Chadwick W, Brenneman R, Martin B, Maudsley S. Complex and multidimensional lipid raft alterations in a murine model of Alzheimer's disease. Int J Alzheimers Dis 2010;2010:604792.##Miyata S, Kurachi M, Okano Y, Sakurai N, Kobayashi A, Harada K, et al. Blood transcriptomic markers in patients with late-onset major depressive disorder. PLoS One 2016;11(2):e0150262.##Wang Y, Cella M, Mallinson K, Ulrich JD, Young KL, Robinette ML, et al. TREM2 lipid sensing sustains microglia response in an Alzheimer’s disease model. Cell 2015;160(6):1061-1071.##Vélez JI, Lopera F, Sepulveda-Falla D, Patel HR, Johar AS, Chuah A. APOE*E2 allele delays age of onset in PSEN1 E280A Alzheimer's disease. Mol Psychiatry 2016;21(7):916-924.##Heinzen EL, Yoon W, Weale ME, Sen A, Wood NW, Burke JR, et al. Alternative ion channel splicing in mesial temporal lobe epilepsy and Alzheimer's disease. Genome Biol 2007;8(3):R32.##De Iudicibus S, Franca R, Martelossi S, Ventura A, Decorti G. Molecular mechanism of glucocorticoid resistance in inflammatory bowel disease. World J Gastroenterol 2011;17(9):1095-1108.##Oka S, Leon J, Sakumi K, Ide T, Kang D, LaFerla FM, et al. Human mitochondrial transcriptional factor a breaks the mitochondria-mediated vicious cycle in Alzheimer’s disease. Sci Rep 2016;6:37889.##Zimmer DB, Keeling D, Campbell K, Campbell K, Afanador L. S100A1 modulates inflammation and PI3/Akt signaling. FASEB J 2013;27(1Suppl):lb516.##Gautam V, D’Avanzo C, Hebisch M, Kovacs DM, Kim DY. BACE1 activity regulates cell surface contactin-2 levels. Mol Neurodegener 2014;9:4.##Gray AL, Hyde TM, Deep-Soboslay A, Kleinman JE, Sodhi MS. Sex differences in glutamate receptor gene expression in major depression and suicide. Mol Psychiatry 2015;20(9):1139.##Olgiati P, Politis AM, Papadimitriou GN, Ronchi DD, Serretti A. Genetics of late-onset Alzheimer's disease: update from the alzgene database and analysis of shared pathways. Int J Alzheimers Dis 2011;2011:832379.##Bandopadhyay R. Sequential extraction of soluble and insoluble alpha-synuclein from parkinsonian brains. J Vis Exp 2016;(107).##Winkler EA, Nishida Y, Sagare AP, Rege SV, Bell RD, Perlmutter D, et al. GLUT1 reductions exacerbate Alzheimer’s disease vasculoneuronal dysfunction and degeneration. Nat Neurosci 2015;18(4):521-530.##Teunissen CE, Veerhuis R, De Vente J, Verhey FR, Vreeling F, van Boxtel MP, et al. Brain-specific fatty acid-binding protein is elevated in serum of patients with dementia-related diseases. Eur J Neurol 201;18(6):865-871.##Ke Y, Dramiga J, Schütz U, Kril JJ, Ittner LM, Schröder H, et al. Tau-mediated nuclear depletion and cytoplasmic accumulation of SFPQ in Alzheimer's and Pick's disease. PLoS One 2012;7(4):e35678.##Piacentini S, Polimanti R, Squitti R, Ventriglia M, Cassetta E, Vernieri F, et al. GSTM1 null genotype as risk factor for late-onset Alzheimer's disease in Italian patients. J Neurol Sci 2012;317(1-2):137-140.##Hemming ML, Elias JE, Gygi SP, Selkoe DJ. Identification of beta-secretase (BACE1) substrates using quantitative proteomics. PLoS One 2009;4(12):e8477.##Xu Y, Yue W, Shugart YY, Li S, Cai L, Li Q, et al. Exploring transcription factors-microRNAs Co-regulation networks in Schizophrenia. Schizophr Bull 2016;42(4):1037-1045.##Oldham MC, Horvath S, Geschwind DH. Conservation and evolution of gene co-expression networks in human and chimpanzee brains. Proc Natl Acad Sci USA 2006;103(47):17973-17978.##Okamura Y, Aoki Y, Obayashi T, Tadaka S, Ito S, Narise T, et al. COXPRESdb in 2015: coexpression database for animal species by DNA- microarray and RNAseq-based expression data with multiple quality assessment systems. Nucleic Acids Res 2015;43(Database issue):D82-86.##de la Fuente A. From ‘differential expression’ to ‘differential networking’ - identification of dysfunctional regulatory networks in diseases. Trends Genet 2010;26(7):326-333.##Torkamani A, Dean B, Schork NJ, Thomas EA. Coexpression network analysis of neural tissue reveals perturbations in developmental processes in schizophrenia. Genome Res 2010;20:403-412.##de Jong S, Boks MP, Fuller TF, Strengman E, Janson E, de Kovel CG, et al. A gene co-expression network in whole blood of schizophrenia patients is independent of antipsychotic-use and enriched for brain-expressed genes. PLoS One 2012;7(6):e39498.##Ponomarev I, Wang S, Zhang L, Harris RA, Mayfield RD. Gene co-expression networks in human brain identify epigenetic modifications in alcohol dependence. J Neurosci 2012;32(5):1884-1897.##Chen C, Cheng L, Grennan K, Pibiri F, Zhang C, Badner JA, et al. Two gene co-expression modules differentiate psychotics and controls. Mol Psychiatry 2013;18(12):1308-1314.##El Gaamouch F, Jing P, Xia J, Cai D. Alzheimer's disease risk genes and lipid regulators. J Alzheimers Dis 2016;23;53(1):15-29.##Yuan H, Low CM, Moody OA, Jenkins A, Traynelis SF. Ionotropic GABA and Glutamate receptor mutations and human neurologic diseases. Mol Pharmacol 2015;88(1):203-217.##Hamilton A, Zamponi GW, Ferguson SS. Glutamate receptors function as scaffolds for the regulation of β-amyloid and cellular prion protein signaling complexes. Mol Brain 2015;8:18.##Takenouchi T, Hashida N, Torii C, Kosaki R, Takahashi T, Kosaki K. 1p34.3 deletion involving GRIK3: further clinical implication of GRIK family glutamate receptors in the pathogenesis of developmental delay. Am J Med Genet A 2014;164A(2):456-460.##Schnack C, Danzer KM, Hengerer B, Gillardon F. Protein array analysis of oligomerization-induced changes in alpha-synuclein protein-protein interactions points to an interference with Cdc42 effector proteins. Neuroscience 2008;154(4):1450-1457.##Min D, Guo F, Zhu S, Xu X, Mao X, Cao Y, et al. The alterations of Ca2+/calmodulin/CaMKII/CaV1.2 signaling in experimental models of Alzheimer’s disease and vascular dementia. Neurosci Lett 2013;538:60-65.##Rangaraju S, Gearing M, Jin LW, Levey A. Potassium channel Kv1.3 is highly expressed by microglia in human Alzheimer’s disease. J Alzheimers Dis 2015;44(3):797-808.##Hokama M, Oka S, Leon J, Honda H, Sasaki K, Nakabeppu Y, et al. Altered expression of diabetes-related genes in Alzheimer’s disease brains: the Hisayama study. Cereb Cortex 2014;24(9):2476-2488.##Rogers J. The inflammatory response in Alzheimer's disease. J Periodontol 2008;79(8 Suppl):1535-1543.##Satoh J, Yamamoto Y, Asahina N, Kitano S, Kino Y. RNA-Seq data mining: downregulation of neuroD6 serves as a possible biomarker for Alzheimer’s disease brains. Dis Markers 2014;2014:123165.##Yamada M, Shida Y, Takahashi K, Tanioka T, Nakano Y, Tobe T, et al. Prg1 is regulated by the basic helix-loop-helix transcription factor Math2. J Neurochem 2008;106(6):2375-2384.##Fowler KD, Funt JM, Artyomov MN, Zeskind B, Kolitz SE, Towfic F. Leveraging existing data sets to generate new insights into Alzheimer’s disease biology in specific patient subsets. Sci Rep 2014;5:14324.##

Association of G/C (rs638405) Polymorphism in β-secretase Gene with Alzheimer’s Disease 2 2 Background: Alzheimer's Disease (AD) is a neurodegenerative disorder, which is the most common cause of dementia in the elderly. Accumulation of β-amyloid plaques outside neurons is the most important pathological hallmark of AD, which is produced by cleavage of amyloid precursor protein by the Alzheimer's β-secretase (BACE1). Since BACE1 is a key enzyme in the formation of β-amyloid peptides, the purpose of this study was to assess the association between polymorphisms of G/C (rs638405) BACE1 gene with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran. Methods: Genotypes were determined by the PCR–Restriction Fragment Length Polymorphism (PCR–RFLP) technique in two groups including 89 sporadic AD patients and 73 healthy subjects. Results: The findings of the BACE1 G/C (rs638405) polymorphism revealed that there was no significant difference between AD patients and controls in men group; however, there was a weak difference in the frequency of CC genotype between patients and controls in women group (χ2=3.333, df=1, p=0.068). Conclusion: The results of this study suggest that the G/C (rs638405) polymorphism of BACE1 gene might not be related with sporadic AD in Khuzestan, Isfahan and Fars provinces in Iran. However, our results do not support a genetic risk factor of this polymorphism for developing AD in male group of this study. 242 247 Mostafa Chashmpoosh Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Iran Hossein Babaahmadi Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Iran Rouhollah Mousavidehmordi Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Iran Bita Shalbafan Department of Neurology, Faculty of Medical, Ahvaz Jundishapur University of Medical Sciences Department of Neurology, Faculty of Medical, Ahvaz Jundishapur University of Medical Sciences Iran Asma Mohammadi Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Iran Alireza Kheirollah Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Department of Biochemistry, Faculty of Medical, Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences Iran Alzheimer’s diseaseAmyloidogenic proteinsBACE1 geneGenotypeIran 331.pdf De Strooper B, Annaert W. Proteolytic processing and cell biological functions of the amyloid precursor protein. J Cell Sci 2000;113:1857-1870.##Zhang X, Li Y, Xu H, Zhang YW. The γ-secretase complex: from structure to function. Front Cell Neurosci 2013;8:427.##Glenner GG, Wong CW. Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Alzheimer Dis Assoc Disord 1988;2(2):134.##De-Paula VJ, Radanovic M, Diniz BS, Forlenza OV. Alzheimer's disease. Subcell Biochem 2012;65:329-352.##Younkin SG. The role of Aβ42 in Alzheimer's disease. J Physiol Paris 1998;92(3-4):289-292.##Vassar R. beta-Secretase, APP and Abeta in Alzheimer's disease. Subcell Biochem 2005;38:79-103.##Vassar R. BACE1: the beta-secretase enzyme in Alzheimer's disease. J Mol Neurosci 2004;23(1-2):105-114.##Tran L, Ha-Duong T. Exploring the Alzheimer amyloid-β peptide conformational ensemble: A review of molecular dynamics approaches. Peptides 2015;69:86-91.##Vassar R, Citron M. Aβ-generating enzymes: recent advances in β-and γ-secretase research. Neuron 2000;27(3):419-422.##Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiolog Rev 2001;81(2):741-766.##Zhang X, Zhou K, Wang R, Cui J, Lipton SA, Liao FF, et al. Hypoxia-inducible factor 1alpha (HIF-1alpha)-mediated hypoxia increases BACE1 expression and beta-amyloid generation. J Biol Chem 2007;282(15):10873-10880.##Lahiri DK, Maloney B, Ge YW. BACE1 gene promoter is differentially regulated: detection of a novel promoter region for its cell type-specific regulation. J Mol Neurosci 2006;28(2):193-210.##Bourne KZ, Ferrari DC, Lange‐Dohna C, Rossner S, Wood TG, Perez‐Polo JR. Differential regulation of BACE1 promoter activity by nuclear factor‐κB in neurons and glia upon exposure to β‐amyloid peptides. J Neurosci Res 2007;85(6):1194-1204.##Cho HJ, Kim SK, Jin SM, Hwang EM, Kim YS, Huh K, et al. IFN‐gamma‐induced BACE1 expression is mediated by activation of JAK2 and ERK1/2 signaling pathways and direct binding of STAT1 to BACE1 promoter in astrocytes. Glia 2007;55(3):253-262.##Yu M, Liu Y, Shen J, Lv D, Zhang J. Meta-analysis of BACE1 gene rs638405 polymorphism and the risk of Alzheimer’s disease in Caucasion and Asian population. Neurosci lett 2016;616:189-196.##Todd S, McKnight AJ, Liu WW, Carson R, Heggarty S, McGuinness B, et al. BACE1 polymorphisms do not influence platelet membrane β-secretase activity or genetic susceptibility for Alzheimer’s disease in the northern Irish population. Neuromolecular Med 2008;10(4):368-376.##Cai L, Tang G, Chen L, Zhang B, Jiang S, Ren D. Genetic studies of A2M and BACE1 genes in Chinese Han Alzheimer's disease patients. Neuroreport 2005;16(9):1023-1026.##Clarimón J, Bertranpetit J, Calafell F, Boada M, Tàrraga L, Comas D. Association study between Alzheimer’s disease and genes involved in Abeta biosynthesis, aggregation and degradation: suggestive results with BACE1. J Neurol 2003;250(8):956-961.##Nowotny P, Kwon JM, Chakraverty S, Nowotny V, Morris JC, Goate AM. Association studies using novel polymorphisms in BACE1 and BACE2. Neuroreport 2001;12(9):1799-1802.##Kan R, Wang B, Zhang C, Jin F, Yang Z, Ji S, et al. Genetic association of BACE1 gene polymorphism C786G with late-onset Alzheimer's disease in Chinese. J Mol Neurosci 2005;25(2):127-131.##Bekris LM, Galloway NM, Millard S, Lockhart D, Li G, Galasko D, et al. Amyloid precursor protein (APP) processing genes and cerebrospinal fluid APP cleavage product levels in Alzheimer's disease. Neurobiol Aging 2011;32(3):556. e13-e23.##Liu HC, Leu SJ, Chang JG, Sung SM, Hsu WC, Lee LS, et al. The association of beta-site APP cleaving enzyme (BACE) C786G polymorphism with Alzheimer’s disease. Brain Res 2003;961(1):88-91.##Jo SA, Ahn K, Kim E, Kim HS, Jo I, Kim DK, et al. Association of BACE1 gene polymorphism with Alzheimer’s disease in Asian populations: meta-analysis including Korean samples. Dement Geriatr Cogn Disord 2008;25(2):165-169.##Murphy T, Yip A, Brayne C, Easton D, Evans JG, Xuereb J, et al. The BACE gene: genomic structure and candidate gene study in late-onset Alzheimer's disease. Neuroreport 2001;12(3):631-634.##Llorca J, Rodríguez-Rodríguez E, Dierssen-Sotos T, Delgado-Rodríguez M, Berciano J, Combarros O. Meta‐analysis of genetic variability in the beta‐amyloid production, aggregation and degradation metabolic pathways and the risk of Alzheimer’s disease. Acta Neurol Scand 2008;117(1):1-14.##Wang M, Yang J, Su J. Relationship between the polymorphism in exon 5 of BACE1 gene and Alzheimer's disease. Aging Clin Exp Res 2017;29(2):105-113.##Mousavidehmordi R, Babaahmadi H, Shalbafan B, Mohammadzadeh G, Afsharmanesh M, Kheirollah A. Polymorphism of CYP46A1 gene and Alzheimer’s disease in the Iranian population. Shiraz E-Med J 2016;17(9):e41218.##Yamagata H, Chen Y, Akatsu H, Kamino K, Ito JI, Yokoyama S, et al. Promoter polymorphism in fibroblast growth factor 1 gene increases risk of definite Alzheimer’s disease. Biochem Biophys Res Commun 2004;321(2):320-323.##Kawas C, Katzman R. Epidemiology of dementia and Alzheimer disease. Alzheimer Disease 1999;2:95-116.##Chashmpoosh M, Babaahmadi H, Mosavidehmordi R, Mohammadi A, Kheirollah A. The Association between G/A (rs34011) Polymorphism of the FGF1 Gene and Alzheimer’s Disease. J Mazandaran Univ Med Sci 2016;25(134):89-97.##Raygani AV, Zahrai M, Raygani AV, Doosti M, Javadi E, Rezaei M, et al. Association between apolipoprotein E polymorphism and Alzheimer disease in Tehran, Iran. Neurosci Lett 2005;375(1):1-6.##He XM, Zhang ZX, Zhang JW, Zhou YT, Wu CB, Tang MN, et al. An intronic CYP46A1 polymorphism is associated with Alzheimer disease in a Chinese Han population. J Mol Neurosci 2012;47(3):514-518.##Bian JT, Zhang JW, Zhang ZX, Zhao HL. Association analysis of brain-derived neurotrophic factor (BDNF) gene 196 A/G polymorphism with Alzheimer's disease (AD) in mainland Chinese. Neurosci Lett 2005;387(1): 11-16.##Tsai A, Huang CC, Yang AC, Liu ME, Tu PC, Hong CJ, et al. Association of BACE1 gene polymorphism with cerebellar volume but not cognitive function in normal individuals. Dement Geriatr Cogn Dis Extra 2012;2(1): 632-637.##Bodendorf U, Fischer F, Bodian D, Multhaup G, Paganetti P. A splice variant of β-secretase deficient in the amyloidogenic processing of the amyloid precursor protein. J Biological Chem 2001;276(15):12019-12023.##Sinha S, Anderson JP, Barbour R, Basi GS, Caccavello R, Davis D, et al. Purification and cloning of amyloid precursor protein beta-secretase from human brain. Nature 1999;402(6761):537-540.##Gold G, Blouin JL, Herrmann FR, Michon A, Mulligan R, Duriaux Sail G, et al. Specific BACE1 genotypes provide additional risk for late‐onset alzheimer disease in APOE epsilon 4 carriers. Am J Med Genet B Neuropsychiatr Genet 2003;119B(1):44-47.##Shi J, Zhang S, Tang M, Liu X, Li T, Wang Y, et al. The 1239G/C polymorphism in exon 5 of BACE1 gene may be associated with sporadic Alzheimer's disease in Chinese Hans. Am J Med Genet B Neuropsychiatr Genet 2004;124B(1):54-57.##

Interaction Effect of RsaI and BamHI Polymorphisms of TGFα, BMP2 and BMP4 on the Occurrence of Non-Syndromic Cleft Lip and Palate in Iranian Patients 2 2 Background: Orofacial cleft is the most common congenital defect of the maxillofacial region. Its non-syndromic type is multi-factorial, and several genes are involved in its occurrence. This study aimed to assess the interaction effect of Rsal and BamHI polymorphisms of Transforming Growth Factor-alpha (TGFα) gene and Bone Morphogenetic Protein-2 (BMP2) and BMP4 variants on the occurrence of Non-Syndromic Cleft Lip and Palate (NSCLP) in the Iranian population. Methods: This case-control study was conducted on 120 children with NSCLP and 215 healthy children. Genotyping of the TGFA/BamHI (rs11466297), TGFA/RsaI (rs3732248), BMP4 (rs17563) and BMP2 (rs235768) was performed by Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) methods. Logistic regression was applied to determine the effective factors and the interaction effect of different variants on the occurrence of NSCLP.  Results: Gender of patients had no significant association with the occurrence of NSCLP (p=0.335). Multiple logistic regression showed that the interaction effect of the aforementioned polymorphisms on the occurrence of NSCLP was not statistically significant (p=1.000). Conclusion: Although the individual effect of each of the BMP4, BMP2, RsaI and BamHI variants on the occurrence of NSCLP in the Iranian population has been previously confirmed, their interaction does not play a role in this respect. 248 252 Saba Samadi General Dentist General Dentist Iran Asghar Ebadifar Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Department of Orthodontic, Faculty of Dentistry, Shahid Behehsti University of Medical Sciences Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Department of Orthodontic, Faculty of Dentistry, Shahid Behehsti University of Medical Sciences Iran Hamid Reza Khorram Khorshid Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Koorosh Kamali Department of Public Health, Faculty of Public Health, Zanjan University of Medical Sciences Department of Public Health, Faculty of Public Health, Zanjan University of Medical Sciences Iran Mohammadreza Badiee Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Iran Bone morphogenetic proteinCleft Lip and palatePolymorphism 332.pdf Dixon MJ, Marazita ML, Beaty TH, Murray JC. Cleft lip and palate: understanding genetic and environmental influences. Nat Rev Genet 2011;12(3):167-178.##Conway JC, Taub PJ, Kling R, Oberoi K, Doucette J, Jabs EW. Ten-year experience of more than 35,000 orofacial clefts in Africa . BMC Pediatr 2015;15:8.##Mirfazeli A, Kaviany N, Hosseinpour KR, Golalipour MJ. Incidence of cleft lip and palate in gorgan-northern iran: an epidemiological study. Oman Med J 2012;27(6):461-464.##Kianifar H, Hasanzadeh N, Jahanbin A, Ezzati A, Kianifar H. Cleft lip and palate: A 30-year epidemiologic study in north-east of Iran. Iran J Otorhinolaryngol 2015;27(78):35-41.##Cooper ME, Ratay JS, Marazita ML. Asian oral-facial cleft birth prevalence. Cleft Palate Craniofac J 2006;43(5):580-589.##Mossey PA, Little J, Munger RG, Dixon MJ, Shaw WC. Cleft lip and palate. Lancet 2009;374(9703):1773-1785.##Setó-Salvia N, Stanier P.Genetics of cleft lip and/or cleft palate: association with other common anomalies. Eur J Med Genet 2014;57(8):381-393.##Vieira AR. Association between the transforming growth factor alpha gene and nonsyndromic oral clefts: a HuGE review. Am J Epidemiol 2006;163(9):790-810.##Lu XC, Yu W, Tao Y, Zhao PL, Li K, Tang LJ, et al. Contribution of transforming growth factor α polymorphisms to nonsyndromic orofacial clefts: a HuGE review and meta-analysis. Am J Epidemiol 2014;179(3):267-281.##Ardinger HH, Buetow KH, Bell GI, Bardach J, VanDemark DR, Murray JC. Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. Am J Hum Genet 1989;45(3):348-353.##Ebadifar A, Hamedi R, Khorram Khorshid HR, Saliminejad, Kamali K, Aghakhani Moghadam F, et al. Association of transforming growth factor alpha polymorphisms with nonsyndromic cleft lip and palate in Iranian population. Avicenna J Med Biotechnol 2015;7(4):168-172.##Nie X, Luukko K, Kettunen P. BMP signalling in craniofacial development. Int J Dev Biol 2006;50(6):511-521.##Kempa I, Ambrozaitytė L, Stavusis J, Akota I, Barkane B, Krumina A, et al. Association of BMP4 polymorphisms with non-syndromic cleft lip with or without cleft palate and isolated cleft palate in Latvian and Lithuanian populations. Stomatologija 2014;16(3):94-101.##Araújo TK, Simioni M, Félix TM, de Souza LT, Fontes MÍ, Monlleó IL, et al. Preliminary analysis of the nonsynonymous polymorphism rs17563 in BMP4 gene in Brazilian population suggests protection for nonsyndromic cleft lip and palate. Plast Surg Int 2012;2012:247104.##Saket M, Saliminejad K, Kamali K, Moghadam FA, Anvar NE, Khorram Khorshid HR. BMP2 and BMP4 variations and risk of non-syndromic cleft lip and palate. Arch Oral Biol 2016;72:134-137.##Blanco R, Colombo A, Pardo R, Suazo J. Haplotype-based gene-gene interaction of bone morphogenetic protein 4 and interferon regulatory factor 6 in the etiology of non-syndromic cleft lip with or without cleft palate in a chilean population. Eur J Oral Sci 2017;125(2):102-109.##Lee SH, Fu KK, Hui JN, Richman JM. Noggin and retinoic acid transform the identity of avian facial prominences. Nature 2001;414(6866):909-912.##Ashique AM, Fu K, Richman JM. Endogenous bone morphogenetic proteins regulate outgrowth and epithelial survival during avian lip fusion. Development 2002;129(19):4647-4660.##Hu D, Marcucio RS, Helms JA. A zone of frontonasal ectoderm regulates patterning and growth in the face. Development 2003;130(9):1749-1758.##Mehrotra D. Genomic expression in non syndromic cleft lip and palate patients: a review. J Oral Biol Craniofac Res 2015;5(2):86-91.##Song T, Wu D, Wang Y, Li H, Yin N, Zhao Z. SNPs and interaction analyses of IRF6, MSX1 and PAX9 genes in patients with non-syndromic cleft lip with or without palate. Mol Med Rep 2013;8(4):1228-1234.##Lidral AC, Romitti PA, Basart AM, Doetschman T, Leysens NJ, Daack-Hirsch S, et al. Association of MSX1 and TGFB3 with nonsyndromic clefting in humans. Am J Hum Genet 1998;63(2):557-568.##Jugessur A, Lie RT, Wilcox AJ, Murray JC, Taylor JA, Saugstad OD, et al. Variants of developmental genes (TGFA, TGFB3, and MSX1) and their associations with orofacial clefts: a case-parent triad analysis. Genet Epidemiol 2003;24(3):230-239.##Koshy L, Anju AL, Harikrishnan S, Kutty VR, Jissa VT, Kurikesu I, et al. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays. Mol Biol Rep 2017;44(1):97-108.##Sivertsen A, Wilcox AJ, Skjaerven R, Vindenes HA, Abyholm F, Harville E, et al. Familial risk of oral clefts by morphological type and severity: population based cohort study of first degree relatives. BMJ 2008,336(7641):432-434.##Leslie EJ, Marazita ML. Genetics of cleft lip and cleft palate. Am J Med Genet C Semin Med Genet 2013,163C(4):246-258.##Leslie EJ, Murray JC. Evaluating rare coding variants as contributing causes to non-syndromic cleft lip and palate. Clin Genet 2013,84(5):496-500.##Lidral AC, Murray JC, Buetow KH, Basart AM, SchearerH, Shiang R, et al. Studies of the candidate genes TGFB2, MSX1, TGFA, and TGFB3 in the etiology of cleft lip and palate in the Philippines. Cleft Palate Craniofac J 1997;34(1):1-6.##Jianyan L, Zeqiang G, Yongjuan C, Kaihong D, Bing D, Rongsheng L. Analysis of interactions between genetic variants of BMP4 and environmental factors with nonsyndromic cleft lip with or without cleft palate susceptibility. Int J Oral Maxillofac Surg 2010;39(1):50-56.##Wang H, Zhou X, Cui Y, Liu J, Wang W. Relationship between nonsyndromic cleft lip with or without cleft palate (NSCL/P) and genetic polymorphisms of BMP4. Jiangsu Med J 2012,38(8):897-900.##Hu YY, Qin CQ, Deng MH, Niu YM, Long X. Association between BMP4 rs17563 polymorphism and NSCL/P risk: a meta-analysis. Dis Markers 2015;2015:763090.##Sull JW, Liang KY, Hetmanski JB, Wu T, Fallin MD, Ingersoll RG, et al. Evidence that TGFA influences risk to cleft lip with/without cleft palate through unconventional genetic mechanisms. Hum Genet 2009;126(3):385-394.##

Retraction: The Association of PON1 192 Q/R Polymorphism with the Risk of Idiopathic Male Infertility in Northern Iran 2 2 Background: Infertility is defined as the inability to achieve pregnancy after 12 months of regular unprotected sexual intercourse. Environmental and genetic factors are involved in male infertility. The polymorphism studies have a crucial role in disease recognition. Paraoxonase (PON) is an oxidant enzyme which is associated with inflammation, oxidative stress and lipid metabolism. The present study aimed to evaluate the relationship between PON1 192 Q/R polymorphism and the susceptibility to idiopathic male infertility.  Methods: Samples were collected from 220 patients diagnosed with male infertility and 230 controls genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP).  Results: A significant difference in genotype distributions of PON1 192 Q/R polymorphism was observed between patients and controls (p=0.001). Our findings revealed that individuals with the variant QR had a significant decreased risk of idiopathic male infertility (OR=0.49, 95%CI=0.33–0.73, p=0.0004). Moreover, analyses showed that R allele may have a protective effect on susceptibility of idiopathic male infertility (OR=0.31, 95%CI=0.21-0.47, p=0.0001).  Conclusion: The data from this study indicates that the PON1 192 Q/R polymorphism is associated with decreased risk of idiopathic male infertility. However, more studies should be considered with larger number of patients and control subjects to confirm our results. 253 256 Setareh Behrouzi Department of Biology, Faculty of Sciences, University of Guilan Department of Biology, Faculty of Sciences, University of Guilan Iran Farhad Mashayekhi Department of Biology, Faculty of Sciences, University of Guilan Department of Biology, Faculty of Sciences, University of Guilan Iran Mohammad Hadi Bahadori Cellular and Molecular Research Center, Faculty of Medical Sciences, Guilan University of Medical Sciences Cellular and Molecular Research Center, Faculty of Medical Sciences, Guilan University of Medical Sciences Iran InfertilityPON1Polymorphism 10358.pdf Kara E, Simoni M. Genetic screening for infertility: when should it be done? Middle East Fertil Soc J 2010;15(3):139-145.##Avenarius MR, Hildebrand MS, Zhang Y, Meyer NC, Smith LL, Kahrizi K, et al. Human male infertility caused by mutationsin the CATSPER1 channel protein. Am J Hum Genet 2009;84(4):505-510.##Hildebrand MS, Avenarius MR, Fellous M, Zhang Y, Meyer NC, Auer J, et al. Genetic male infertility and mutation of CATSPER ion channels. Eur J Hum Genet 2010;18(11):1178-1184.##Hamada A, Esteves S, Agarwal A. The role of contemporary andrology in unraveling the mystery of unexplained male infertility. Open Reprod Sci J 2011;4:27-41.##Plaseska-Karanfilska D, Noveski P, Plaseski T, Maleva I, Madjunkova S, Moneva Z. Genetic causes of male infertility. Balkan J Med Genet 2012;15(Suppl):31-34.##Jungwirth (chair) A, Diemer T, Dohle GR, Giwercman A, Kopa Z, Krausz C, et al. Guidelines on male infertility. European Association of Urology 2013; p. 176-226.##Oberley TD. Oxidative damage and cancer. Am J Pathol. 2000;160(2):403-408.##Olayemi FO. A review on some causes of male infertility. Afr J Biotechnol 2010;9(20):2834-2842.##Lewis SE, Sterling ES, Young IS, Thompson W. Comparison of individual antioxidants of sperm and seminal plasma in fertile and infertile men. Fertil Steril 1997;67(1):142-147.##Lazaros L, Markoula S, Kkyritsis A, Georgiou I. Paraoxonase gene polymorphisms and stroke severity. Eu J Neurol 2010;17(5):757-759.##Lazaros LA, Xita VN, Hatzi EG, Kaponis AI, Stefos TJ, Plachouras NI, et al. Association of paraoxonase gene polymorphisms with sperm parameters. J Androl 2011;32(4):394-401.##Kim DS, Burt AA, Ranchalis JE, Richter RJ, Marshall JK, Eintracht JF, et al. Additional common polymorphisms in the PON gene cluster predict PON1 activity but not vascular disease. J Lipids 2012;2012:476316.##She ZG, Chen HZ, Yan Y, Li H, Liu DP. The human paraoxonase gene cluster as a targetin the treatment of atherosclerosis. Antioxid Redox Signal 2012;16(6):597-632.##Bahrehmand F, Vaisi-Raygani A, Ahmadi R, Kiani A, Rahimi Z, Tavilani H, et al. Paraoxonase (PON1) 55 polymorphism and association with systemic lupus erythematosus. Iran J Allergy Asthma Immunol 2013;12(3):211-219.##Eom SY, Kim YS, Lee CJ, Lee CH, Kim YD, Kim H. Effects of intronic and exonic polymorphisms of paraoxonase 1(PON1) gene on serum PON1 activity in a Korean population. J Korean Med Sci 2011;26(6):720-725.##World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Geneva: World Health Organization. 1999. 271 p.##Ferlin A, Arredi B, Foresta C. Genetic causes of male infertility. Reprod Toxicol 2006;22(2):133-141.##Mackness MI, Arrol S, Durrington PN. Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein. FEBS Lett 1991;286(1-2):152-154.##Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, et al. Human paraoxonase-3 is an HDL-associated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vasc Biol 2001;21(4):542-547.##Mahadesh Prasad AJ, Kemparaju K, Elizabeth AF, Jaichander P, Arun A, Cletus JM, et al. Lack of correlation of paraoxonase (PON1) activity with smoking among the south Indians and risk of cardiovascular disease. World Appl Sci J 2010;9:194-198.##Haj Mouhamed D, Ezzaher A, Mechri A, Neffati F, Om-ezzine A, Bouslama A, et al. Effect of cigarette smoking on paraoxonase 1 activity according to PON1 L55M and PON1 Q192R gene polymorphisms. Environ Health Prev Med 2012;17:316-321.##Bhattacharyya T, Nicholls SJ, Topol EJ, Zhang R, Yang X, Schmitt D, et al. Relationship of paraoxonase 1 (PON1) gene polymorphisms and functional activity with systemic oxidative stress and cardiovascular risk. JAMA 2008;299(11):1265-1276.##Aydin M, Gencer M, Cetinkaya Y, Ozkok E, Ozbek Z, Kilic G, et al. PON1 55/192 polymorphism, oxidative stress, type, prognosis and severity of stroke. IUBMB Life 2006;58(3):165-172.##Erlich PM, Lunetta KL, Cupples LA, Huyck M, Green RC, Baldwin CT, at al. Polymorphisms in the PON gene cluster are associated with Alzheimer disease. Hum Mol Genet 2006;15(1):77-85.##Khoshi A, Mortazavi Y, Sokhanvar S, Tanzifi A, Akbari A, Kalantari S. Determination of PON1 gene polymorphisms in Iranian individuals with high LDL/HDL ratios. Indian J Clin Biochem 2015;30(4):449-456.##Asefi M, Vaisi-Raygani A, Bahrehmand F, Kiani A, Rahimi Z, Nomani H, et al. Paraoxonase 1 (PON1) 55 polymorphism, lipid profiles and psoriasis. Br J Dermatol 2012;167(6):1279-1286.##Marsillach J, Lafuente R, Checa MA, Maestre-Martínez C, Fabián E, Brassesco M, et al. Paraoxonase-1 is only present in traceable amounts in seminal fluid and does not show any relationship with male subfertility. BJU Int 2010;108(4):566-570.##

Pattern Analysis of Short Tandem Repeats Allele Frequencies among the Population of Khuzestan Province, South of Iran 2 2 Background: The basis of genetic fingerprinting and DNA profiling in forensic laboratories is the use of Short Tandem Repeats (STRs) according to local and ethnical genetics characteristics. Methods: Forensic parameters and allele frequencies for 15 autosomal STRs in 100 unrelated individuals from Khuzestan province, south Iran were determined. PCR was carried out for amplification of STRs and GeneMapper ID software was used for genotyping and allelic analyzing.  Results: The Power of Exclusion (PE) varied between 0.332 (TPOX) and 0.768 (FGA). With exception of the THO1 (0.020), TPOX (0.014) and D18S51 (0.003), other STRs showed no deviation from the Hardy-Weinberg equilibrium (p>0.05). Conclusion: Out of 15 STRs, 12 repeats seemed to be more useful and more powerful tools in identity and paternity determination for our studied population. Variation in our data analysis revealed that effective use of these 15 STR loci in forensic cases needed to be localized by collection and analysis of population data from the general population. 257 260 Seyed Farzad Hosseini Department of Forensic Medicine, Khuzestan Legal Medicine Organization Department of Forensic Medicine, Khuzestan Legal Medicine Organization Iran Mahdi Bijanzadeh Department of Medical Genetics, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences Department of Medical Genetics, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences Iran Elham Modheji Department of Forensic Medicine, Khuzestan Legal Medicine Organization Department of Forensic Medicine, Khuzestan Legal Medicine Organization Iran DNA finger printingForensic sciencesGenotypePolymerase chain reaction 10344.pdf Butler JM, Buel E, Crivellente F, McCord BR. Forensic DNA typing by capillary electrophoresis using the ABI Prism 310 and 3100 genetic analyzers for STR analysis. Electrophoresis 2004;25(10-11):1397-1412.##DuVall JA, Roux DL, Thompson BL, Birch C, Nelson DA, Li J. et al. Rapid multiplex DNA amplification on an inexpensive microdevice for human identification via short tandem repeat analysis. Anal Chim Acta 2017;980:41-49.##Brown SM, Hopkins MS, Mitchell SE, Senior M, Wang TY, Duncan R, et al. Multiple methods for the identification of polymorphic simple sequence repeats (SSRs) in sorghum [Sorghum bicolor (L.) Moench]. Theor Appl Genet 1996;93(1-2):190-198.##Jäger AC, Alvarez ML, Davis CP, Guzmán E, Han Y, Way L et al. Developmental validation of the MiSeq FGx Forensic genomics system for targeted next generation sequencing in forensic DNA casework and database laboratories. Forensic Sci Int Genet 2017;28:52-70.##Dobbs LJ, Madigan MN, Carter AB, Earls L. Use of FTA gene guard filter paper for the storage and transportation of tumor cells for molecular testing. Arch Pathol Lab Med 2002;126(1):56-63.##Kutanan W, Kitpipit T, Phetpeng S, Thanakiatkrai P. Forensic STR loci reveal common genetic ancestry of the Thai-Malay Muslims and Thai Buddhists in the deep Southern region of Thailand. J Hum Genet 2014;59(12):675-681.##Excoffier L, Laval G, Schneider S. Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 2005;1:47-50.##Tereba A. Tools for analysis of population statistics. Profiles DNA 1999;2:14-16.##Hildebrand EC, Torney DC, Wagner RP. Informativeness of polymorphic DNA markers. In: Cooper NG, editor. The human genome project: deciphering the blueprint of heredity. Calfornia: Palace Press; 1992. p. 99-102.##Hammond H A, Jin L, Zhong Y, Thomas Caskey C, Chakraborty R. Evaluation of 13 short tandem repeat loci for use in personal identification applications. Am J Hum Genet 1994;55(1):175-189.##Bosch E, Calafell F, Pérez-Lezaun A, Clarimón J, Comas D, Mateu E, et al. Genetic structure of north-west Africa revealed by STR analysis. Eur J Hum Genet 2000;8(5):360-366.##Hedjazi A, Nikbakht A, Hosseini M , Hoseinzadeh A, Hosseini SM. Allele frequencies for 15 autosomal STR loci in Fars province population, southwest of Iran. Leg Med (Tokyo) 2013;15(4):226-228.##Stanciu F, Stoian IM, Popescu OR. Population data for 15 short tandem repeat loci from Wallachia region, south Romania. Croat Med J 2009;50(3):321-325.##

A study on Association Between CCRΔ32 Mutation and HCV Infection in Iranian Patients 2 2 Background: Mutations in the coding region of the Chemokine Receptor 5 (CCR5) genes reduce or eliminate CCR5 expression in immune cells and progression of HCV infection. This study aimed to investigate the role of this mutation in HCV infection in Iranian patients in comparison with healthy individuals. Methods: 100 HCV infected patients and 100 healthy individuals were randomly selected. The CCR5Δ32 genotypes were determined using specific primers and PCR method. Results: The agarose gel electrophoresis showed a189-bp fragment from wild type for both alleles of CCR5 gene. The CCR5-Δ32 allele was not found in any HCV infected and healthy subjects. Conclusion: The mutation in CCR5 gene was not detected in any of the two groups; therefore, the role of CCR5 gene expression in immune cells and progression of HCV infection needs to be studied in larger samples in our country. 261 264 Farahnaz Bineshian Department of Parasitology & Mycology, Faculty of Medicine, Semnan University of Medical Sciences Department of Parasitology & Mycology, Faculty of Medicine, Semnan University of Medical Sciences Iran Asieh Hosseini Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Iran Zohreh Sharifi Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Iran Afsaneh Aghaie Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Iran CCR5 proteinHepatitic CHumanInfectionMutation 10334.pdf Sharma SD. Hepatitis C virus: molecular biology & current therapeutic options. Indian J Med Res 2010;131:17-34.##Larrubia JR, Benito-Martínez S, Calvino M, Sanz-de-Villalobos E, Parra-Cid T. Role of chemokines and their receptors in viral persistence and liver damage during chronic hepatitis C virus infection. World J Gastroenterol 2008;14(47):7149-7159.##[No authors listed]. Hepatitis C--global prevalence (update). Wkly Epidemiol Rec 1999;74(49):425-427.##Mohd Hanafiah K, Groeger J, Flaxman AD, Wiersma ST. Global epidemiology of hepatitis C virus infection: new estimates of age specific antibody to HCV seroprevalence. Hepatology 2013;57(4):1333-1342.##Petruzziello A, Marigliano S, Loquercio G, Cozzolino A, Cacciapuoti C. Global epidemiology of hepatitis C virus infection: an up-date of the distribution and circulation of hepatitis C virus genotypes. World J Gastroenterol 2016;22(34):7824-7840.##Daw MA, El-Bouzedi AA, Ahmed MO, DauAA, Agnan MM, Drah AM. Geographic integration of hepatitis C virus: A global threat. Word J Virol 2016;5(4):170-182.##Alavian SM, Adibi P, Zali MR. Hepatitis C virus in Iran: epidemiology of an emerging infection. Arch Iranian Med 2005;8:84-90.##Ahlenstiel G, Woitas RP, Rockstroh J, Spengler U. CC-chemokine receptor 5 (CCR5) in hepatitis C--at the crossroads of the antiviral immune response. J Antimicrob Chemother 2004;53(6):895-898.##Konishi I, Horiike N, Hiasa Y, Michitaka K, Onji M. CCR5 promoter polymorphism influences the interferon response of patients with chronic hepatitis C in Japan. Intervirology 2004;47(2):114-120.##Coenen M, Nattermann J. The role of CCR5 in HCV infection. Eur J Med Res 2010;15(3):97-101.##Wierda RJ, van den Elsen PJ. Genetic and epigenetic regulation of CCR5 transcription. Biology (Basel) 2012;1(3):869-879.##Ahlenstiel G, Berg T, Woitas RP, Grünhage F, Iwan A, Hess L, et al. Effects of the CCR5-Δ32 mutation on antiviral treatment in chronic hepatitis C. J Hepatol 2003;39(2):245-252.##Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia growth and development study, multicenter AIDS cohort study, multicenter hemophilia cohort study, San Francisco city cohort, ALIVE study. Science 1996;273(5283):1856-1862.##Goulding C, Murphy A, MacDonald G, Barrett S, Crowe J, Hegarty J, et al. The CCR5Δ32 mutation: impact on disease outcome in individuals with hepatitis C infection from a single source. Gut 2005;54(8):1157-1161.##Woitas RP, Ahlenstiel G, Iwan A, Rockstroh JK, Brackmann HH, kupfer B, et al. Frequency of the HIV-protective CC chemokine receptor 5-Delta32/Delta32 genotype is increased in hepatitis C. Gastroenterology 2002;122(7):1721-1728.##Hosseini A, Sharifi Z, Baghbani-Arani F. Survey on the association of CCR5 promoter -59353T/C polymorphism with HCV infection. J Mazandaran Univ Med Sci 2014;24:45-53.##Jalali S, Sharifi Z, Sanati MH, Fazeli AS. Survey on CCR5-Δ32 mutation in healthy individuals and patients with chronic hepatitis B referred to the clinical laboratory of Iranian blood transfusion organization. Koomesh 2014;15(3):359-364.##Ahlenstiel G, Woitas RP, Iwan A, Nattermann J, Feldmann G, Rock¬stroh JK, et al. Effects of the CCR5-Delta32 mutation on hepatitis C virus-specific immune responses in patients with haemophilia. Immunol Invest 2009;38(3-4):284-296.##Morard I, Clément S, Calmy A, Mangia A, Cerny A, De Gottardi A, et al. Clinical significance of the CCR5delta32 allele in hepatitis C. Plos One 2014;9(9):e106424.##Khorram Khorshid HR, Manoochehri M, Nasehi L, Ohadi M, Rahgozar M, Kamali K. Ccr2-64i and Ccr5 Δ32 Polymorphisms in Patients with Late-Onset Alzheimer's disease; A study from Iran (Ccr2-64i And Ccr5 Δ32 Polymorphisms in Alzheimer's disease). Iran J Basic Med Sci 2012;15(4):937-944.##Abdel-Wahab K, Foda M, El-salakawy A, Abdel-Moneim M, El-Attar G, Harada Sh, et al. Possible association between the chemokine receptor gene CCR5-delta-32 mutation and hepatitis C virus pathogenesis. Egyptian J Hospital Med 2004;17:58-62.##El-Moamly AA, El-Sweify MA, Rashad RM, Abdalla EM, Ragheb MM, Awad MM. Role of CCR5D32 mutation in protecting patients with Schistosoma mansoni infection against hepatitis C viral infection or progression. Parasitol Res 2013;112(7):2745-2752.##Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature 1996;382(6593):722-725.##Arnaiz-Villena A, Karin M, Bendikuze N, Gomez-Casado E, Moscoso J, Silvera C, et al. HLA alleles and haplotypes in the Turkish population: relatedness to Kurds, Armenians and other Mediterraneans. Tissue Antigens 2001;57(4):308-317.##

A Study of Recombinant Factor IX in Drosophila Insect S2 Cell Lines Through Transient Gene Expression Technology 2 2 Background: Since the mass production of recombinant proteins requires the development of stable cell lines which is a time-consuming complex process, the use of transient expression on a large scale can be a comparatively useful alternative. Although various cell lines have been used for the expression of recombinant proteins, only a limited number of cells enjoy a high transfection characteristic and the ability to adapt to serum-free suspension culture easily. In the present study, the S2 cells from Drosophila insect with the ability to grow in suspension and serum-free cultures were used for the expression of factor IX (FIX) using Transient Gene Expression (TGE) technique. Methods: Drosophila Schneider (S2) cells were seeded in special roller bottles, and then, the cells were transfected with pMT-hFIX plasmid employing the calcium phosphate co-precipitation method. The stable S2-hFIX cells were also seeded in special roller bottles, separately. After the induction, recombinant FIX was quantified in conditioned media employing an ELISA. Moreover, its functional activity was examined using an aPTT assay. Results: The results showed that the expression of FIX through TGE technology was 1.6 times as high as that obtained through S2-FIX stable cells. Furthermore, the comparison of the FIX expression in S2 cells through TGE techniques with that obtained in previous studies in HEK cells or CHO cells revealed that S2 cells were more efficient in terms of FIX expression. Conclusion: The S2 cells with the capability to grow in suspension and serum-free cultures are a suitable alternative for transient expression for the large scale production of proteins. 265 268 Jafar Vatandoost Department of Biology, Hakim Sabzevari University Department of Biology, Hakim Sabzevari University Iran Kambiz Kafi Sani Department of Biotechnology, Sabzevar Branch, Islamic Azad University Department of Biotechnology, Sabzevar Branch, Islamic Azad University Iran Drosophila S2 cellFactor IXTransient gene expression 321.pdf Meissner P, Pick H, Kulangara A, Chatellard P, Friedrich K, Wurm FM. Transient gene expression: recombinant protein production with suspension‐adapted HEK293‐ EBNA cells. Biotechnol Bioeng 2001;75(2):197-203.##Geisse S, Henke M. Large-scale transient transfection of mammalian cells: a newly emerging attractive option for recombinant protein production. J Struct Funct Genomics 2005;6(2-3):165-170.##Shi C, Shin YO, Hanson J, Cass B, Loewen MC, Durocher Y. Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells. Biochemistry 2005;44(48):15705-15714.##Graham F, Smiley J, Russell W, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977;36(1):59-74.##de Jongh WA, Salgueiro S, Dyring C. The use of drosophila S2 cells in R&d and bioprocessing. Pharm Bioprocess 2013;1(2):197-213.##Bernard AR, Kost TA, Overton L, Cavegn C, Young J, Bertrand M, et al. Recombinant protein expression in a Drosophila cell line: comparison with the baculovirus system. Cytotechnology 1994;15(1-3):139-144.##Cherbas L, Cherbas P. Transformation of Drosophila cell lines: an alternative approach to exogenous protein expression. Baculovirus and Insect Cell Expression Protocols 2007:317-340.##Vatandoost J, Bos MH. Efficient expression of functional human coagulation factor IX in stably-transfected Drosophila melanogaster S2 cells; comparison with the mammalian CHO system. Biotechnol Lett 2016;38(10):1691-1698.##Moraes AM, Jorge SA, Astray RM, Suazo CA, Calderón Riquelme CE, Augusto EF, et al. Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development. Biotechnol Adv 2012;30(3):613-628.##Bandyopadhyay PK, Clark K, Stevenson BJ, Rivier JE, Olivera BM, Golic KG, et al. Biochemical characterization of Drosophila gamma‐glutamyl carboxylase and its role in fly development. Insect Mol Biol 2006;15(2):147-156.##Vatandoost J, Zomorodipour A, Sadeghizadeh M, Aliyari R, Bos MH, Ataei F. Expression of biologically active human clotting factor IX in Drosophila S2 cells: γ-carboxylation of a human vitamin K-dependent protein by the insect enzyme. Biotechnol Prog 2012;28(1):45-51.##Baldi L, Muller N, Picasso S, Jacquet R, Girard P, Thanh HP, et al. Transient gene expression in suspension HEK‐293 cells: application to large‐scale protein production. Biotechnol Prog 2005;21(1):148-153.##Davami F, Eghbalpour F, Barkhordari F, Mahboudi F. Effect of peptone feeding on transient gene expression process in CHO DG44. Avicenna J Med Biotech 2014;6(3):147-155.##Girard P, Derouazi M, Baumgartner G, Bourgeois M, Jordan M, Jacko B, et al. 100-liter transient transfection. Cytotechnology 2002;38(1-3):15-21.##Derouazi M, Girard P, Van Tilborgh F, Iglesias K, Muller N, Bertschinger M, et al. Serum‐free large‐scale transient transfection of CHO cells. Biotechnol Bioeng 2004;87(4):537-545.##Azimifar M, Vatandoost J. Large-scale production of recombinant FIX in stable HEK-FIX cells: Islamic Azad University-Sabzevar Branch; 2016. http://theses.iaus.ac.ir/Abstract/thesis-3837.html##Baghani A, Vatandoost J. Comparison of recombinant FIX expression in stable CHO-FIX cells in small and large-scale: Islamic Azad University-Sabzevar Branch; 2016. http://theses.iaus.ac.ir/Abstract/thesis-3835.html##Schlaeger EJ, Christensen K. Transient gene expression in mammalian cells grown in serum-free suspension culture. Cytotechnology 1999;30(1-3):71-83.##

Rapid and Simple Detection of Escherichia coli by Loop-Mediated Isothermal Amplification Assay in Urine Specimens 2 2 Background: To improve urinary tract infection detection, we evaluated the specificity and sensitivity of Loop-mediated isothermal Amplification Method (LAMP) for detection of the Eschericia coli (E. coli) in urine samples, for the first time.  Methods: Primers were designed to target the malB gene of Escherichia coli. LAMP assay was performed on urine specimens collected from patients with urinary tract infection symptoms. Results: As expected, LAMP was more specific and sensitive than direct microscopic tests. LAMP assay showed the best detection limit of DNA copies with 1.02 copies. Conclusion: LAMP method offers several advantages in terms of sensitivity, rapidness and simplicity for detection of E. coli infection in urine samples. The LAMP method would be highly suitable for the early detection of the UTIs and also comfort quick diagnosis of UTI in clinical laboratories with limited equipment. 269 272 Reihaneh Ramezani Department of Biomedical sciences, Women Research Center, Alzahra University Department of Biomedical sciences, Women Research Center, Alzahra University Iran Zahra Kardoost Parizi Department of Biotechnology, Faculty of Biological Sciences, Alzahra University Department of Biotechnology, Faculty of Biological Sciences, Alzahra University Iran Nassim Ghorbanmehr Department of Biotechnology, Faculty of Biological Sciences, Alzahra University Department of Biotechnology, Faculty of Biological Sciences, Alzahra University Iran Hamideh Mirshafiee Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University Iran Escherichia coliIsothermal amplificationLAMPUrinary tract infection 333.pdf Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28(12):E63. ##Gill P, Ramezani R, Amiri MV, Ghaemi A, Hashempour T, Eshraghi N, Ghalami M, et al. Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis. Biochem Biophys Res Commun 2006;347(4):1151-1157. ##Hill J, BeriwalSh, Chandra I, Paul VK, Paul VK, Kapil A, Singh T, et al. Loop-mediated isothermal amplification assay for rapid detection of common strainsof Escherichia coli. J Clin Microbiol 2008;46(8):2800-2804. ##Minogue T, Daligault HA, Davenport KW, Bishop-Lilly KA, Broomall SM, Bruce DC, et al. Complete genome assembly of Escherichia coli ATCC 25922, a Serotype O6 reference strain. Genome Announc 2014;2(5):e00969-14.##Khleifat KhM, Abboud MM , Omar ShS, Al-Kurishy JH. Urinary tract infection in South Jordanian population. J Med Sci 2006;6(1):5-11. ##Chander Y, Koelbl J, Puckett J, Moser MJ, Klingele AJ, Liles MR, et al. A novel thermostable polymerase for RNA and DNA loop-mediated isothermal amplification (LAMP). Front Microbiol 2014;1;5:395. ##Ushikubo H. [Principle of LAMP method-a simple and rapid gene amplification method]. Uirus 2004;54(1):107-112. Japanese##Wang F, Jiang L, Ge B. Loop-mediated isothermal amplification assays for detecting shiga toxin-producing Escherichia coli in ground beef and human stools. J Clin Microbiol 2012; 50(1):91-97. ##Yokoyama E, Uchimura M, Ito K. Detection of enteroaggregative Escherichia coli by loop-mediated isothermal amplification. J Food Prot 2010;73(6):1064-1072. ##Song T, Toma C, Nakasone N, Iwanaga M. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol Lett 2005;243(1):259-263. ##Yano A, Ishimaru R, Hujikata R. Rapid and sensitive detection of heat-labile I and heat-stable I enterotoxin genes of enterotoxigenic Escherichia coli by Loop-Mediated Isothermal Amplification. J Microbiol Methods 2007;68(2):414-420. ##

KIF21A Gene c.2860C>T Mutation in CFEOM1A: The First Report from Iran 2 2 Congenital Fibrosis of the Extra Ocular Muscles1 (CFEOM1) is an autosomal dominant condition, caused by mutation in the KIF21A and TUBB3. It is characterized by congenital non-progressive restrictive ophthalmoplegia and ptosis. Mutational analysis of the known genes in such rare diseases by Sanger sequencing not only prevents wasting the time and expenses but also speeds diagnosis process, genetic counseling, and the possibility of prenatal diagnosis. Here, for the first time, association of pathogenic variant c.2860C>T in KIF21A gene in an Iranian family with positive history of CFEOM1A was reported. 273 276 Masoomeh Ramahi Department of Biology, Sabzevar branch, Islamic Azad University Department of Biology, Sabzevar branch, Islamic Azad University Iran Abolfazl Rad Cellular and Molecular Research Center, Sabzevar University of Medical Sciences Cellular and Molecular Research Center, Sabzevar University of Medical Sciences Iran Ebrahim Shirzadeh Department of Ophthalmology, Sabzevar University of Medical Sciences Department of Ophthalmology, Sabzevar University of Medical Sciences Iran Maryam Najafi Genome Research Division, Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 KL Genome Research Division, Department of Human Genetics, Radboud University Medical Center, Geert Grooteplein Zuid 10, 6525 KL Netherlands Fibrosis of extra ocular musclesIranMutationPrenatal diagnosis 10357.pdf Engle EC, Goumnerov BC, McKeown CA, Schatz M, Johns DR, Porter JD, et al. Oculomotor nerve and muscle abnormalities in congenital fibrosis of the extraocular muscles. Ann Neurol 1997;41(3):314-325.##Nakano M, Yamada K, Fain J, Sener EC, Selleck CJ, Awad AH, et al. Homozygous mutations in ARIX(PHOX2A) result in congenital fibrosis of the extraocular muscles type 2. Nat Genet 2001;29(3):315-320.##Tischfield MA, Baris HN, Wu C, Rudolph G, Van Maldergem L, He W, et al. Human TUBB3 mutations perturb microtubule dynamics, kinesin interactions, and axon guidance. Cell 2010;140(1):74-87.##Sener EC, Lee BA, Turgut B, Akarsu AN, Engle EC. A clinically variant fibrosis syndrome in a Turkish family maps to the CFEOM1 locus on chromosome 12. Arch Ophthalmol 2000;118(8):1090-1097.##Aubourg P, Krahn M, Bernard R, Nguyen K, Forzano O, Boccaccio I, et al. Assignment of a new congenital fibrosis of extraocular muscles type 3 (CFEOM3) locus, FEOM4, based on a balancedtranslocation t(2;13) (q37.3;q12.11) and identification of candidate genes. J M Genet 2005;42(3):253-259.##Tukel T, Uzumcu A, Gezer A, Kayserili H, Yuksel-Apak M, Uyguner O, et al. A new syndrome, congenital extraocular muscle fibrosiswith ulnar hand anomalies, maps to chromosome 21qter. J M Genet 2005;42(5):408-415.##Shinwari JM, Khan A, Awad S, Shinwari Z, Alaiya A, Alanazi M, et al. Recessive mutations in COL25A1 are a cause of congenital cranial dysinnervationdisorder. Am J Hum Genet 2015;96(1):147-152.##Yamada K, Andrews C, Chan WM, McKeown CA, Magli A, de Berardinis T, et al. Heterozygous mutations of the kinesin KIF21A in congenital fibrosis of the extraocular muscles type 1 (CFEOM1). Nat Genet 2003;35(4):318-321.##Whitman M, Hunter DG, Engle E. Congenital fibrosis of the extraocular muscles (CFEOM). Washington, Seattle; 1993-2018. 2004 Apr 27 [updated 2016 Jan 14].##Lu S, Zhao C, Zhao K, Li N, Larsson C. Novel and recurrent KIF21A mutations in congenital fibrosis of the extraocular musclestype 1 and 3. Arch Ophthalmol 2008;126(3):388-394.##Kaçar Bayram A, Per H, Quon J, Canpolat M, Ülgen E, Doğan H, et al. A rare case of congenital fibrosis of extraocular muscle type 1A due to KIF21A mutation with Marcus Gunnjaw-winking phenomenon. Eur J Paediatr Neurol 2015;19(6):743-746.##Luk HM, Lo IF, Lai CW, Ma LC, Tong TM, Chan DH, et al. Congenital fibrosis of extraocular muscle type 1A due to KIF21A mutation: first case report from Hong Kong. Hong Kong Med J 2013;19(2):182-185.##Tiab L, d'Allèves Manzi V, Borruat FX, Munier F, Schorderet D. Mutation analysis of KIF21A in congenital fibrosis of the extraocular muscles (CFEOM) patients. Ophthalmic Genet 2004;25(4):241-246.##Chen H, Liu T, Zeng Z, Wang Y, Lin Y, Cheng L, et al. Clinical characteristics of a KIF21A mutation in a Chinese family with congenital fibrosis of the extraocular muscles type 1. Medicine (Baltimore) 2017;96(38):e8068.##Wang P, Li S, Xiao X, Guo X, Zhang Q. KIF21A novel deletion and recurrent mutation in patients with congenital fibrosis of the extraocular muscles-1. Int J Mol Med 2011;28(6):973-975.##Chan WM, Andrews C, Dragan L, Fredrick D, Armstrong L, Lyons C, et al. Three novel mutations in KIF21A highlight theimportance of the third coiled-coil stalk domain in the etiology of CFEOM1. BMC Genet 2007;8:26.##

Letter to: Arylamine N-acetyltransferase 2 Polymorphisms and the Risk of Endometriosis 2 2 No Abstract 277 278 Fabio Barra Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Italy Lorenzo Ferro Desideri Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Italy Carolina Scala Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10 Italy Simone Ferrero Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10, 16121 Academic Unit of Obstetrics and Gynaecology IRCCS Ospedale Policlinico San Martino, University of Genoa Largo Rosanna Benzi 10, 16121 Italy Editorial 10386.pdf Fayez D, Saliminejad K, Irani S, Kamali K, Memariani T, Khorram Khorshid HR. Arylamine N-acetyltrans-ferase 2 Polymorphisms and the Risk of Endometriosis. Avicenna J Med Biotechnol 2018;10(3):163-167.##Hein DW, Doll MA, Fretland AJ, Leff MA, Webb SJ, Xiao GH, et al. Molecular genetics and epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol Biomarkers Prev 2000;9(1):29-42.##Augoulea A, Alexandrou A, Creatsa M, Vrachnis N, Lambrinoudaki I. Pathogenesis of endometriosis: the role of genetics, inflammation and oxidative stress. Arch Gynecol Obstet 2012;286(1):99-103.##Deguchi M, Yoshida S, Kennedy S, Ohara N, Motoyama S, Maruo T. Lack of association between endometriosis and N-acetyl transferase 1 (NAT1) and 2 (NAT2) polymorphisms in a Japanese population. J Soc Gynecol Investig 2005;12(3):208-213.##Babu KA, Rao KL, Reddy NG, Kanakavalli MK, Zondervan KT, Deenadayal M, et al. N-acetyl transferase 2 polymorphism and advanced stages of endometriosis in South Indian women. Reprod Biomed Online 2004;9(5):533-540.##Nakago S, Hadfield RM, Zondervan KT, Mardon H, Manek S, Weeks DE, et al. Association between endo-metriosis and N-acetyl transferase 2 polymorphisms in a UK population. Mol Hum Reprod 2001;7(11):1079-1083.##Vercellini P, Vigano P, Somigliana E, Fedele L. Endo-metriosis: pathogenesis and treatment. Nat Rev Endo-crinol 2014;10(5):261-275.##Ferrero S, Alessandri F, Racca A, Leone Roberti Mag-giore U. Treatment of pain associated with deep endo- metriosis: alternatives and evidence. Fertil Steril 2015;104(4):771-792.##Barra F, Scala C, Mais V, Guerriero S, Ferrero S. Inves-tigational drugs for the treatment of endometriosis, an update on recent developments. Expert Opin Investig Drugs 2018;27(5):445-458.##Ferrero S, Evangelisti G, Barra F. Current and emerging treatment options for endometriosis. Expert Opin Pharmacother 2018;19(10):1109-1125.##