Medical Biotechnology and Alzheimer's Disease: New Hopes 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 1-3. Devastating nature of AD leads to serious social and economic impacts on the healthcare systems which implies the necessity of its proper management 1-3. 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. Progress to a cure for AD has been hampered by the lack of information about the biology of the disease. The therapies currently approved for Alzheimer’s disease work by treating the patients’ symptoms, improving their cognitive and overall functions 4-8. Increasingly, however, experts are intent on slowing or halting the disease process, before it has ravaged patients’ brains. A lot of data is being generated on changes in imaging biomarkers before patients really become clinically significantly impaired. For example, there has been a lot of great work done in identifying patients early based on these biomarkers. The current therapeutic market is valued at $3 to $4 billion, shared among drugs that temporarily delay disease progression or address the symptoms but do not alter the underlying disease. Currently, medical biotechnology has brought new hopes in the treatment of Alzheimer's disease. For example one of the ongoing trials is related to bapineuzumab. Bapineuzumab is a monoclonal antibody (mAb) to target and clear ß-amyloid. This vaccine is the first new drug aimed at slowing or even halting AD progression. 47 47 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Psychiatric Research Center, Roozbeh Hospital, South Kargar Street Iran Editorial 233.pdf Akhondzadeh S. Hippocampal synaptic plasticity and cognition. J Clin Pharm Ther 1999;24(4):241-248.##Akhondzadeh S, Noroozian M. Alzheimer's disease: pathophysiology and pharmacotherapy. IDrugs 2002;5(11):1062-1069.##Akhondzadeh S, Abbasi SH. Herbal medicine in the treatment of Alzheimer's disease. Am J Alzheimers Dis Other Demen 2006;21(2):113-118.##Akhondzadeh S, Noroozian M, Mohammadi M, Ohadinia S, Jamshidi AH, Khani M. Salvia officinalis extract in the treatment of patients with mild to moderate Alzheimer's disease: a double blind, randomized and placebo-controlled trial. J Clin Pharm Ther 2003;28(1):53-59.##Akhondzadeh S, Noroozian M, Mohammadi M, Ohadinia S, Jamshidi AH, Khani M. Melissa officinalis extract in the treatment of patients with mild to moderate Alzheimer's disease: a double blind, randomised, placebo controlled trial. J Neurol Neurosurg Psychiatry 2003;74(7):863-866.##Akhondzadeh S, Shafiee Sabet M, Harirchian MH, Togha M, Cheraghmakani H, Razeghi S, et al. A 22-week, multicenter, randomized, double-blind controlled trial of Crocus sativus in the treatment of mild-to-moderate Alzheimer's disease. Psychopharmacology (Berl) 2010;207(4):637-643.##Akhondzadeh S, Sabet MS, Harirchian MH, Togha M, Cheraghmakani H, Razeghi S, et al. Saffron in the treatment of patients with mild to moderate Alzheimer's disease: a 16-week, randomized and placebo-controlled trial. J Clin Pharm Ther 2010;35(5):581-588.##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.##

Role of Superoxide Dismutase 2 Gene Ala16Val Polymorphism and Total Antioxidant Capacity in Diabetes and its Complications 2 2 Diabetes Mellitus (DM) is a chronic heterogeneous disorder and oxidative stress is a key participant in the development and progression of it and its complications. Anti-oxidant status can affect vulnerability to oxidative damage, onset and progression of diabetes and diabetes complications. Superoxide dismutase 2 (SOD2) is one of the major antioxidant defense systems against free radicals. SOD2 is encoded by the nuclear SOD2 gene located on the human chromosome 6q25 and the Ala16Val polymorphism has been identified in exon 2 of the human SOD2 gene. Ala16Val (rs4880) is the most commonly studied SOD2 single nucleotide polymorphism (SNP) in SOD2 gene. This SNP changes the amino acid at position 16 from valine (Val) to alanine (Ala), which has been shown to cause a conformational change in the target sequence of manganese superoxide dismutase (MnSOD) and also affects MnSOD activity in mitochondria. Ala16Val SNP and changes in the activity of the SOD2 antioxidant enzyme have been associated with altered progression and risk of different diseases. Association of this SNP with diabetes and some of its complications have been studied in numerous studies. This review evaluated how rs4880, oxidative stress and antioxidant status are associated with diabetes and its complications although some aspects of this line still remain unclear. 48 56 Katayoun Pourvali Department of Basic Sciences and Cellular and Molecular Nutrition, Faculty of Nutrition Sciences and Food Technology and National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences Department of Basic Sciences and Cellular and Molecular Nutrition, Faculty of Nutrition Sciences and Food Technology and National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences Iran Mehrnaz Abbasi Department of Basic Sciences and Cellular and Molecular Nutrition, Faculty of Nutrition Sciences and Food Technology and National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences Department of Basic Sciences and Cellular and Molecular Nutrition, Faculty of Nutrition Sciences and Food Technology and National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences Iran Azadeh Mottaghi Obesity Research Center, Nutrition and Endocrine Research Center, Research Institute of Endocrine Sciences, Shahid Beheshti University of Medical Sciences Obesity Research Center, Nutrition and Endocrine Research Center, Research Institute of Endocrine Sciences, Shahid Beheshti University of Medical Sciences Iran Diabetes complicationsDiabetes mellitusPolymorphismSuperoxide dismutase 2 234.pdf Banerjee M, Vats P. 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Construction and Production of Foxp3- Fc (IgG) DNA Vaccine/Fusion Protein 2 2 Background: It seems that the success of vaccination for cancer immunotherapy such as Dendritic Cell (DC) based cancer vaccine is hindered through a powerful network of immune system suppressive elements in which regulatory T cell is the common factor. Foxp3 transcription factor is the most specific marker of regulatory T cells. In different studies, targeting an immune response against regulatory cells expressing Foxp3 and their removal have been assessed. As these previous studies could not efficiently conquer the suppressive effect of regulatory cells by their partial elimination, an attempt was made to search for constructing more effective vaccines against regulatory T cells by which to improve the effect of combined means of immunotherapy in cancer. In this study, a DNA vaccine and its respective protein were constructed in which Foxp3 fused to Fc(IgG) can be efficiently captured and processed by DC via receptor mediated endocytosis and presented to MHCII and I (cross priming). Methods: DNA construct containing fragment C (Fc) portion of IgG fused to Foxp3 was designed. DNA construct was transfected into HEK cells to investigate its expression through fluorescent microscopy and flow cytometry. Its specific expression was also assessed by western blot. For producing recombinant protein, FOXP3-Fc fusion construct was inserted into pET21a vector and consequently, Escherichia coli (E. coli) strain BL21 was selected as host cells. The expression of recombinant fusion protein was assayed by western blot analysis. Afterward, fusion protein was purified by SDS PAGE reverse staining. Results: The expression analysis of DNA construct by flow cytometry and fluorescent microscopy showed that this construct was successfully expressed in eukaryotic cells.  Moreover, the Foxp3-Fc expression was confirmed by SDS-PAGE followed by western blot analysis. Additionally, the presence of fusion protein was shown by specific antibody after purification. Conclusion: Due to successful expression of Foxp3-Fc (IgG), it would be expected to develop vaccines in tumor therapies for removal of regulatory cells as a strategy for increasing the efficiency of other immunotherapy means. 57 64 Neda Mousavi Niri Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Iran Arash Memarnejadian Department of Hepatitis and AIDS, Pasteur Institute of Iran Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Jamshid Hadjati Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences Iran Mohammad Reza Aghasadeghi Department of Hepatitis and AIDS, Pasteur Institute of Iran Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Mehdi Shokri Department of Hepatitis and AIDS, Pasteur Institute of Iran Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Yones Pilehvar-soltanahmadi Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Iran Abolfazl Akbarzadeh Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Department of Medical Nanotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences Iran Nosratollah Zarghami Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical SciencesDepartment of Clinical Biochemistry, Radiopharmacy Lab, Drug Applied Research Center, Tabriz University of Medical Sciences Department of Medical Biotechnology, Faculty of Advanced Medical Sciences, Tabriz University of Medical SciencesDepartment of Clinical Biochemistry, Radiopharmacy Lab, Drug Applied Research Center, Tabriz University of Medical Sciences IranIran FOXP3 proteinFusion proteinImmunoglobulin G (IgG) 235.pdf Powell DJ Jr, Felipe-Silva A, Merino MJ, Ahmadzadeh M, Allen T, Levy C, et al. 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J Immunother 2010;33(2):211-218.##Lutsiak ME, Semnani RT, De Pascalis R, Kashmiri SV, Schlom J, Sabzevari H. Inhibition of CD4(+)25+ T regulatory cell function implicated in enhanced immune response by low-dose cyclophosphamide. Blood 2005;105(7):2862-2868.##Rosalia RA, Štěpánek I, Polláková V, Šímová J, Bieblová J, Indrová M, et al. Administration of anti-CD25 mAb leads to impaired alpha-galactosylceramide-mediated induction of IFN-gamma production in a murine model. Immunobiology 2013;218(6):851-859.##Rech AJ, Vonderheide RH. Clinical use of anti‐CD25 antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory T cells. Ann N Y Acad Sci 2009;1174:99-106.##Litzinger MT, Fernando R, Curiel TJ, Grosenbach DW, Schlom J, Palena C. IL-2 immunotoxin denileukin diftitox reduces regulatory T cells and enhances vaccine-mediated T-cell immunity. Blood 2007;110(9):3192-3201.##Klages K, Mayer CT, Lahl K, Loddenkemper C, Teng MW, Ngiow SF, et al. 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Hypoglycemic Effect of Aquatic Extract of Stevia in Pancreas of Diabetic Rats: PPARγ -dependent Regulation or Antioxidant Potential 2 2 Background: Traditional medicines with anti-diabetic effects are considered suitable supplements to treat diabetes. Among medicinal herbs, Stevia Rebaudiana Bertoni is famous for its sweet taste and beneficial effect in regulation of glucose. However, little is known about the exact mechanism of stevia in pancreatic tissue. Therefore, this study investigated the possible effects of stevia on pancreas in managing hyperglycemia seen in streptozotocin-induced Sprague-Dawley rats. Methods: Sprague-Dawley rats were divided into four groups including normoglycemic, diabetic and two more diabetic groups in which, one was treated with aquatic extract of stevia (400 mg/kg) and the other with pioglitazone (10 mg/kg) for the period of 28 days. After completion of the experimental duration, rats were dissected; blood samples and pancreas were further used for detecting biochemical and histopathological changes. FBS, TG, cholestrol, HDL, LDL, ALT and AST levels were measured in sera. Moreover, MDA (malondialdehyde) level, catalase activity, levels of insulin and PPARγ mRNA expression were also measured in pancreatic tissue. Results: Aquatic extract of stevia significantly reduced the FBS, triglycerides, MDA, ALT, AST levels and normalized catalase activity in treated rats compared with diabetic rats (p<0.05). In addition to this, stevia surprisingly, increased PPARγ and insulin mRNA levels in treated rats (p<0.05). Furthermore, stevia compensated for the histopathological damage in diabetic rats. Conclusion: It is concluded that stevia acts on pancreatic tissue to elevate the insulin level and exerts beneficial anti-hyperglycemic effects through the PPARγ-dependent mechanism and stevia’s antioxidant properties. 65 74 Raheleh Assaei Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences Iran Pooneh Mokarram Gasteroenterohepatology Research Center, Nemazee Hospital, School of Medicine, Shiraz University of Medical SciencesDepartment of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Gasteroenterohepatology Research Center, Nemazee Hospital, School of Medicine, Shiraz University of Medical SciencesDepartment of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences IranIran Sanaz Dastghaib Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical SciencesDepartment of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical SciencesDepartment of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences IranIran Sara Darbandi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mahsa Darbandi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Fatemeh Zal Reproductive Biology Group, Graduate School of Biomedical Sciences, Shiraz University of Medical Sciences Reproductive Biology Group, Graduate School of Biomedical Sciences, Shiraz University of Medical Sciences Iran Masoumeh Akmali Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Iran Gholam Hossein Ranjbar Omrani Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences Endocrinology and Metabolism Research Center, Nemazee Hospital, Shiraz University of Medical Sciences Iran Diabetes mellitusInsulinPancreasPPAR gammaStevia 236.pdf Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27(5):1047-1053.##Goyal SK, Samsher, Goyal RK. Stevia (Stevia rebaudi-ana) a bio-sweetener: a review. Int J Food Sci Nutr 2010;61(1):1-10.##Puri M. Extraction and safety of stevioside; response to the article Stevia rebaudiana Bertoni, source of a high potency natural sweetener: a comprehensive review on the biochemical, nutritional and functional aspects. Food Chem 2012;135(3):1861-1862.##Wölwer-Rieck U. The leaves of Stevia rebaudiana (Bertoni), their constituents and the analyses thereof: a review. J Agric Food chem 2012;60(4):886-895.##Thiyagarajan M, Venkatachalam P. Large scale in vitro propagation of Stevia rebaudiana (bert) for commercial application: Pharmaceutically important and antidiabetic medicinal herb. Ind Crops Prod 2012;37(1):111-117.##Kujur RS, Singh V, Ram M, Yadava HN, Singh KK, Kumari S, et al. Antidiabetic activity and phytochemical screening of crude extract of stevia rebaudiana in alloxan-induced diabetic rats. Pharmacognosy Res 2010;2(4):258-263.##Shivanna N, Naika M, Khanum F, Kaul VK. Antioxidant, anti-diabetic and renal protective properties of Stevia rebaudiana. J Diabetes Complications 2013;27(2):103-113.##Román-Ramos R, Flores-Sáenz JL, Partida-Hernández G, Lara-Lemus A, Alarcón-Aguilar F. Experimental study of the hypoglycemic effect of some antidiabetic plants. Arch Invest Med 1991;22(1):87-93.##German MS. Glucose sensing in pancreatic islet beta cells: the key role of glucokinase and the glycolytic intermediates. Proc Natl Acad Sci USA 1993;90(5):1781-1785.##Kim HI, Ahn YH. Role of peroxisome proliferator-activated receptor-gamma in the glucose-sensing apparatus of liver and beta-cells. Diabetes 2004;53 Suppl 1:S60-65.##Huang TH, Peng G, Kota BP, Li GQ, Yamahara J, Roufogalis BD, et al. Anti-diabetic action of Punica granatum flower extract: activation of PPARγ and identification of an active component. Toxicol Appl Pharmacol 2005;207(2):160-169.##Staels B, Fruchart JC. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes 2005;54 (8):2460-2470.##Gupta D, Kono T, Evans-Molina C. The role of peroxi-some proliferator-activated receptor γ in pancreatic β cell function and survival: therapeutic implications for the treatment of type 2 diabetes mellitus. Diabetes Obes Metab 2010;12(12):1036-1047.##Sharma R, Yadav R, Manivannan E. Study of effect of Stevia rebaudiana bertoni on oxidative stress in type-2 diabetic rat models. Biomed Aging Pathol 2012;2(3):126-131.##Singh S, Garg V. Antidiabetic, antidyslipidymic and anti-oxidative potential of methanolic root extract of Stevia rebaudiana (Bertoni) on alloxan induced diabetic. J Pharm Pharm Sci 2014;3(7):1859-1872.##Ozbayer C, Degirmenci I, Hulyam K, Ozden H, Kısmet C, Basaran A, et al. Antioxidant and free radical-scavenging properties of Stevia rebaudiana (Bertoni) extracts and L-NNA in streptozotocine-nicotinamide induced diabetic rat liver. Turk Klin J Med Sci 2011;31(1):51-60.##Murali R, Saravanan R. Antidiabetic effect of d-limonene, a monoterpene in streptozotocin-induced diabetic rats. Biomed Prev Nutr 2012;2(4):269-275.##Lu YX, Zhang Q, Li J, Sun YX, Wang LY, Cheng WM, et al. Antidiabetic effects of total flavonoids from Litsea Coreana leve on fat-fed, streptozotocin-induced type 2 diabetic rats. Am J Chin Med 2010;38(4):713-725.##Misra H, Soni M, Silawat N, Mehta D, Mehta B, Jain D. Antidiabetic activity of medium-polar extract from the leaves of Stevia rebaudiana Bert.(Bertoni) on alloxan-induced diabetic rats. J Pharm Bioallied Sci 2011;3(2):242-248.##Taleie N, Hamidoghli Y, Rabiei B, Hamidoghli S. Effects of plant density and transplanting date on herbage, stevioside, phenol and flavonoid yield of Stevia rebaudiana Bertoni. Intl J Agri Crop Sci 2012;4(6):298-302.##Makapugay HC, Nanayakkara NPD, Soejarto DD, Kinghorn AD. High-performance liquid chromatographic analysis of the major sweet principle to Lo Han Kuo fruits. J Agric Food Chem 1985;33(3):348-350.##Dastgheib S, Irajie C, Assaei R, Koohpeima F, Mokarram P. Optimization of RNA extraction from rat pancreatic tissue. Iran J Med Sci 2014;39(3):282-288.##Hagar HH, El Etter E, Arafa M. Taurine attenuates hypertension and renal dysfunction induced by cyclosporine A in rats. Clin Exp Pharmacol Physiol 2006;33(3):189-196.##Goth L. A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 1991;196(2-3):143-151.##Yuan JS, Reed A, Chen F, Stewart CN Jr. Statistical analysis of real-time PCR data. BMC bioinformatics 2006;7:85.##Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29(9):e45.##Yan MX, Li YQ, Meng M, Ren HB, Kou Y. Long-term high-fat diet induces pancreatic injuries via pancreatic microcirculatory disturbances and oxidative stress in rats with hyperlipidemia. Biochem Biophys Res Commun 2006;347(1):192-199.##Brim H, Mokarram P, Naghibalhossaini F, Saberi-Firoozi M, Al-Mandhari M, Al-Mawaly K, et al. Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study. Mol Cancer 2008;7:68.##Gharaibeh MN, Elayan HH, Salhab AS. Hypoglycemic effects of Teucrium polium. J Ethnopharmacol 1988;24(1):93-99.##Aybar MJ, Sánchez Riera AN, Grau A, Sánchez SS. Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats. J Ethnopharmacol 2001;74(2):125-132.##Amin A, Lotfy M, Shafiullah M, Adeghate E. The protective effect of Tribulus terrestris in diabetes. Ann N Y Acad Sci 2006;1084:391-401.##Del Rio D, Stewart AJ, Pellegrini N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress. Nutr Metab Cardiovasc Dis 2005;15(4):316-328.##Rahimi R, Nikfar S, Larijani B, Abdollahi M. A review on the role of antioxidants in the management of diabetes and its complications. Biomed Pharmacother 2005;59(7):365-373.##Madan S, Ahmad S, Singh GN, Kohli K, Kumar Y, Singh R, et al. Stevia rebaudiana (Bert.) Bertonia review. Indian J Nat Prod Resour 2010;1(3):267-286.##Bracht AK, Alvarez M, Bracht A. Effects of stevia rebaudiana natural products on rat liver mitochondria. Biochem Pharmacol 1985;34(6):873-882.##Benford D, Hill MF, Schlatter J, DiNovi M. Steviol glycosides (addendum). Safety evaluation of certain food additives 2009:183.##Rafiq K, Sherajee SJ, Sufiun M, Mostofa M, Alam A, Barman B. Comparative efficacy of stevia leaf (stevia rebaudiana bertoni), methi seeds (trigonella foenum- graecum) and glimepiride in streptozotocin induced rats. Int J Phytopharmacol 2011;2(1):9-14.##Spiegelman BM. PPAR-gamma: adipogenic regulator and thiazolidinedione receptor. Diabetes 1998;47(4):507-514.##Wein S, Behm N, Petersen RK, Kristiansen K, Wolffram S. Quercetin enhances adiponectin secretion by a PPAR-gamma independent mechanism. Eur J Pharm Sci 2010;41(1):16-22.##Staels B, Fruchart JC. Therapeutic roles of peroxisome proliferator-activated receptor agonists. Diabetes 2005;54(8):2460-2470.##Ferreira EB, de Assis Rocha Neves F, da Costa MA, do Prado WA, de Araújo Funari Ferri L, Bazotte RB. Comparative effects of Stevia rebaudiana leaves and stevioside on glycaemia and hepatic gluconeogenesis. Planta Med 2006;72(8):691-696.##Atten MJ, Verma A, Liu K, Walter R, Attar BM, Gattuso P, et al. Antioxidants up-regulate PPARγ and decrease fibrosis in chronic pancreatitis. Am J Gastroenterol 2003;98:S64.##Ahmed I, Adeghate E, Sharma AK, Pallot DJ, Singh J. Effects of Momordica charantia fruit juice on islet morphology in the pancreas of the streptozotocin-diabetic rat. Diabetes Res Clin Pract 1998;40(3):145-151.##Bolkent S, Yanardağ R, Tabakoğlu-Oğuz A, Ozsoy-Saçan O. Effects of chard (Beta vulgaris L. var. Cicla) extract on pancreatic B cells in streptozotocin-diabetic rats: a morphological and biochemical study. J Ethnopharmacol 2000;73(1-2):251-259.##Geuns JM. Stevioside. Phytochemistry 2003;64(5):913-921.##Jeppesen PB, Gregersen S, Poulsen CR, Hermansen K. Stevioside acts directly on pancreatic beta cells to secrete insulin: actions independent of cyclic adenosine monophosphate and adenosine triphosphate-sensitivie K+-channel activity. Metabolism 2000;49(2):208-214.##Hossain MS, Alam MB, Asadujjaman M, Islam MM, Rahman MA, Islam M, et al. Antihyperglycemic and anti-hyperlipidemic effects of different fractions of Stevia rebaudiana leaves in alloxan induced diabetic rats. 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Induction of Apoptosis by Green Synthesized Gold Nanoparticles Through Activation of Caspase-3 and 9 in Human Cervical Cancer Cells 2 2 Background: Gold Nanoparticles (GNPs) are used in imaging and molecular diagnostic applications. As the development of a novel approach in the green synthesis of metal nanoparticles is of great importance and a necessity, a simple and safe method for the synthesis of GNPs using plant extracts of Zataria multiflora leaves was applied in this study and the results on GNPs’ anticancer activity against HeLa cells were reported. Methods: The GNPs were characterized by UV-visible spectroscopy, FTIR, TEM, DLS and Zeta-potential measurements. In addition, the cellular up-take of nanoparticles was investigated using Dark Field Microscopy (DFM). Induction of apoptosis by high dose of GNPs in HeLa cells was assessed by MTT assay, Acridin orange, DAPI staining, Annexin V/PI double-labeling flow cytometry and caspase activity assay. Results: UV-visible spectroscopy results showed a surface plasmon resonance band for GNPs at 530 nm. FTIR results demonstrated an interaction between plant extract and nanoparticles. TEM images revealed different shapes for GNPs and DLS results indicated that the GNPs range in size from 10 to 42 nm. The Zeta potential values of the synthesized GNPs were between 30 to 50 Mev, indicating the formation of stable particles. As evidenced by MTT assay, GNPs inhibit proliferation of HeLa cells in dose- dependent GNPs and cytotoxicity of GNPs in Bone Marrow Mesenchymal Stem Cell (BMSCs) was lower than cancerous cells. At nontoxic concentrations, the cellular up-take of the nanoparticles took place. Acridin orange and DAPI staining showed morphological changes in the cell’s nucleus due to apoptosis. Finally, caspase activity assay demonstrated HeLa cell’s apoptosis through caspase activation. Conclusion: The results showed that GNPs have the ability to induce apoptosis in HeLa cells. 75 83 Javad Baharara Animal Developmental Biology, Research Center for Animal Development Applied Biology and Department of Biology, Mashhad Branch, Islamic Azad University Animal Developmental Biology, Research Center for Animal Development Applied Biology and Department of Biology, Mashhad Branch, Islamic Azad University Iran Tayebe Ramezani Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Department of Animal Biology, Faculty of Biological Sciences, Kharazmi University Iran Adeleh Divsalar Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University Department of Cell and Molecular Biology, Faculty of Biological Sciences, Kharazmi University Iran Marzieh Mousavi Animal Developmental Biology, Research Center for Animal Development Applied Biology and Department of Biology, Mashhad Branch, Islamic Azad University Animal Developmental Biology, Research Center for Animal Development Applied Biology and Department of Biology, Mashhad Branch, Islamic Azad University Iran Arefeh Seyedarabi Institute of Biochemistry and Biophysics, University of Tehran Institute of Biochemistry and Biophysics, University of Tehran Iran BiosynthesisCaspaseGold nanoparticlesHeLa cellsZataria multiflora 237.pdf Kanipandian N, Kannan S, Ramesh R, Subramanian P, Thirumurugan R. Characterization, antioxidant and cytotoxicity evaluation of green synthesized silver nanoparticles using Cleistanthus collinus extract as surface modifier. Mater Res Bull 2014;49:494-502.##Liu X, Atwater M, Wang J, Huo Q. Extinction coefficient of gold nanoparticles with different sizes and different capping ligands. Colloids Surf B Biointerfaces 2007;58(1):3-7.##Abdelhalim MAK, Mady MM, Ghannam MM. Physical properties of different gold nanoparticles: Ultraviolet-Visible and fluorescence measurements. J Nanomed Nanotechnol 2012;3(03):2-5.##Chueh PJ, Liang RY, Lee YH, Zeng ZM, Chuang SM. Differential cytotoxic effects of gold nanoparticles in different mammalian cell lines. J Hazard Mater 2014;264:303-312.##Haratifar EAD, Shahverdi HR, Shakibaie M, Mollazadeh Moghaddam K, Amini M, Montazeri H, et al. Semi-biosynthesis of magnetite-gold composite nanoparticles using an ethanol extract of eucalyptus camaldulensis and study of the surface chemistry. J Nanomater 2009;2009:1-5.##Motevasel M, Okhovat MA, Zomorodian K, Farshad S. A study of the effect of Zataria multiflora extract on methicillin resistant staphylococcus aureus. Jundishapur J Microbiol 2013;6(5).##Shokri H, Asadi F, Bahonar AR, Khosravi AR. The role of Zataria multiflora essence (Iranian herb) on innate immunity of animal model. Iran J Immunol 2006;3(4):164-168.##Yahyaraeyat R, Khosravi AR, Shahbazzadeh D, Khalaj V. The potential effects of Zataria multiflora Boiss essential oil on growth, aflatoxin production and transcription of aflatoxin biosynthesis pathway genes of toxigenic Aspergillus parasiticus. Braz J Microbiol 2013;44(2):643-649.##Pulit J, Banach M, Kowalski Z. Nanosilver-making difficult decisions. Ecol Chem Eng S 2011;18(2):185-196.##Gericke M, Pinches A. Microbial production of gold nanoparticles. Gold Bull 2006;39(1):22-28.##Geetha R, Ashokkumar T, Tamilselvan S, Govindaraju K, Sadiq M, Singaravelu G. Green synthesis of gold nanoparticles and their anticancer activity. Cancer Nanotechnol 2013;4(4-5):91-98.##Lan MY, Hsu YB, Hsu CH, Ho CY, Lin JC, Lee SW. Induction of apoptosis by high-dose gold nanoparticles in nasopharyngeal carcinoma cells. Auris Nasus Larynx 2013;40(6):563-568.##He C, Hu Y, Yin L, Tang C, Yin C. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010;31(13):3657-3666.##Alkilany AM, Murphy CJ. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res 2010;12(7):2313-2333.##Huang X, Qian W, El-Sayed IH, El-Sayed MA. The potential use of the enhanced nonlinear properties of gold nanospheres in photothermal cancer therapy. Lasers Surg Med 2007;39(9):747-753.##Ying TH, Yang SF, Tsai SJ, Hsieh SC, Huang YC, Bau DT, et al. Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway. Arch Toxicol 2012;86(2):263-273.##Klekotko M, Matczyszyn K, Siednienko J, Olesiak-Banska J, Pawlik K, Samoc M. Bio-mediated synthesis, characterization and cytotoxicity of gold nanoparticles. Phys Chem Chem Phys 2015;17(43):29014-29019.##Huang X, Jain PK, El-Sayed IH, El-Sayed MA. Determination of the minimum temperature required for selective photothermal destruction of cancer cells with the use of immunotargeted gold nanoparticles. Photochem Photobiol 2006;82(2):412-417.##Hitchman A, Smith GH, Ju-Nam Y, Sterling M, Lead JR. The effect of environmentally relevant conditions on PVP stabilised gold nanoparticles. Chemosphere 2013;90(2):410-416.##Mytych J, Lewinska A, Zebrowski J, Wnuk M. Gold nanoparticles promote oxidant-mediated activation of NF-κB and 53BP1 recruitment-based adaptive response in human astrocytes. Biomed Res Int 2015;2015:304575.##Bhowmik T, Saha PP, Dasgupta A, Gomes A. Antileukemic potential of PEGylated gold nanoparticle conjugated with protein toxin (NKCT1) isolated from Indian cobra (Naja kaouthia) venom. Cancer Nanotechnol 2013;4(1-3):39-55.##

Chimeric External Control to Quantify Cell Free DNA in Plasma Samples by Real Time PCR 2 2 Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA (cfDNA) is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control (EDC) is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time. Methods: A DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/ 500 μl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples. Results: Comparison of real time PCR threshold cycle (Ct) for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them. Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis. 84 90 Maryam Eini Department of Medical Biotechnology, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesStudent Research Committee, Shiraz University of Medical Sciences Department of Medical Biotechnology, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesStudent Research Committee, Shiraz University of Medical Sciences IranIran Abbas Behzad-Behbahani Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Mohammad Ali Takhshid Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Amin Ramezani Department of Medical Biotechnology, Faculty of Advanced Medical Sciences and Technology, Shiraz University of Medical SciencesInstitute for Cancer Research, Shiraz University of Medical Sciences Department of Medical Biotechnology, Faculty of Advanced Medical Sciences and Technology, Shiraz University of Medical SciencesInstitute for Cancer Research, Shiraz University of Medical Sciences IranIran Gholam Reza Rafiei Dehbidi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Mohammad Ali Okhovat Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Ali Farhadi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran Parniyan Alavi Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences Iran DNAReal time polymerase chain reactionReference standards 238.pdf Anker P, Mulcahy H, Stroun M. Circulating nucleic acids in plasma and serum as a noninvasive investigation for cancer: Time for large‐scale clinical studies? Int J Cancer 2003;103(2):149-152.##Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62(4):768-775.##Ziegler A, Zangemeister-Wittke U, Stahel RA. Circulating DNA: a new diagnostic gold mine? Cancer Treat Rev 2002;28(5):255-271.##Fleischhacker M, Schmidt B. Circulating nucleic acids (CNAs) and cancer--a survey. Biochim Biophys Acta 2007;1775(1):181-232.##Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YM. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002;48(3):421-427.##Chiu RW, Poon LL, Lau TK, Leung TN, Wong EM, Lo YM. Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 2001;47(9):1607-1613.##Swarup V, Rajeswari M. Circulating (cell-free) nucleic acids--a promising, non-invasive tool for early detection of several human diseases. FEBS letters 2007;581(5):795-799.##Vandenbroucke II, Vandesompele J, Paepe AD, Messiaen L. Quantification of splice variants using real-time PCR. Nucleic Acids Res 2001;29(13):E68.##Mengelle C, Pasquier C, Rostaing L, Sandres‐Sauné K, Puel J, Berges L, et al. Quantitation of human cytomegalovirus in recipients of solid organ transplants by real‐time quantitative PCR and pp65 antigenemia. J Med Virol 2003;69(2):225-231.##Rose’Meyer RB, Mellick AS, Garnham BG, Harrison GJ, Massa HM, Griffiths LR. The measurement of adenosine and estrogen receptor expression in rat brains following ovariectomy using quantitative PCR analysis. Brain Res Brain Res Protoc 2003;11(1):9-18.##Dworkin LL, Gibler TM, Van Gelder RN. Real-time quantitative polymerase chain reaction diagnosis of infectious posterior uveitis. Arch Ophthalmol 2002;120 (11):1534-1539.##Hufnagl C, Stöcher M, Moik M, Geisberger R, Greil R. A modified phenol-chloroform extraction method for isolating circulating cell free DNA of tumor patients. J Nucleic Acids Investig 2013;4(1):e1.##Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29(9):e45.##Pinzani P, Salvianti F, Pazzagli M, Orlando C. Circulating nucleic acids in cancer and pregnancy. Methods 2010;50(4):302-307.##Anker P, Lyautey J, Lederrey C, Stroun M. Circulating nucleic acids in plasma or serum. Clin Chim Acta 2001;313(1-2):143-146.##Gormally E, Caboux E, Vineis P, Hainaut P. Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res 2007;635(2-3):105-117.##Suzuki N, Kamataki A, Yamaki J, Homma Y. Characterization of circulating DNA in healthy human plasma. 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LC-MS Method for Studying the Pharmacokinetics and Bioequivalence of Clonidine Hydrochloride in Healthy Male Volunteers 2 2 Background: A simple and sensitive high performance liquid chromatography-electrospray ionization mass spectrometry method has been evaluated for the assignment of clonidine hydrochloride in human plasma. Methods: The mobile phase composed of acetonitrile-water 60:40 (v/v) and 0.2% formic acid 20 µl of sample was chromatographically analyzed using a repacked ZORBAX-XDB-ODS C18 column (2.1 mmx30 mm, 3.5 μ). Detection of analytes was achieved by tandem mass spectrometry with Electrospray Ionization (ESI) interface in positive ion mode operated under the multiple-reaction monitoring mode (m/z 230.0 →213). Sample pretreatment consisted of a one-step Protein Precipitation (PPT) with methanol and perchloric acid (HClO4) of 0.10 ml plasma. Results: Standard curve was linear (r=0.998) over the concentration range of 0.01-10.0 ng/ml and showed suitable accuracy and precision. The Limit of Quantification (LOQ) was 0.01 ng/ml. The mean (SD) Cmax, Tmax, AUC0–t and AUC0–∞ values after administration of the test and reference formulations, respectively, were in this manner: 6.16 (0.32) versus 6.21 (0.07) ng/ml, 30.12 (0.86) versus 30.13 (0.73) hr, 290.37 (1.13) versus 293.39 (1.22) ng/ml/hr, and 350.17 (1.98) versus 352.96 (1.67) ng/ml/hr. The mean (SD) t1/2 was 120.12 (1.90) hr for the test formulation and 120.96 (1.54) hr for the reference formulation. No statistical differences were showed for Cmax and the area under the plasma concentration-time curve for test and reference tablets. Conclusion: The method is rapid, simple, very steady and precise for the separation, assignment, pharmacokinetic and bioavailability evaluation of clonidine in healthy Iranian adult male volunteers. 91 98 Hossein Danafar Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical SciencesDepartment of Medicinal Chemistry, Faculty of Pharmacy, Zanjan University of Medical Sciences Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical SciencesDepartment of Medicinal Chemistry, Faculty of Pharmacy, Zanjan University of Medical Sciences IranIran Mehrdad Hamidi Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical SciencesDepartment of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences Zanjan Pharmaceutical Nanotechnology Research Center, Zanjan University of Medical SciencesDepartment of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences IranIran Clonidine hydrochlorideHigh performance liquid chromatographyPharmacokinetics 239.pdf Neil MJ. Clonidine: clinical pharmacology and therapeutic use in pain management. Curr Clin Pharmacol 2011;6:280-287.##Fujimura A, Ebihara A, Ohashi K, Shiga T, Kumagai Y, Nakashima H, et al. Comparison of the pharmacokinetics, pharmacodynamics, and safety of oral (Catapres) and transdermal (M-5041T) clonidine in healthy subjects. J Clin Pharmacol 1994;34(3):260-265.##Ke GM, Zhang EH, Wang L, Zhang Q, Du HG, Guo HY. [Determination of clonidine in rabbit plasma by HPLC-MS]. Yao Xue Xue Bao 2004;39(5):367-369. Chinese.##Ke GM, Wang L, Xue HY, Lu WL, Zhang X, Zhang Q, et al. In vitro and in vivo characterization of a newly developed clonidine transdermal patch for treatment of attention deficit hyperactivity disorder in children. Biol Pharm Bull 2005;28(2):305-310.##Zhao L, Ding L, Wei X. Determination of moxonidine in human plasma by liquid chromatography-electrospray ionization-mass spectrometry. J Pharm Biomed 2006;40 (1):95-99.##Hercegová A, Sádecká J, Polonský J. Isotachophoretic determination of bisoprolol, clonidine, disopyramide and tolazoline in human fluids. Acta Pol Pharm 1998;55(3):167-171.##Larsson P, Nordlinder A, Bergendahl HT, Lönnqvist PA, Eksborg S, Almenrader N, Anderson BJ. Oral bioavailability of clonidine in children. Paediatr Anaesth 2011;21(3):335-340.##Ghosh C, Singh RP, Inamdar S, Mote M, Chakraborty BS. Sensitive, selective, precise and accurate LC-MS method for determination of clonidine in human plasma. Chromatographia 2009;69(11):1227-1232.##Wolf CE, Poklis A. A rapid HPLC procedure for analysis of analgesic pharmaceutical mixtures for quality assurance and drug diversion testing. J Anal Toxicol 2005;29(7):711-714.##Walters SM, Stonys DB. Determination of chlorthalidone and clonidine hydrochloride in tablets by HPLC. J Chromatogr Sci 1983;21(1):43-45.##Arrendale RF, Stewart JT, Tackett RL. Determination of clonidine in human plasma by cold on-column injection capillary gas chromatography-selected-ion monitoring-mass spectrometry. J Chromatogr 1988;432:165-175.##Todesco LM, Thoma JJ, Barth RD, Myers NJ, White R, Ward RM. Quantitative determination of tolazoline in human serum by high performance liquid chromatography. Ther Drug Monit 1987;9(1):78-84.##Yamahata T, Dote S, Ozawa Y, Nishikawa H, Maeda S. Determination of clonidine in human plasma by gas chromatography-electron-impact mass spectrometry. J Chromatogr B Biomed Appl 1994;653(1):92-97.##Wilczyńska-Wojtulewicz I, Sadlej-Sosnowska N. Determination of clonidine hydrochloride in pharmaceutical preparations by high-performance liquid chromatography. J Chromatogr 1986;367(2):434-437.##Muller C, Ramic M, Harlfinger S, Hunseler C, Theisohn M, Roth B. Sensitive and convenient method for the quantification of clonidine in serum of pediatric patients using liquid chromatography/tandem mass spectrometry. J Chromatogr A 2007;1139:221-227.##Li F, McMahon C, Li F, Zulkoski J. LC-MS/MS sensitivity enhancement using 2D-SCX/RPLC and its application in the assessment of pharmacokinetics of clonidine in dried blood spots. Bioanalysis 2011;3(14):1577-1586.##Parekh SA, Pudage A, Joshi SS, Vaidya VV, Gomes NA. Rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of clonidine in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2008;867(2):172-178.##Danafar H, Hamidi M. Pharmacokinetics and bioequivalence study of amlodipine and atorvastatin in healthy male volunteers by LC-MS. Pharm Sci 2015;21(Suppl 1):167-174.##Danafar H, Hamidi M. Simple and sensitive high-performance liquid chromatography (HPLC) method with UV detection for mycophenolic acid assay in human plasma. Application to a bioequivalence study. Adv Pharm Bull 2015;5(4):563-568.##Danafar H, Hamidi M. Liquid chromatography-tandem mass spectrometry (LC-MS) method for the assignment of enalapril and enalaprilat in human plasma. Pharm Biomed Res 2015;1(3):47-58.##Danafar H, Hamidi M. Simple and sensitive high performance liquid chromatographic method for the simultaneous quantitation of the phenylalanine in human plasma. Pharm Biomed Res 2015;1(3):11-19.##Chorilli M, Bonfilio R, Louvandini CR, Goncalves FARM, Salgado HRN. Development and validation of an LC-MS/MS method for quantitative analysis of mirtazapine in human plasma. Am J Analyt Chem 2011;2:650-657.##Danafar H, Hamidi M. A rapid and sensitive LC-MS method for determination of ezetimibe concentration in human plasma: Application to a bioequivalence study. Chromatographia 2013;76(23):1667-1675.##Boroujerdi M. Pharmacokinetics, principles and applications. 1st ed. New York: McGraw-Hill; 2002. 528 p.##Asiri YA, Al-Said MS, Al-Khamis KI, Niazy EM, Al-Sayed YM, Al-Rashood KA, et al. 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Distribution and Diversity of hmw1A Among Invasive Nontypeable Haemophilus influenzae Isolates in Iran 2 2 Background: The pathogenesis of nontypeable Haemophilus influenzae (NTHi) begins with adhesion to the rhinopharyngeal mucosa. Almost 38-80% of NTHi clinical isolates produce proteins that belong to the High Molecular Weight (HMW) family of adhesins, which are believed to facilitate colonization. Methods: In the present study, the prevalence of hmwA, which encodes the HMW adhesin, was determined for a collection of 32 NTHi isolates. Restriction Fragment Length Polymorphism (RFLP) was performed to advance our understanding of hmwA binding sequence diversity. Results: The results demonstrated that hmwA was detected in 61% of NTHi isolates. According to RFLP, isolates were divided into three groups. Conclusion: Based on these observations, it is hypothesized that some strains of nontypeable Haemophilus influenzae infect some specific areas more than other parts. 99 102 Milad Shahini Shams abadi Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Seyed Davar Siadat Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Farzam Vaziri Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Mehdi Davari Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Abolfazl Fateh Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Shahin Pourazar Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Farid Abdolrahimi Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran Morteza Ghazanfari Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran Iran AdhesinsHaemophilus influenzaHMW1 241.pdf Barenkamp SJ, St Geme JW 3rd. Identification of a second family of high‐molecular‐weight adhesion proteins expressed by non‐typable Haemophilus influenzae. Mol Microbiol 1996;19(6):1215-1223.##Davis GS, Patel M, Hammond J, Zhang L, Dawid S, Marrs CF, et al. Prevalence, distribution, and sequence diversity of hmwA among commensal and otitis media non-typeable Haemophilus influenzae. Infect Genet Evol 2014;28:223-232.##Davis GS. Nontypeable Haemophilus influenzae high molecular weight adhesins: molecular epidemiology, evolution, and within-host population dynamics [dissertation]. [USA]: University of Michigan; 2013. 203 p.##Rao VK, Krasan GP, Hendrixson DR, Dawid S, St Geme JW 3rd. Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae. FEMS Microbiol Rev 1999;23(2):99-129.##Erwin AL, Sandstedt SA, Bonthuis PJ, Geelhood JL, Nelson KL, Unrath WC, et al. Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing. J Bacteriol 2008;190(4):1473-1483.##Buscher AZ, Burmeister K, Barenkamp SJ, St Geme JW 3rd. Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins. J Bacteriol 2004;186(13):4209-4217.##Barenkamp SJ, St Geme JW 3rd. Genes encoding high-molecular-weight adhesion proteins of nontypeable Haemophilus influenzae are part of gene clusters. Infect Immun 1994;62(8):3320-3328.##Ecevit IZ, McCrea KW, Marrs CF, Gilsdorf JR. Identification of new hmwA alleles from nontypeable Haemophilus influenzae. Infect Immun 2005;73(2):1221-1225.##Grass S, St Geme JW 3rd. Maturation and secretion of the non‐typable Haemophilus influenzae HMW1 adhesin: roles of the N‐terminal and C‐terminal domains. Mol Microbiol 2000;36(1):55-67.##Xie J, Juliao PC, Gilsdorf JR, Ghosh D, Patel M, Marrs CF. Identification of new genetic regions more prevalent in nontypeable Haemophilus influenzae otitis media strains than in throat strains. J Clin Microbiol 2006;44(12):4316-4325.##Dawid S, Grass S, St Geme JW 3rd. Mapping of binding domains of nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins. Infect Immun 2001;69(1):307-314.##Giufrè M, Muscillo M, Spigaglia P, Cardines R, Mastrantonio P, Cerquetti M. Conservation and diversity of HMW1 and HMW2 adhesin binding domains among invasive nontypeable Haemophilus influenzae isolates. Infect Immun 2006;74(2):1161-1170.##St Geme JW 3rd. The pathogenesis of nontypable Haemophilus influenzae otitis media. Vaccine 2000;19 Suppl 1:S41-50.##van Schilfgaarde M, van Alphen L, Eijk P, Everts V, Dankert J. Paracytosis of Haemophilus influenzae through cell layers of NCI-H292 lung epithelial cells. Infect Immun 1995;63(12):4729-4737.##Hardy GG, Tudor SM, St Geme JW 3rd. The pathogenesis of disease due to nontypeable Haemophilus influenzae. Methods Mol Med 2003;71:1-28.##Erwin AL, Smith AL. Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior. Trends Microbiol 2007;15(8):355-362.##Barenkamp SJ, Leininger E. Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis. Infect Immun 1992;60(4):1302-1313.##Cholon DM, Cutter D, Richardson SK, Sethi S, Murphy TF, Look DC, et al. Serial isolates of persistent Haemophilus influenzae in patients with chronic obstructive pulmonary disease express diminishing quantities of the HMW1 and HMW2 adhesins. Infect Immun 2008;76(10):4463-4468.##Davis GS, Marino S, Marrs CF, Gilsdorf JR, Dawid S, Kirschner DE. Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts. J Theor Biol 2014;355:208-218.##Dawid S, Barenkamp SJ, St Geme JW 3rd. Variation in expression of the Haemophilus influenzae HMW adhesins: a prokaryotic system reminiscent of eukaryotes. Proc Natl Acad Sci USA 1999;96(3):1077-1082.##Noel GJ, Love DC, Mosser DM. High-molecular-weight proteins of nontypeable Haemophilus influenzae mediate bacterial adhesion to cellular proteoglycans. Infect Immun 1994;62(9):4028-4033.##