New Hopes for Treatment of Alzheimer’s Disease 2 2 Alzheimer’s Disease (AD), the leading cause of dementia worldwide, is an irreversible progressive neurodegenerative disorder characterized by cognitive impairment and functional disability 1,2. Devastating nature of AD leads to serious social and economic impacts on the healthcare systems which implies the necessity of its proper management. It has been demonstrated that patients’ quality of life and their overall prognosis has a significant negative correlation with the severity of AD. Patients with severe AD need full-time care and assistance with some basic activities of daily living such as feeding and dressing in addition to severe deterioration in various domains of their cognitive functioning. Moreover, behavioral aberrancy and neuropsychiatric symptoms such as depression, apathy, psychosis, agitation, and aggression are observed more frequently in moderate to severe AD. Despite such an enormous burden, most practical guidelines focus on mild to moderate stages of the illness and there is still a serious lack of evidence regarding the management of severe AD. Among currently FDA-approved drugs, very few medications have shown to be effective in attenuating some of the AD-related symptoms in severe stages. Memantine, an N-methyl-D-aspartate (NMDA) receptor antagonist, and donepezil, an acetylcholinesterase inhibitor (ACEI) are the most widely accepted agents in this regard. Unfavorable side effects of these agents along with lack of optimal efficacy have led to many researches trying to find novel pharmacologic strategies for AD based on its underlying pathophysiological defects 3. More than 400 clinical trials are currently looking at new treatments for AD and many of them are actively recruiting. Many of these studies are based on decreasing the harmful effects of a toxic protein called amyloid-beta in the brain, but others reflect a broadening range of possible treatment approaches based on other theories about AD. For example, the importance of inflammation in exacerbation of amyloid-beta’s neuron-destroying effects has led to trials of medications with anti-inflammatory properties 4. Serotonin neurotransmission failure is a demonstrated aspect of AD, and several experimental medications attempt to correct that problem. RVT-101 and LuAE58054 are two examples of medications that are in clinical trials. Altering the brain’s serotonin activity seems to help cognitive difficulties in schizophrenia, and may also prove helpful in cognitive difficulties associated with AD 5. In conclusion, a number of biotechnologists are working on effective drugs for treatment of AD and in particular severe type. 1 1 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Iran Editorial 296.pdf Bhardwaj D, Mitra C, Narasimhulu CA, Riad A, Doomra M, Parthasarathy S. Alzheimer's disease-current status and future directions. J Med Food 2017. [Epub ahead of print].##Akhondzadeh S. Hippocampal synaptic plasticity and cognition. J Clin Pharm Ther 1999;24(4):241-248.##Farokhnia M, Shafiee Sabet M, Iranpour N, Gougol A, Yekehtaz H, Alimardani R, et al. Comparing the efficacy and safety of Crocus sativus L. with memantine in patients with moderate to severe Alzheimer's disease: a double-blind randomized clinical trial. Hum Psychopharmacol 2014;29(4):351-359.##Chitnis T, Weiner HL. CNS inflammation and neurodegeneration. J Clin Invest 2017;127(10):3577-3587.##Akhondzadeh S. The 5-HT hypothesis of schizophrenia. IDrugs 2001;4(3):295-300.##

Evaluation of Immune Responses Induced by GRA7 and ROP2 Genes by DNA Vaccine Cocktails Against Acute Toxoplasmosis in BALB/c Mice 2 2 Background: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, GRA7 plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice. Methods: In this study, 733 bp of GRA7 gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containing GRA7 and ROP2 genes were administered via IM according to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis. Results: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-γ levels were significantly higher than controls (p<0.05), whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group (p<0.01). In contrast, IgG1 antibodies were similar between the two groups (p>0.5). So, survival time in the immune groups was significantly prolonged in comparison to control ones (p<0.01). Conclusion: The immunized mice by DNA vaccine produce higher titration of IFNγ, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktail GRA7/ROP2 is a potential vaccine candidate against toxoplasmosis. 2 8 Hossein Vazini Department of Nursing, Faculty of Basic Sciences, Hamedan Branch, Islamic Azad University Department of Nursing, Faculty of Basic Sciences, Hamedan Branch, Islamic Azad University Iran Fatemeh Ghafarifar Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University 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 Abdolhossein Dalimi Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University Iran BALB/cDNA vaccineGRA7ROP2Toxoplasma gondii 297.pdf Halonen SK, Weiss LM. Toxoplasmosis. Handb Clin Neurol 2013;114:125-145.##Robert-Gangneux F, Dardé ML. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 2012;25(2):264-296.##Dubey JP. Transmission of toxoplasma gondii-From land to sea, a personal perspective. In: Janovy J, Esch GW, editors. A century of parasitology: Discoveries, ideas and lessons learned by scientists who published in the journal of parasitology. US: John Wiley & Sons, Inc; 2016. 162-191.##Lopes FMR, Gonçalves DD, Mitsuka-Bregano R, Freire RL, Navarro IT. Toxoplasma gondii infection in pregnancy. Braz J Infect Dis 2007;11(5):496-506.##Torgerson PR, de Silva NR, Fèvre EM, Kasuga F, Rokni MB, Zhou XN, et al. The global burden of foodborne parasitic diseases: an update. Trends Parasitol 2014;30(1):20-26.##Boothroyd JC. Toxoplasma gondii: 25 years and 25 major advances for the field. Int J Parasitol 2009;39(8):935-946.##Kur J, Holec-Gasior L, Hiszczyńska-Sawicka E. Current status of toxoplasmosis vaccine development. Expert Rev Vaccines 2009;8(6):791-808.##Jongert E, Melkebeek V, De Craeye S, Dewit J, Verhelst D, Cox E. An enhanced GRA1-GRA7 cocktail DNA vaccine primes anti-Toxoplasma immune responses in pigs. Vaccine 2008;26(8):1025-1031.##Aliberti J. Host persistence: exploitation of anti-inflammatory pathways by Toxoplasma gondii. Nat Rev Immunol 2005;5(2):162-170.##Frölich S, Entzeroth R, Wallach M. Comparison of protective immune responses to apicomplexan parasites. J Parasitol Res. 2012;2012:852591.##Khosroshahi KH, Ghaffarifar F, Sharifi Z, D'Souza S, Dalimi A, Hassan ZM, et al. Comparing the effect of IL-12 genetic adjuvant and alum non-genetic adjuvant on the efficiency of the cocktail DNA vaccine containing plasmids encoding SAG-1 and ROP-2 of Toxoplasma gondii. Parasitol Res 2012;111(1):403-411.##Hoseinian Khosroshahi K, Ghaffarifar F, D'Souza S, Sharifi Z, Dalimi A. Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis. Vaccine 2011;29(4):778-783.##Hosseinian Khosroshahi K, Ghaffarifar F, Sharifi Z, Dalimi A. Expression of complete rhoptry protein 2 (ROP2) gene of Toxoplasma gondii in eukaryotic cell. Afr J Biotechnol 2008;7(24):4432-4436.##Zhang NZ, Chen J, Wang M, Petersen E, Zhu XQ. Vaccines against Toxoplasma gondii: new developments and perspectives. Expert Rev Vaccines 2013;12(11):1287-1299.##Sturge CR, Yarovinsky F. Complex immune cell interplay in the gamma interferon response during Toxoplasma gondii infection. Infect Immun 2014;82(8):3090-3097.##Dimier-Poisson I, Aline F, Mévélec MN, Beauvillain C, Buzoni-Gatel D, Bout D. Protective mucosal Th2 immune response against Toxoplasma gondii by murine mesenteric lymph node dendritic cells. Infect Immun 2003;71(9):5254-5265.##Burrells A, Benavides J, Cantón G, Garcia JL, Bartley PM, Nath M, et al. Vaccination of pigs with the S48 strain of Toxoplasma gondii--safer meat for human consumption. Vet Res 2015;46:47.##Kutzler MA, Weiner DB. DNA vaccines: ready for prime time? Nat Rev Genet 2008;9(10):776-788.##Liu S, Wang S, Lu S. DNA immunization as a technology platform for monoclonal antibody induction. Emerg Microbes Infect 2016;5:e33.##Gong P, Cao L, Guo Y, Dong H, Yuan S, Yao X, et al. Toxoplasma gondii: Protective immunity induced by a DNA vaccine expressing GRA1 and MIC3 against toxoplasmosis in BALB/c mice. Exp Parasitol 2016;166:131-136.##Zhang NZ, Huang SY, Xu Y, Chen J, Wang JL, Tian WP, et al. Evaluation of immune responses in mice after DNA immunization with putative Toxoplasma gondii calcium-dependent protein kinase 5. Clin Vaccine Immunol 2014;21(7):924-929.##Naserifar R, Ghaffarifar F, Dalimi A, Sharifi Z, Solhjoo K, Hosseinian Khosroshahi K. Evaluation of immunogenicity of Cocktail DNA vaccine containing plasmids encoding complete GRA5, SAG1, and ROP2 antigens of toxoplasma gondii in BALB/C mice. Iran J Parasitol 2015;10(4):590-598.##Ghaffarifar F, Naserifar R, Jafari Madrak M. Eukaryotic plasmids with toxoplasma gondii dense granule antigen (GRA 5) and microneme 3 (MIC3) genes as a cocktail DNA vaccine and evaluation of immune responses in BALB/C mice. J Clin Med Genom 2014;3:121.##Ghaffarifar F. Strategies of DNA vaccines against toxoplasmosis. Rev Med Microbiol 2015;26(3):88-90.##

Novel Recombinant Traceable c-Met Antagonist-Avimer Antibody Mimetic Obtained by Bacterial Expression Analysis 2 2 Background: Avimers are originally types of artificial proteins with multiple binding sites for specific binding to certain antigens. Various radioisotopes and nanoparticles link these molecules, which are widely used in early detection in tissue imaging, treatment and study on carcinogenesis. Among these, c-Met antagonist avimer (C426 avimer), with ability to bind the c-Met receptor of tyrosine kinase (RTK) is an attractive candidate for targeted cancer therapy. In this study, a novel traceable C426 avimer gene was designed and introduced by adding the 12nt tracer binding site encoded four specific amino acid residues at the C-terminal region of C426 avimer coding sequence. Methods: The 282 bp DNA sequence encoded 94aa avimer protein was synthesized and sub-cloned into prokaryotic pET26b expression vector. The expression of the mature peptide encoding the traceable avimer molecule was carried out in Escherichia coli strain BL21 using IPTG (Isopropyl β-D-1-thiogalactopyranoside) induction process. The expression level of the 11 kDa traceable avimer was  studied by SDS-PAGE, western blot and ELISA analysis. Results: Docking analysis of C426 avimer protein and its ligand c-Met showed that the traceability related changes happened at the best conformation and optimal energy. The SDS-PAGE, western blotting and ELISA analysis results demonstrated that the expression of the 11 kDa C426 avimer molecule was detectable without any degradation compared with the control group. Conclusion: Concerning the consequences of this work, this new approach can be widely used in the medical field and provide an opportunity to evaluate the affinity and traceability features. 9 14 Bahram Baghban Kohnehrouz Department of Plant Breeding & Biotechnology, University of Tabriz Department of Plant Breeding & Biotechnology, University of Tabriz Iran Afsaneh Talischian Department of Biological Sciences, Higher Education Institute of Rab-Rashid Department of Biological Sciences, Higher Education Institute of Rab-Rashid Iran Alireza Dehnad Department of Biotechnology, East Azerbaijan Research and Education Center Agricultural and Natural Resources, AREEO, Tabriz; Higher Education Institute of Rab-Rashid Department of Biotechnology, East Azerbaijan Research and Education Center Agricultural and Natural Resources, AREEO, Tabriz; Higher Education Institute of Rab-Rashid Iran Shahnoush Nayeri Department of Biotechnology, Shahid Beheshti University Department of Biotechnology, Shahid Beheshti University Iran Antibody avidityE. coli strain BL21Enzyme linked immunosorbent assayMolecular docking analysis 298.pdf Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, et al. Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 2005;23(12):1556-1561.##Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man, Multiple functions of the large family members via interaction with complex ligands. Biol Chem 1998;379(8-9):951-964.##Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu Rev Biochem 1994;63:601-637.##Mammen M, Choi SK, Whitesides GM. Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem 1998;37:2755-2794.##Jeong KJ, Mabry R, Georgiou G. Avimers hold their own. Nat Biotechnol 2005;23(12):1493-1494.##Rothe A, Hosse RJ, Power BE. In vitro display technologies reveal novel biopharmaceutics. FASEB J 2006;20(10):1599-1610.##North CL, Blacklow SC. Structural independence of ligand-binding modules five and six of the LDL receptor. Biochemistry 1999;38:3926-3935.##Cho S, Smith R, Duguay A, Li P, Bakker A, Alba B, et al. c-Met antagonist Avimer™ polypeptide that exhibits potent in vivo and in vitro inhibitor activity. Mol Cancer Ther 2009;8(12):B233.##Ahlgren S, Wållberg H, Tran TA, Widström C, Hjertman M, Abrahmsén L, et al. Targeting of HER2-expressing tumors with a site-specifically 99mTc-labeled recombinant affibody molecule, ZHER2:2395, with C-terminally engineered cysteine. J Nucl Med 2009;50(5):781-789.##Gebauer M, Skerra A. Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 2009;13(3):245-255.##Binz HK, Plückthun A. Engineered proteins as specific binding reagents. Curr Opin Biotechnol 2005;16(4):459-469.##Montvale NJ. Physicians' Desk Reference. 59th ed. USA: Medical Economics Co; 2005. p. 1125-1130.##

RNA Loading on Nano-Structured Hyperbranched β-Cyclodextrin 2 2 Background: β-Cyclodextrin functionalized hyper-branched polyglycerol (HBCD: β-CD-g-PG), a biocompatible polymer, has recently been proposed for delivery of poorly water soluble compounds. Methods: The present study examines the interaction of HBCD with RNA, utilizing a constant concentration of RNA and different HBCD/RNA ratios of 1/16 to 1/1, at physiological condition in an aqueous solution. Circular Dichroism (CD), UV-visible, FTIR spectroscopic methods, zeta potential and Dynamic Light Scattering (DLS) were used to analyze the particle formation, particle charge, particle size, aggregation, RNA conformation, binding constant and mode, and the effect of polymer complexation on RNA stability. Results: The results indicate that the interaction of RNA with HBCD leads to the formation of a linear dendritic supramolecule biopolymer with an overall binding constant of KHBCD/RNA= 1.25 × 103.  Conclusion: The small sized synthesized polymer can be considered as an appropriate system for preventing RNA aggregation and protecting the gene by host-guest interaction. 15 21 Sorina Hirbod Department of Chemistry, Central Tehran Branch, Islamic Azad University Department of Chemistry, Central Tehran Branch, Islamic Azad University Iran Shohreh Nafisi Department of Chemistry, Central Tehran Branch, Islamic Azad UniversityDepartment of Dermatology, University of California Department of Chemistry, Central Tehran Branch, Islamic Azad UniversityDepartment of Dermatology, University of California IranUSA Howard I Maibach Department of Dermatology, University of California Department of Dermatology, University of California USA AggregationGene deliveryParticle size Polymer 301.pdf Wang R, Zhou L, Zhou G, Li G, Zhu B, Gu H, et al. Synthesis and gene delivery of poly(amido amine)s with different branched architecture. Biomacromolecules 2010;11(2):489-495.##Fant K, Esbjörner EK, Jenkins A, Grossel MC, Lincoln P, Nordén B. Effects of PEGylation and acetylation of PAMAM dendrimers on DNA binding, cytotoxicity and in vitro transfection efficiency. Mol Pharm 2010;7(5):1743-1746.##Yuan Q, Yeudall WA, Yang H. PEGylated poly-amidoamine dendrimers with bis-aryl hydrazone linkages for enhanced gene delivery. Biomacromolecules 2010;11(8):1940-1947.##Klajnert B, Bryszewska M. Dendrimers as delivery systems in gene therapy. In: Klajnert B, Bryszewska M, editors. New developments in mutation research. New York: Nova Science Publishers; 2007. p. 217-240.##Templeton NS, Lasic DD. Gene therapy: Therapeutic mechanisms and strategies. 2nd ed. New York: Dekker; 2004. 584 p.##Yao H, Ng SS, Tucker WO, Tsang YK, Man K, Wang XM, et al. The gene transfection efficiency of a folate-PEI600-cyclodextrinnanopolymer. Biomaterials 2009;30(29):5793-5803.##Choi SH, Geckeler KE. Synthesis of a novel poly (oxyethylene)-bridged β-cyclodextrin dimer. J Incl Phenom Macrocycl Chem 2007;57(1-4):257-260.##Harada A, Kawaguchi Y, Hoshino T. Supramolecular polymers formed by modified cyclodextrins. J Incl Phenom Macrocycl Chem 2001;41(1-4):115-121.##Agüeros M, Areses P, Campanero MA, Salman H, Quincoces G, Peñuelas I, et al. Bioadhesive properties and biodistribution of cyclodextrin-poly(anhydride) nanoparticles. Eur J Pharm Sci 2009;37(3-4):231-240.##Liu YY, Fan XD, Hu H, Tang ZH. Release of chlorambucil from poly(N-isopropylacrylamide) hydrogels with beta-cyclodextrin moieties. Macromol Biosci 2004;4(8):729-736.##Asanuma H, Hishiya T, Komiyama M. Tailor-made receptors by molecular imprinting. Adv Mater 2000;12 (14):1019-1030.##Bilensoy E, Gürkaynak O, Doğan AL, Hincal AA. Safety and efficacy of amphiphilic beta-cyclodextrin nanoparticles for paclitaxel delivery. Int J Pharm 2008;347(1-2):163-170.##Lee SC, Huh KM, Lee J, Cho YW, Galinsky RE, Park K. Hydrotropic polymeric micelles for enhanced paclitaxel solubility: in vitro and in vivo characterization. Biomacromolecules 2007;8(1):202-208.##Avadi MR, Sadeghi AM, Mohammadpour N, Abedin S, Atyabi F, Dinarvand R, et al. Preparation and characterization of insulin nanoparticles using chitosan and arabic gum with ionic gelation method. Nanomedicine 2010;6(1):58-63.##Gupta U, Agashe HB, Asthana A, Jain NK. Dendrimers: novel polymeric nanoarchitectures for solubility enhancement. Biomacromolecules 2006;7(3):649-658.##Sharma US, Balasubramanian SV, Straubinger RM. Pharmaceutical and physical properties of paclitaxel (Taxol) complexes with cyclodextrins. J Pharm Sci 1995;84(10):1223-1230.##Li J, Chen B, Wang X, Goh S. H. Preparation and characterization of inclusion complexes formed by biodegradable poly(ε-caprolactone)-poly(tetrahydrofur an)-poly(ε-caprolactone)triblock copolymer and cyclodex-trins. Polymer 2004;45(6):1777-1787.##Wang JH, Cai Z. Incorporation of the antibacterial agent, miconazole nitrate into a cellulosic fabric grafted with β-cyclodextrin. Carbohydr Polym 2008;72(4):695-700.##Connors KA. The stability of cyclodextrin complexes in solution. Chem Rev 1997;97(5):1325-1358.##Zarrabi A, Adeli M, Vossoughi M, Shokrgozar MA. Design and synthesis of novel polyglycerol hybrid nanomaterials for potential applications in drug delivery systems. Macromol Biosci 2011;11(3):383-390.##Sunder A, Hanselmann R, Frey H, Mulhaupt R. Controlled synthesis of hyperbranched polyglycerols by ring-opening multibranching polymerization. Macromolecules 1999;32(13):4240-4246.##Sunder A, Mulhaupt R, Haag R, Frey H. Hyperbranched polyether polyols: a modular approach to complex polymer architectures. Adv Mater 2000;12(3):235-239.##Tao W, Liu Y, Jiang B, Yu S, Huang W, Zhou Y, et al. A linear-hyperbranched supramolecular amphiphile and its self-assembly into vesicles with great ductility. J Am Chem Soc 2012;134(2):762-764.##Zhang X, Zhang X, Wu Z, Gao X, Shu Sh, Wang Z, et al. β-Cyclodextrin grafting hyperbranchedpolyglycerols as carriers for nasal insulin delivery. Carbohydr Polym 2011;84(4):1419-1425.##Zhang X, Zhang X, Wu Z, Gao X, Cheng C, Wang Z, et al. A hydrotropic β-cyclodextrin grafted hyperbranched polyglycerol co-polymer for hydrophobic drug delivery. Acta Biomater 2011;7(2):585-592.##Yao H, Ng SS, Tucker WO, Tsang YK, Man K, Wang XM, et al. The gene transfection efficiency of a folate-PEI600-cyclodextrin nanopolymer. Biomaterials 2009;30(29):5793-5803.##Pun SH, Bellocq NC, Liu A, Jensen G, Machemer T, Quijano E, et alCyclodextrin-modified polyethylenimine polymers for gene delivery. Bioconjug Chem 2004;15(4):831-840.##Loftsson T, Duchêne D. Cyclodextrins and their pharmaceutical applications. Int J Pharm 2007;329(1-2):1-11.##Adeli M, Fard AK, Abedi F, Chegeni BK, Bani F. Thermo- and pH-sensitive dendrosomes as bi-phase drug delivery systems. Nanomedicine 2013;9(8):1203-1213.##Adeli M, Hakimpoor F, Parsamanesh M, Kalantari M, Sobhani Z, Attyabi F. Quantum dot-pseudopolyrotax-anesupramolecules as anticancer drug delivery systems. Polymer 2011;52(11):2401-2413.##Marmur J, Doty P. Thermal renaturation of deoxribo-nucleic acids. J Mol Biol 1961;3(5):585-594.##Reichmann ME, Rice SA, Thomas CA, Doty PJ. A further examination of the molecular weight and size of desoxypentose nucleic acid. J Am Chem Soc 1954;76(11):3047-3053.##Vijayalakshmi R, Kanthimathi M, Subramanian V, Nair BU. DNA cleavage by a Chromium(III) complex. Biochem Biophys Res Commun 2000;271(3):731-734.##Kanakis CD, Tarantilis PA, Polissiou MG, Diamantoglou S, Tajmir-Riahi HA. DNA interaction with naturally occurring antioxidant flavonoids quercetin, kaempferol, and delphinidin. J Biomol Struct Dyn 2005;22(6):719-724.##Kanakis CD, Tarantilis PA, Polissiou MG, Diamantoglou S, Tajmir-Riahi HA. An overview of DNA and RNA bindings to antioxidant flavonoids. Cell Biochem Biophys 2007;49(1):29-36.##Arakawa H, Ahmad R, Naoui M, Tajmir-Riahi HA. A comparative study of calf thymus DNA binding to Cr(III) and Cr(VI) ions. Evidence for the guanine N-7-chromium-phosphate chelate formation. J Biol Chem 2000;275(14):10150-10153.##Froehlich E, Mandeville JS, Kreplak L, Tajmir-Riahi HA. A. Aggregation and particle formation of tRNA by dendrimers. Biomacromolecules 2011;12(7):2780-2787.##Connors KA. Binding constants: The measurement of molecular complex stability. 1st ed. New York: Wiley & Sons; 1997. 432 p.##N'soukpoé-Kossi CN, Descôteaux C, Asselin E, Tajmir-Riahi HA, Bérubé G. DNA interaction with novel antitumor estradiol-platinum(II) hybrid molecule: a comparative study with cisplatin drug. DNA Cell Biol 2008;27(2):101-107.##Nafisi Sh, Shadaloi A, Feizbakhsh A, Tajmir-Riahi HA. RNA binding to antioxidant flavonoids. J. Photochem Photobiol B 2009;94(1):1-7.##Kypr J, Vorlícková M. 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Microwave-Assisted Hydro-Distillation of Essential Oil from Rosemary: Comparison with Traditional Distillation 2 2 Background: Hydro-distillation (HD) method is a traditional technique which is used in most industrial companies. Microwave-assisted Hydro-distillation (MAHD) is an advanced HD technique utilizing a microwave oven in the extraction process.  Methods: In this research, MAHD of essential oils from the aerial parts (leaves) of rosemary (Rosmarinus officinalis L.) was studied and the results were compared with those of the conventional HD in terms of extraction time, extraction efficiency, chemical composition, quality of the essential oils and cost of the operation.  Results: Microwave hydro-distillation was superior in terms of saving energy and extraction time (30 min, compared to 90 min in HD). Chromatography was used for quantity analysis of the essential oils composition. Quality of essential oil improved in MAHD method due to an increase of 17% in oxygenated compounds.  Conclusion: Consequently, microwave hydro-distillation can be used as a substitute of traditional hydro-distillation. 22 28 Sara Moradi Department of Chemical Engineering, Arak University Department of Chemical Engineering, Arak University Iran Alireza Fazlalia Department of Chemical Engineering, Arak University Department of Chemical Engineering, Arak University Iran Hamid Hamedi Department of Chemical Engineering, University of Science and Technology Department of Chemical Engineering, University of Science and Technology Iran Essential OilHydro distillationMicrowave distillationRosemary 300.pdf Ban L, Narasimhamoorthy B, Zhao L, Greaves JA, Schroeder WD. Antioxidant activities from different rosemary clonal lines. Food Chem 2016;201(15):259-263.##Yang Y, Song X, Sui X, Qi B, Wang Z, Li Y, et al. Rosemary extract can be used as a synthetic antioxidant to improve vegetable oil oxidative stability. Ind Crops Prod 2016;80:141-147.##Berdahl DR, McKeague J. Rosemary and sage extracts as antioxidants for food preservation. In: Shahidi F, editor. Handbook of antioxidants for food preservation. UK: Woodhead Publishing; 2015. p. 177-217.##Visentin A, Rodríguez-Rojo S, Navarrete A, Maestri D, Cocero MJ. Precipitation and encapsulation of rosemary antioxidants by supercritical antisolvent process. J Food Eng 2012;109(1):9-15.##Oluwatuyi M, Kaatz GW, Gibbons S. Antibacterial and resistance modifying activity of Rosmarinus officinalis. PhytoChemistry 2004;65(24):3249-3254.##Hernández MD, Sotomayor JA, Hernández A, Jordán MJ. Rosemary (Rosmarinus officinalis L.) Oils. In: Preedy VR, editor. Essential oils in food preservation, flavor and safety. UK: Academic Press; 2016. p. 677-688.##Zeng QH, Zhao JB, Wang JJ, Zhang XW, Jiang JG. Comparative extraction processes, volatile compounds analysis and antioxidant activities of essential oils from Cirsium japonicum Fisch. ex DC and Cirsium setosum (Willd.) M.Bieb. Food Sci Technol 2016;68:595-605.##Memarzadeh SM, Ghasemi Pirbalouti A, AdibNejad M. Chemical composition and yield of essential oils from Bakhtiari savory (Satureja bachtiarica Bunge.) under different extraction methods. Ind Crops Prod 2015;76(15):809-816.##Seidi Damyeh M, Niakousari M, Saharkhiz MJ. Ultrasound pretreatment impact on Prangos ferulacea Lindl. and Satureja macrosiphonia Bornm. essential oil extraction and comparing their physicochemical and biological properties. Ind Crops Prod 2016;87:105-115.##Benmoussa H, Farhat A, Romdhane M, Bouajil J. Enhanced solvent-free microwave extraction of Foeniculum vulgare Mill. essential oil seeds using double walled reactor. Arab J Chem vol. 2016. In Press.##Carvalho RN, Moura LS, Rosa PTV, Meireles MAA. Supercritical fluid extraction from rosemary (Rosmarinus officinalis): Kinetic data, extract’s global yield, composition, and antioxidant activity. J Supercrit Fluids 2005;35(3):197-204.##Vian MA, Fernandez X, Visinoni F, Chemat F. Microwave hydrodiffusion and gravity, a new technique for extraction of essential oils. J Chromatogr A 2008;1190(1-2):14-17.##Bousbia N, Vian MA, Ferhat MA, Petitcolas E, Melakati BY, Chemat F. Comparison of two isolation methods for essential oil from rosemary leaves: Hydrodistillation and microwave hydrodiffusion and gravity. Food Chem 2009;114(1):355-362.##Liu Z, Chen Z, Han F, Kang X, Gu H, Yang L. Microwave-assisted method for simultaneous hydrolysis and extraction in obtaining ellagic acid, gallic acid and essential oil from Eucalyptus globulus leaves using Brönsted acidic ionic liquid [HO3S(CH2)4mim]HSO4. Ind Crops Prod 2016;81:152-161.##Thakker MR, Parikh JK, Desai MA. Microwave assisted extraction of essential oil from the leaves of Palmarosa: Multi-response optimization and predictive modelling. Ind Crops Prod 2016;86:311-319.##Stashenko EE, Jaramillo BE, Martinez JR. Analysis of volatile secondary metabolites from Colombian Xylopia aromatica (Lamarck) by different extraction and headspace methods and gas chromatography. J Chromatogr A 2004;1025(1):105-113.##Stashenko EE, Jaramillo BE, Martinez JR. Comparison of different extraction methods for the analysis of volatile secondary metabolites of Lippia alba (Mill) N.E. Brown, grown in Colombia, and evaluation of its in vitro antioxidant activity. J Chromatogr A 2004;1025(1):93-103.##Tigrine-Kordjani N, Meklati BY, Chemat F. Microwave ‘dry’ distillation as a useful tool for extraction of edible essential oils. Int J Aroma 2006;16(3-4):141-147.##Sui X, Liu T, Ma C, Yang L, Zu Y, Zhang L. 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Relative Expression of PBMC MicroRNA-133a Analysis in Patients Receiving Warfarin After Mechanical Heart Valve Replacement 2 2 Background: MicroRNAs (miRNAs) are implicated in various biological processes including anticoagulation. However, the modulation of miRNA by pharmacological intervention such as warfarin treatment in patients receiving warfarin has not been disclosed yet. The aim of this study work was to assess the effect of warfarin drug on expression level of mir-133a-3p in patients with mechanical heart valve replacement. Methods: In this research, the expression level of miRNA-133a-3p was analyzed in Peripheral Blood Mononuclear cells (PBMCs) from mechanical valve replacement patients who had received warfarin for at least 3 months continuously. Quantitative RT-PCR method was used for this assay. Results: Our findings indicated a significant diffrence between the rate of miR-133a-3p expression in individuals receiving warfarin and the control group (p<0.01). There was also a statistically significant difference in miR-133a-3p expression in patients with different ages (p<0.05) suggesting that the rate of miR-133a-3p expression in persons receiving warfarin is related to age. However, other variables like warfarin dose, International Normalized Ratio (INR), gender, and Body Mass Index (BMI) were not significantly effective on the miR-133a-3p experssion rate in individuals receving warfarin.  Conclusion: Based on our results, it can be concluded that miR-133a-3p is involved in the coagulation pathway. The recent result indicates that warfarin affects the expression of miR-133a. This expression may be potentially important for treatment by anticoagulants. Awareness of the time course of miRNA expression profile can improve efficiency of response to warfarin. 29 33 Hamid Kabiri Rad International Campus, Shahid Sadoughi University of Medical Sciences International Campus, Shahid Sadoughi University of Medical Sciences Iran Mahta Mazaheri Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Department of Medical Genetics, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Iran Ali Dehghani Firozabadi Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences Yazd Cardiovascular Research Center, Shahid Sadoughi University of Medical Sciences Iran AnticoagulationMicroRNAsQuantitative reverse transcription-PCRWarfarin 299.pdf Cannegieter SC, Rosendaal F, Wintzen A, Van der Meer FJ, Vandenbroucke JP, Briët E. Optimal oral anticoagulant therapy in patients with mechanical heart valves. N Engl J Med 1995;333(1):11-17.##Li Y, Zhu J, Ding JQ. VKORC1 rs2359612 and rs9923231 polymorphisms correlate with high risks of cardiovascular and cerebrovascular diseases. Genet Mol Res 2015;14(4):14731-14744.##[No authors listed]. Effect of long-term oral anticoagulant treatment on mortality and cardiovascular morbidity after myocardial infarction. Anticoagulants in the secondary prevention of events in coronary thrombosis (ASPECT) research group. Lancet 1994;343(8896):499-503.##Drapkin A, Merskey C. Anticoagulant therapy after acute myocardial infarction. Relation of therapeutic benefit to patient's age, sex, and severity of infarction. JAMA 1972;222(5):541-548.##Oldenburg J, Watzka M, Rost S, Müller C. VKORC1: molecular target of coumarins. J Thromb Haemost 2007;5 Suppl 1:1-6.##Sabir I, Khavandi K, Brownrigg J, Camm AJ. Oral anticoagulants for asian patients with atrial fibrillation. ‎Nat Rev Cardiol 2014;11(5):290-303.##Lane DA, Lip GY. Maintaining therapeutic anticoagulation: the importance of keeping "within range". Chest 2007;131(5):1277-1279.##Sconce EA, Kamali F. Appraisal of current vitamin K dosing algorithms for the reversal of over anticoagulation with warfarin: the need for a more tailored dosing regimen. Eur J Heamathol 2006;77(6):457-462.##International Warfarin Pharmacogenetics Consortium, Klein TE, Altman RB, Eriksson N, Gage BF, Kimmel SE, et al. Estimation of the warfarin dose with clinical and pharmacogenetic data. N Engl J Med 2009;360(8):753-764.##Pérez-Andreu V, Roldán V, Antón AI, García-Barberá N, Corral J, Vicente V, et al. Pharmacogenetic relevance of CYP4F2 V433M polymorphism on acenocoumarol therapy. Blood 2009;113(20):4977-4979.##Esteller M. Non-coding RNAs in human disease. Nat Rev Genet 2011;12(12):861-874.##Almeida MI, Reis RM, Calin GA. MicroRNA history: Discovery, recent applications, and next frontiers. Mutat Res 2011;717(1-2):1-8.##Huang Y, Wu Y, Dong J, Han D, Yang S, J Lin. MicroRNA-133a-3p exerts inhibitory effects on gallbladder carcinoma via targeting RBPJ. Am J Cancer Res 2016;6(11):2448-2462.##Wang GK, Zhu JQ, Zhang JT, Li Q, Li Y, He J, et al. Circulating microRNA: a novel potential biomarker for early diagnosis of acute myocardial infarction in humans. Eur Heart J 2010;31(6):659-666.##Flockhart DA, O'Kane D, Williams MS, Watson MS, Flockhart DA, Gage B, et al. Pharmacogenetic testing of CYP2C9 and VKORC1 alleles for warfarin. Genet Med 2008;10(2):139-150.##Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acid Res 2001;29(9):e45.##Borgiani P, Ciccacci C, Forte V, Sirianni E, Novelli L, Bramanti P, et al. CYP4F2 genetic variant (rs2108622) significantly contributes to warfarin dosing variability in the Italian population. Pharmacogenomics 2009;10(2):261-266.##Sconce EA, Khan TI, Wynne HA, Avery P, Monkhouse L, King BP, et al. The impact of CYP2C9 and VKORC1 genetic polymorphism and patient characteristics upon warfarin dose requirements: proposal for a new dosing regimen. Blood 2005;106(7):2329-2333.##Li J, Chen H, Ren J, Song J, Zhang F, Zhang J, et al. Effects of statin on circulating microRNAome and predicted function regulatory network in patients with unstable angina. BMC Med Genomics 2015;8:12.##Tu Y, Wan L, Bu L, Zhao D, Dong D, Huang T, et al. MicroRNA-22 downregulation by atorvastatin in a mouse model of cardiac hypertrophy: a new mechanism for antihypertrophic intervention. Cell Physiol Biochem 2013;31(6):997-1008.##Simon LM, Edelstein LC, Nagalla S, Woodley AB, Chen ES, Kong X, et al. Human platelet microRNA-mRNA networks associated with age and gender revealed by integrated plateletomics. Blood 2014;123(16):e37-45.##Okuhara A, Nakasa T, Shibuya H, Niimoto T, Adachi N, Deie M, et al. Changes in microRNA expression in peripheral mononuclear cells according to the progression of osteoarthritis. Mod Rheumatol 2012;22(3):446-457.##Noren Hooten N, Abdelmohsen K, Gorospe M, Ejiogu N, Zonderman AB, Evans MK. microRNA expression patterns reveal differential expression of target genes with age. PLoS One 2010;5(5):e10724.##McDermott AM, Kerin MJ, Miller N. Identification and validation of miRNAs as endogenous controls for RQ-PCR in blood specimens for breast cancer studies. PLoS One 2013;8(12):e83718.##Peltier HJ, Latham GJ. Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitablereference RNA targets in normal and cancerous human solid tissues. RNA 2008;14(5):844-852.##Han S, Zhou V, Pan S, Liu Y, Hornsby M, McMullan D, et al. Identification of coumarin derivatives as a novel class of allosteric MEK1 inhibitors. Bioorg Med Chem Lett 2005;15(24):5467-5473.##Kater AP, Peppelenbosch MP, Brandjes DP, Lumbantobing M. Dichotomal effect of the coumadin derivative warfarin on inflammatory signal transduction. 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Molecular Characterization and Functional Analysis of the PilQ380-705: a Novel Secretin Domain in Pseudomonas aeruginosa 2 2 Background: Type 4 pili (T4P) is an important virulence factor of Pseudomonas aeruginosa (P. aeruginosa). T4P pass the outer membrane through a large oligomeric channel made of a single PilQ protein that is most highly conserved at their C-termini. To develop a functional vaccine that can be used in clinical application, the secretin domain of the PilQ (PilQ380-706) was produced as a recombinant protein. Methods: A 981 bp fragment of C-terminal of the pilQ secretin (pilQ1138-2118) from was designed into the prokaryotic expression vector pET28a. The presence of the pilQ1138-2118 gene in the recombinant construct (pET28a/pilQ) was assessed by double digestion and PCR. After transformation, expression of the recombinant PilQ was induced by addition of IPTG. The expressed recombinant protein was purified by a modified method using a HisTrap affinity column and finally confirmed by SDS-PAGE. The functional activities of the produced PilQ380-706 confirmed by Western blot analysis and twitching inhibition assay.  Results: The PCR and enzymatic digestion results showed the presence of the pilQ1138-2118 gene in the construct. The protein electrophoresis showed that the molecular weight of the recombinant PilQ380-706 is approximately 37 kDa. The Western blot analysis confirmed the specificity of specific IgG against the PilQ380-706 protein. The PilQ380-706 protein showed high biological activity in all of these standard assays. Conclusion: Since, the PilQ380-706 protein plays an important role in the biogenesis of pili; and thus, the primary establishment of P. aeruginosa; it seems that it can be used as a candidate vaccine or an adjuvant in the future studies. 34 40 Sobhan Faezi Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical SciencesDepartment of Mycobacteriology and Pulmonary Research, Pasteur Institute of IranMycobacteriology Research Center (MRC) Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical SciencesDepartment of Mycobacteriology and Pulmonary Research, Pasteur Institute of IranMycobacteriology Research Center (MRC) IranIranIran Iraj Nikokar Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical SciencesLaboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical SciencesLaboratory of Microbiology and Immunology of Infectious Diseases, Faculty of Paramedicine, Guilan University of Medical Sciences IranIran Ali Elmi Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Iran Yusuf Ghasemi Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Iran Mojtaba Farahbakhsh Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Medical Biotechnology Research Center, Faculty of Paramedicine, Guilan University of Medical Sciences Iran Alireza Salimi Chirani Department of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science Department of Medical Microbiology, Faculty of Medicine, Shahid Beheshty University of Medical Science Iran Mehdi Mahdavi Department of Immunology, Pasteur Institute of Iran Department of Immunology, Pasteur Institute of Iran Iran Pseudomonas aeruginosaProteinsSecretin 302.pdf Sousa AM, Pereira MO. Pseudomonas aeruginosa diversification during infection development in cystic fibrosis lungs-A review. Pathogens 2014;3(3):680-703.##Hirsch EB, Tam VH. Impact of multidrug-resistant Pseudomonas aeruginosa infection on patient outcomes. Expert Rev Pharmacoecon Outcomes Res 2010;10(4):441-451.##Bucior I, Pielage JF, Engel JN. Pseudomonas aeruginosa pili and flagella mediate distinct binding and signaling events at the apical and basolateral surface of airway epithelium. PLoS Pathog 2012;8(4):e1002616.##Wehbi H, Portillo E, Harvey H, Shimkoff AE, Scheurwater EM, Howell PL, et al. The peptidoglycan-binding protein FimV promotes assembly of the Pseudomonas aeruginosa type IV pilus secretin. J Bacteriol 2011;193(2):540-550.##Farinha MA, Conway BD, Glasier LM, Ellert NW, Irvin RT, Sherburne R, et al. Alteration of the pilin adhesin of Pseudomonas aeruginosa PAO results in normal pilus biogenesis but a loss of adherence to human pneumocyte cells and decreased virulence in mice. Infect Immun 1994;62(10):4118-4123.##Ayers M, Sampaleanu LM, Tammam S, Koo J, Harvey H, Howell PL, et al. PilM/N/O/P proteins form an inner membrane complex that affects the stability of the Pseudomonas aeruginosa type IV pilus secretin. J Mol Biol 2009;394(1):128-142.##Burrows LL. Weapons of mass retraction. Mol Microbiol 2005;57(4):878-888.##Tang H, Kays M, Prince A. Role of Pseudomonas aeruginosa pili in acute pulmonary infection. Infect Immun 1995;63(4):1278-1285.##Tammam S, Sampaleanu LM, Koo J, Sundaram P, Ayers M, Chong PA, et al. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex. Mol Microbiol 2011;82(6):1496-1514.##Bohn YS, Brandes G, Rakhimova E, Horatzek S, Salunkhe P, Munder A, et al. Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection. Mol Microbiol 2009;71(3):730-747.##Hackbarth C, Hodges RS. Synthetic peptide vaccine development: designing dual epitopes into a single pilin peptide immunogen generates antibody cross-reactivity between two strains of Pseudomonas aeruginosa. Chem Biol Drug Des 2010;76(4):293-304.##Kus JV, Tullis E, Cvitkovitch DG, Burrows LL. Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients. Microbiology 2004;150(Pt 5):1315-1326.##Tammam S, Sampaleanu LM, Koo J, Manoharan K, Daubaras M, Burrows LL, et al. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA. J Bacteriol 2013;195(10):2126-2135.##Hoang HH, Nickerson NN, Lee VT, Kazimirova A, Chami M, Pugsley AP, et al. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries. MBio 2011;2(6):00246-11.##Pelicic V. Type IV pili: e pluribus unum? Mol Microbiol 2008;68(4):827-837.##Nudleman E, Kaiser D. Pulling together with type IV pili. J Mol Microbiol Biotechnol 2004;7(1-2):52-62.##Castric P, Cassels FJ, Carlson RW. Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan. J Biol Chem 2001;276(28):26479-26485.##Matthey B, Engert A, Klimka A, Diehl V, Barth S. A new series of pET-derived vectors for high efficiency expression of Pseudomonas exotoxin-based fusion proteins. Gene 1999;229(1-2):145-153.##Haghi F, Peerayeh SN, Siadat SD, Zeighami H. Recombinant outer membrane secretin PilQ(406-770) as a vaccine candidate for serogroup B Neisseria meningitidis. Vaccine 2012;30(9):1710-1714.##Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif 2003;28(1):1-8.##Middelberg AP. Preparative protein refolding. Trends Biotechnol 2002;20(10):437-443.##Singh SM, Panda AK. Solubilization and refolding of bacterial inclusion body proteins. J Biosci Bioeng 2005;99(4):303-310.##Gellatly SL, Hancock RE. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 2013;67(3):159-173.##Koo J, Tang T, Harvey H, Tammam S, Sampaleanu L, Burrows LL, et al. Functional mapping of PilF and PilQ in the Pseudomonas aeruginosa type IV pilus system. Biochemistry 2013;52(17):2914-2923.##

Male Pronuclear Formation and Embryo Development Following Intracytoplasmic Injection of Ovine Pretreated Sperm 2 2 Background: Failure of Male Pronucleus (MPN) formation is a major concern in the success of Intracytoplasmic Sperm Injection (ICSI) in some species. Despite the conducted unsuccessful efforts to improve ICSI efficiency in ovine, the present study was aimed to improve MPN formation and embryo development in ovine ICSI procedure through accompaniment of sperm pretreatment with co-injection of some compounds.  Methods: In experiment 1, sperm were treated with either 2-mercaptoethanol (2ME), glutathione (GSH), or DTT (dithiothreitol) in combination with Heparin (Hep). Following DNA integrity and fragmentation assessments, the best sperm pretreatment approach in induction of sperm head decondensation was applied for the second and third experiments. In experiment 2, in vitro matured oocytes were subjected to ICSI using pretreated sperm with or without GSH and Sperm Extract (SE) co-injection. In experiment 3, the procedure was followed as experiment 2 with acrosome reacted sperm.  Results: The highest percentages of oocyte activation were observed in Hep+GSH and Hep+2ME groups. The greatest MPN formations were also observed in the same groups when ICSI procedure was accompanied with GSH co-injection. Despite the higher percentage of MPN formation and oocyte activation in Hep+GSH and Hep+2ME groups, none of the employed strategies could increase the cleavage and blastocyst rates compared to the control.  Conclusion: In our study condition, despite the lack of significant increase in embryo development in treated groups, the significant increase in MPN formation in Hep+GSH+co.GSH and Hep+2ME+co.GSH groups indicates the lower chance of parthenote formation that means a higher chance of normal fertilization compared with control. 41 48 Abolfazl Shirazi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRDepartment of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRDepartment of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University IranIran Arefeh Golestanfar Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Masomeh Bashiri Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Ebrahim Ahmadi Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Naser Shams-Esfandabadi Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Department of Gametes and Cloning, Research Institute of Animal Embryo Technology, Shahrekord University Iran Intracytoplasmic sperm injection (ICSI)MaleOvine Pronucleus 304.pdf Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340(8810):17-18.##García-Roselló E, Matás C, Cánovas S, Moreira PN, Gadea J, Coy P. Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs. J Androl 2006;27(2):268-275.##Ramadan WM, Kashir J, Jones C, Coward K. Oocyte activation and phospholipase C zeta (PLCζ): diagnostic and therapeutic implications for assisted reproductive technology. Cell Commun Signal 2012;10(1):12.##Kren R, Kikuchi K, Nakai M, Miyano T, Ogushi S, Nagai T, et al. Intracytoplasmic sperm injection in the pig: where is the problem? J Reprod Dev 2003;49(4):271-273.##Lee JW, Yang X. Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm. Mol Reprod Dev 2004;68(1):96-102.##Perreault SD, Barbee RR, Slott VL. Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes. Dev Biol 1988;125(1):181-186.##Keefer CL. Fertilization by sperm injection in the rabbit. Gamete Res 1989;22(1):59-69.##Hoshi K, Yanagida K, Sato A. Pretreatment of hamster oocytes with Ca2+ ionophore to facilitate fertilization by ooplasmic micro-injection. Hum Reprod 1992;7(6):871-875.##Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995;52(4):709-720.##Tesarik J, Sousa M. More than 90% fertilization rates after intracytoplasmic sperm injection and artificial induction of oocyte activation with calcium ionophore. Fertil Steril 1995;63(2):343-349.##Horiuchi T, Numabe T. Intracytoplasmic sperm injection (ICSI) in cattle and other domestic animals: Problems and improvements in practical use. J Mammal Ova Res 1999;16(1):1-9.##Shirazi A, Derakhshan-Horeh M, Pilvarian AA, Ahmadi E, Nazari H, Heidari B. Effect of pre-treatment of ovine sperm on male pronuclear formation and subsequent embryo development following intracytoplasmic sperm injection. 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Optimization of Effective Minerals on Riboflavin Production by Bacillus subtilis subsp. subtilis ATCC 6051 Using Statistical Designs 2 2 Background: Riboflavin (vitamin B2) is an essential component of the basic metabo-lism, and an important nutritional and growth factor in humans, animals, plants and micro-organisms. It has been widely used in the fields of pharmaceuticals, feed and food additives. The industrial production of riboflavin mostly relies on the microbial fermentation. Designing an appropriate fermentation medium is of crucial importance to improve the riboflavin production.  Methods: In this study, sequential methodology combining a screening test of minerals by Plackett-Burman (PB) and an optimization test by Central Composite Design (CCD) was applied to enhance riboflavin production by Bacillus subtilis ATCC 6051 in shake flasks. Results: Initially, one-factor-at-a-time approach was applied to evaluate the effect of different carbon sources. The results showed that fructose was significantly most effective on biomass and riboflavin production. After that, 13 minerals [CaCl2, CuCl, FeCl3, FeSO4, AlCl3, Na3MoO4, Co(NO3)2, NaCl, KH2PO4, K2HPO4, MgSO4, ZnSO4, and MnSO4] were studied with the screening test. The results revealed that concentration of MgSO4, K2HPO4, and FeSO4 had greater influence on riboflavin production (p<0.05). A CCD with five factors (concentration of fructose, MgSO4, K2HPO4, FeSO4, and yeast extract) at five levels was then used to determine the maximum riboflavin concentration. The optimal concentrations (g/l) of these variables determined by Response Surface Methodology (RSM) were fructose, 38.10; MgSO4, 0.85; K2HPO4, 2.27; FeSO4, 0.02; and yeast extract, 4.37. Conclusion: Statistical experimental design offers a practicable approach to the implementation of medium optimization. From an industrial view point, our optimum medium, besides fructose and a small amount of yeast extract, is mainly composed of common and cheap inorganic salts, which are available to the industrial riboflavin production. 49 55 Marjan Oraei Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Iran Seyed Hadi Razavi Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Iran Faramarz Khodaiyan Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Bioprocess Engineering Laboratory (BPEL), Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, University of Tehran Iran Bacillus subtilis ATCC 6051MineralsRiboflavin 10335.pdf Liu S, Kang P, Cui Z, Wang Z, Chen T. Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner-Doudoroff pathway in Escherichia coli. Biotechnol Lett 2016;38(8):1307-1314.##Dmytruk K, Lyzak O, Yatsyshyn V, Kluz M, Sibirny V, Puchalski C, et al. Construction and fed-batch cultivation of Candida famata with enhanced riboflavin production. J Biotechnol 2014;172:11-17.##Wu QL, Chen T, Gan Y, Chen X, Zhao XM. Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs. Appl Microbiol Biotechnol 2007;76(4):783-794.##Stahmann KP, Revuelta JL, Seulberger H. Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol 2000;53(5):509-516.##Kato T, Park EY. Riboflavin production by Ashbya gossypii. Biotechnol Lett 2012;34(4):611-618.##Lim SH, Ming H, Park EY, Choi JS. 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Association of Serotonin Transporter Gene Polymorphism with Recurrent Aphthous Stomatitis 2 2 Background: Recurrent Aphthous Stomatitis (RAS) is one of the most common diseases of the oral cavity all over the world (5-66%). RAS has a multifactorial etiology, while psychological factors such as stress and anger play a role in its manifestation. The serotonergic mechanisms particularly the serotonin-transporter gene (5-HTT) may affect the risk of psychological alterations and stress response. The aim of the present study was to evaluate the polymorphism of the promoter region of 5-HTT (5-HTTLPR) in the patients with RAS, compared to that in the control subjects. Methods: In this case-control study, 100 patients with RAS and 100 healthy subjects were enrolled. PCR was performed on DNA of the samples, using a pair of primers capable of distinguishing S/L alleles and replicating 5-HTTLPR. Results: No statistically significant difference existed between LL and LS genotype frequencies in the case and control groups. However, SS genotype frequency was significantly higher in the case group, as compared to the control group (p=0.001). Conclusion: The conclusion of the present study demonstrated that S allele could approximately double the risk of RAS. 56 60 Shamsolmoulouk Najafi Faculty of Dentistry, Tehran University of Medical SciencesInternational Campus, Dental Research Center, Tehran University of Medical Sciences Faculty of Dentistry, Tehran University of Medical SciencesInternational Campus, Dental Research Center, Tehran University of Medical Sciences IranIran Mahsa Mohammadzadeh Dental Branch, Islamic Azad University Dental Branch, Islamic Azad University Iran Amirabbas Zahedi Faculty of Dentistry, Tehran University of Medical Sciences Faculty of Dentistry, Tehran University of Medical Sciences Iran Mansour Heidari Farabi Eye Hospital, Farabi Research Center, Tehran University of Medical Sciences Farabi Eye Hospital, Farabi Research Center, Tehran University of Medical Sciences Iran Nima Rezaei Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical SciencesDepartment of Immunology, School of Medicine, Tehran University of Medical SciencesNetwork of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN) Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical SciencesDepartment of Immunology, School of Medicine, Tehran University of Medical SciencesNetwork of Immunity in Infection, Malignancy and Autoimmunity (NIIMA), Universal Scientific Education and Research Network (USERN) IranIranUSA 5-HTTLPRPCRRecurrent aphthous stomatitis (RAS)Single-nucleotide polymorphism 303.pdf Messadi DV, Younai F. 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