IVF Technology 2 2 The Parliament of Great Britain approved three-parent IVF project with 382 votes for and 128 votes against it on 3rd February, 2015. In case of the project approval in House of Lords, Great Britain would be the first country which legalizes the action. The purpose of this procedure is prevention of genetic diseases with mitochondrial defects in which the defective mitochondria in the cytoplasm of the mother’s egg is transferred to the embryo. The consequence is the death of the child, muscle weakness, blindness and heart diseases. If the healthy mitochondria of another person are used in this procedure, the defects would disappear. This is done in two ways: 1. Nucleus transfer from the mother's egg with mitochondrial defects or egg cytoplasm to the donated egg in which the nucleus has been removed, 2. Transfer of parents’ nuclei from the early embryo (zygote) containing the cytoplasm to a donated early embryo (zygote) in which the parents’ nuclei have been removed. In each of these cases, the mitochondria in the egg or the donated zygote replace defective mitochondria and protect the child from related diseases. The advancement of IVF technology brought up the possibility in replacing the egg of patients with mitochondrial diseases with healthy donated eggs. In this case, the child produced has no genetic relationship with the patient, while in the current practice, 23 pairs of parental chromosomes construct the genetic essence of the child. In this practice, 37 mitochondrial genes, which comprise 1.10% of the total genetic essence of the child and play key roles in production of energy in cytoplasm of the egg, can be replaced with donated mitochondria in case of any defects, and thereby culminate in preventing mitochondrial defects in 1200 cases of children in Great Britain. The support of majority of reproduction scientists from the action and the concern and opposition of the Catholic Church together with some law and ethics specialists for authorizing the birth of engineered children denote the contrary position of the parties in applying this preventive and therapeutic action. The advent and development of reproductive technologies have always been intermingled with theological, ethical, legal and social issues and consequences. While great emphasis has been placed on ethical, legal and social dimensions and implications of cutting edge advances in reproduction, it seems that under supervision and surveillance of law makers and regulations, the possibility for amending inappropriate decisions is always provided. This is the focal point that the Parliament of Great Britain adhered to. However, there is no doubt that if necessary practical measures are not taken for discussing problems and compiling rules and regulations in treatment of reproductive diseases with higher incidence (such as application of IVF in indications for surrogacy in Iran), more theoretical and practical vexing issues will emerge which are complicated enough to be handled optimally. Although further studies are required to evaluate the action approved by the Parliament of Great Britain, the legal aspect of the action is worthy of attention since timely evaluation of it has been done. Also, as a prominent advancement in the field of biotechnology, the potential for its modification and amendment is permitted by the law. 49 49 Mohammad Mehdi Akhondi Editorial 201.pdf ####

Comparison between Platinum-Azidothymidine and Azidothymidine Effects on Bcl-2 and Telomerase Gene Expression in Rats with Hepatocellular Carcinoma 2 2 Background: High expression of telomerase and Bcl-2 are reported in hepatocellular carcinoma. Some anticancer drugs show their effects through reduction of these factors. In this study, it was aimed to investigate the effects of a new synthetic compound, platinum azidothymidine, on inhibition of telomerase and Bcl-2 expression in hepatocellular carcinoma compared to azidothymidine. Methods: To study the effects of Pt-AZT on hepatocellular carcinoma and compare its effects with AZT in inhibition of telomerase and Bcl-2 gene expression, pathogen-free male Wistar rats (n=100) were used. They were randomly divided to 4 groups (n=25). Group A as the control group contained 25 healthy rats; in the rest of animals, preneoplastic lesions were induced in their livers (groups B, C, and D) using Solt-Farber resistant hepatocyte protocol. Cancer development was approved by a pathology laboratory. Group B was negative control (untreated), groups C and D were treated by intraperitoneal injection (IP) of Pt-AZT (0.9 mg/kg/day) and AZT (0.3 mg/kg/day), respectively for 14 days. At the end of the protocol, all rats were sacrificed and Bcl-2 and telomerase gene expression was determined using real -time PCR. Results: No tumor in the livers was found in group A at any point of the study, but it was present in livers of all animals in B, C and D groups. Results showed that telomerase and Bcl-2 expression was significantly lower in group C compared with group B (0.473±0.231 vs. 5.137±5.08, p<0.001, for telomerase expression, and 0.41±0.276 vs. 7.25±11.6, p<0.001, for Bcl-2 expression) and also compared with group D (0.473±0.231 vs. 3.48±4.02, p<0.001, for telomerase expression, and 0.41±0.276 vs. 4.93±18, p<0.001, for Bcl-2 expression). Conclusion: For the first time, it was demonstrated that Pt-AZT has more inhibitory effect on telomerase and Bcl-2 expression than AZT. It effectively inhibits the growth of liver tumor in rats by extending apoptosis. 50 56 Abdolreza Sabokrouh Department of Clinical Biochemistry, Hamadan University of Medical Sciences Department of Clinical Biochemistry, Hamadan University of Medical Sciences Iran Asad Vaisi-Raygani Molecular Diagnostic Research Center, Kermanshah University of Medical Sciences Molecular Diagnostic Research Center, Kermanshah University of Medical Sciences Iran Mohammad Taghi Goodarzi Department of Clinical Biochemistry, Hamadan University of Medical SciencesResearch Center for Molecular Medicine, Hamadan University of Medical Sciences Department of Clinical Biochemistry, Hamadan University of Medical SciencesResearch Center for Molecular Medicine, Hamadan University of Medical Sciences IranIran Shohreh Khatami Department of Clinical Biochemistry, Pasteur Institute Department of Clinical Biochemistry, Pasteur Institute Iran Masoud Taghizadeh-Jahed Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Nahid Shahabadi Department of Chemistry, Razi University of Kermanshah, Kermanshah Department of Chemistry, Razi University of Kermanshah, Kermanshah Iran Niknam Lakpour Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Yadollah Shakiba Molecular Diagnostic Research Center, Kermanshah University of Medical Sciences Molecular Diagnostic Research Center, Kermanshah University of Medical Sciences Iran Gene expressionHepatocellular carcinomaPlatinumTelomerase 202.pdf Cannon G, Gupta P, Gomes F, Kerner J, Parra W, Weiderpass E, et al. 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Bcl-2 family proteins as targets for anticancer drug design. Oncogene 2000;19(56):6627-6631.##Ji HJ, Rha SY, Jeung HC, Yang SH, An SW, Chung HC. Cyclic induction of senescence with intermittent AZT treatment accelerates both apoptosis and telomere loss. Breast Cancer Res Treat 2005;93(3):227-236.##Brown T, Sigurdson E, Rogatko A, Broccoli D. Telomerase inhibition using azidothymidine in the HT-29 colon cancer cell line. Ann Surg Oncol 2003;10(8):910-915.##Tejera AM, Alonso DF, Gomez DE, Olivero OA. Chronic in vitro exposure to 3'-azido-2', 3'-dideoxythymidine induces senescence and apoptosis and reduces tumorigenicity of metastatic mouse mammary tumor cells. Breast Cancer Res Treat 2001;65(2):93-99.##Engelhardt M, Kumar R, Albanell J, Pettengell R, Han W, Moore MA. Telomerase regulation, cell cycle, and telomere stability in primitive hematopoietic cells. Blood 1997;90(1):182-193.##Meeker AK, De Marzo AM. Recent advances in telomere biology: implications for human cancer. Curr Opin Oncol 2004;16(1):32-38.##Mediavilla MD, Sanchez-Barcelo EJ. Doses and time-dependent effects of 3'-azido-3'-deoxythymidine on T47D human breast cancer cells in vitro. Pharmacol Toxicol 2000;87(3):138-143.##Marty R, Ouameur AA, Neault JF, Nafisi S, Tajmir-Riahi HA. AZT-DNA interaction. DNA Cell Biol 2004;23(3):135-140.##Melana SM, Holland JF, Pogo BG. Inhibition of cell growth and telomerase activity of breast cancer cells in vitro by 3'-azido-3'-deoxythymidine. Clin Cancer Res 1998;4(3):693-696.##Sabokrouh A, Goodarzi MT, Vaisi-Raygani A, Khatami S, Taghizadeh-Jahed M. Effects of treatment with platinum azidothymidine and azidothymidine on telomerase activity and bcl-2 concentration in hepatocellular carcinoma- induced rats. Avicenna J Med Biotechnol 2014;6(4):200-209.##Coluccia M, Boccarelli A, Cermelli C, Portolani M, Natile G. Platinum(II)-Acyclovir Complexes: Synthesis, Antiviral and Antitumour Activity. 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The Effects of Ultraviolet Light and Riboflavin on Inactivation of Viruses and the Quality of Platelet Concentrates at Laboratory Scale 2 2 Background: This study investigated the effects of Riboflavin (RB) combined with different doses of UV on Platelet Concentrate (PC) which was infected by three models of virus. Platelet quality after treatment was also assessed. Methods: Three models of virus used in this study were Vesicular Stomatitis Virus (VSV), Herpes Simplex Virus (HSV), and Polio virus, which were added to PC. After photochemical treatment with RB and UV light, residual viral infectivity was titrated using 50% Tissue Culture Infective Dose (TCID50)/ml. This treatment was done with concentration of 50 µM of RB and different doses of UV light (0.24, 0.48, 0.97, 1.29 J/cm2). Platelet quality was assessed by measuring pH, Lactate Dehydrogenase (LDH), MTT assay and cell count after treatments and during 4 days of storage against control groups. Results: Concentration of 50 µM RB with combination of 1.29 J/cm2 dose of UV resulted in the highest titer reduction of VSV (4 log 10) and HSV (4.26 log10) and lowest titer reduction of Polio virus (2.6 log10). No significant difference was observed between different doses in comparison with control groups. In all treatment groups, the storage stability of platelets in PC was in the acceptable range in comparison with control group. Conclusion: This study indicated that RB/UV treatment was a promising pathogen reduction technique in PC and had limited effects on platelet quality. However, further optimization of this method is necessary to deal with blood-borne viruses like non-enveloped viruses. 57 63 Hamideh Mirshafiee Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti 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 Seyed Masoud Hosseini Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University Department of Microbiology, Faculty of Biological Sciences, Shahid Beheshti University Iran Fatemeh Yari 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 Hamed Nikbakht Laser and Plasma Research Institute, Shahid Beheshti University Laser and Plasma Research Institute, Shahid Beheshti University Iran Hamid Latifi Laser and Plasma Research Institute, Shahid Beheshti University Laser and Plasma Research Institute, Shahid Beheshti University Iran Blood plateletRiboflavinUltraviolet lightVirus inactivation 203.pdf Seghatchian J, de Sousa G. Pathogen-reduction systems for blood components: the current position and future trends. Transfus Apher Sci 2006;35(3):189-196.##Kaur P, Basu S. Transfusion-transmitted infections: existing and emerging pathogens. J Postgrad Med 2005;51(2):146-151.##Goodrich R, Keil Sh D. Induction of maintenance of nucleic acid damage in pathogens using riboflavin and light. United States patent US 7,985,588 B2. 2011.##Kumar V, Lockerbie O, Keil SD, Ruane PH, Platz MS, Martin CB, et al. Riboflavin and UV-light based pathogen reduction: extent and consequence of DNA damage at the molecular level. Photochem Photobiol 2004;80:15-21.##George JN. Platelets. Lancet 2000;355(9214):1531-1539.##Saran RK. Transfusion medicine technical manual. 2nd. ed. New Delhi, India: WHO; 2003.##Raback JD, et al. Technical manual. 17th ed. AABB 2011;199-219.##Ruane PH, Edrich R, Gampp D, Keil SD, Leonard RL, Goodrich RP. Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light. Transfusion 2004;44(6):877-885.##Elikaei A, Sharifi Z, Hosseini SM, Latifi H, Hosseini MKM. Inactivation of model viruses suspended in fresh frozen plasma using novel methylene blue based device. Iran J Microbiol 2014;6(1):41-45.##Berridge MV, Herst PM, Tan AS. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 2005;11:127-152.##Heal JM, Blumberg N. Optimizing platelet transfusion therapy. Blood Rev 2004;18(3):149-165.##Goodrich RP, Li J, Pieters H, Crookes R, Roodt J, Heyns Adu P. Correlation of in vitro platelet quality measurements with in vivo platelet viability in human subjects. Vox Sang 2006;90(4):279-285.##Pelletier JP, Transue S, Snyder EL. Pathogen inactivation techniques. Best Pract Res Clin Haematol 2006;19(1):205-242.##Keil SD, Kiser P, Sullivan JJ, Kong AS, Reddy HL, Avery A, et al. Inactivation of Plasmodium spp. in plasma and platelet concentrates using riboflavin and ultraviolet light. Transfusion 2013;53(10):2278-2286.##Roback JD, Combs MR, Grossman BJ, Hillyer CD, eds. Technical manual. 16th ed. Bethesda, MD: American Association of Blood Banks, 2008.##Xia Y, Li J, Bertino A, Kuter DJ. Thrombopoietin and the TPO receptor during platelet storage. Transfusion 2000;40(8):976-987.##Li J, Lockerbie O, de Korte D, Rice J, McLean R, Goodrich RP. Evaluation of platelet mitochondria integrity after treatment with mirasol pathogen reduction technology. Transfusion 2005;45(6):920-926.##Galan AM, Lozano M, Molina P, Navalon F, Marschner S, Goodrich R, et al. Impact of pathogen reduction technology and storage in platelet additive solutions on platelet function. Transfusion 2011;51(4):808-815.##AuBuchon JP, Herschel L, Roger J, Taylor H, Whitley P, Li J, et al. Efficacy of apheresis platelets treated with riboflavin and ultraviolet light for pathogen reduction. Transfusion 2005;45(8):1335-1341.##Reikvam H, Marschner S, Apelseth TO, Goodrich R, Hervig T. The mirasol pathogen reduction technology system and quality of platelets stored in platelet additive solution. Blood Transfus 2010;8(3):186-192.##Johnson L, Winter KM, Reid S, Hartkopf-Theis T, Marschner S, Goodrich RP, et al. The effect of pathogen reduction technology (Mirasol) on platelet quality when treated in additive solution with low plasma carryover. Vox Sang 2011;101(3):208-214.##Marschner S, Goodrich R. Pathogen reduction technology treatment of platelets, plasma and whole blood using riboflavin and UV light. Transfus Med Hemother 2011;38(1):8-18.##

Cobalt Zinc Ferrite Nanoparticles as a Potential Magnetic Resonance Imaging Agent: An In Vitro Study 2 2 Background: Magnetic Nanoparticles (MNP) have been used for contrast enhancement in Magnetic Resonance Imaging (MRI). In recent years, research on the use of ferrite nanoparticles in T2 contrast agents has shown a great potential application in MR imaging. In this work, Co0.5Zn0.5Fe2O4 and Co0.5Zn0.5Fe2O4-DMSA magnetic nanoparticles, CZF-MNPs and CZF-MNPs-DMSA, were investigated as MR imaging contrast agents. Methods: Cobalt zinc ferrite nanoparticles and their suitable coating, DMSA, were investigated under in vitro condition. Human prostate cancer cell lines (DU145 and PC3) with bare (uncoated) and coated magnetic nanoparticles were investigated as nano-contrast MR imaging agents. Results: Using T2-weighted MR images identified that signal intensity of bare and coated MNPs was enhanced with increasing concentration of MNPs in water. The values of 1/T2 relaxivity (r2) for bare and coated MNPs were found to be 88.46 and 28.80 (mM–1 s–1), respectively. Conclusion: The results show that bare and coated MNPs are suitable as T2-weighted MR imaging contrast agents. Also, the obtained r2/r1 values (59.3 and 50) for bare and coated MNPs were in agreement with the results of other previous relevant works. 64 68 Zeinab Ghasemian Department of Medical Physics, Faculty of Medicine, Isfahan University of Medical Sciences Department of Medical Physics, Faculty of Medicine, Isfahan University of Medical Sciences Iran Daryoush Shahbazi-Gahrouei Department of Medical Physics, Faculty of Medicine, Isfahan University of Medical Sciences Department of Medical Physics, Faculty of Medicine, Isfahan University of Medical Sciences Iran Sohrab Manouchehri Department of Physics, Nano-center, Maleke-ashtar University of Technology, Shahin-shahr Department of Physics, Nano-center, Maleke-ashtar University of Technology, Shahin-shahr Iran Magnetite nanoparticles MR imagingProstatic neoplasm 204.pdf Massoud TF, Gambhir SS. Molecular imaging in living subjects: seeing fundamental biological processes in a new light. Genes Dev 2003;17(5):545-580.##Shahbazi-Gahrouei D, Williams M, Rizvi S, Allen BJ. In vivo studies of Gd-DTPA-monoclonal antibody and gd-porphyrins: potential magnetic resonance imaging contrast agents for melanoma. J Magn Reson Imaging 2001;14(2):169-174.##Corti M, Lascialfari A, Micotti E, Castellano A, Donativi M, Quarta A, et al. Magnetic properties of novel superparamagnetic MRI contrast agents based on colloidal nanocrystals. J Magn Magn Mater 2008;320(14):e320-e323.##Abdolahi M, Shahbazi-Gahrouei D, Laurent S, Sermeus C, Firozian F, Allen BJ, et al. Synthesis and in vitro evaluation of MR molecular imaging probes using J591 mAb-conjugated SPIONs for specific detection of prostate cancer. Contrast Media Mol Imaging 2013;8(2):175-184.##Shahbazi-Gahrouei D, Abdolahi M. Superparamagnetic iron oxide-C595: Potential MR imaging contrast agents for ovarian cancer detection. J Med Phys 2013;38(4):198-204.##Laurent S, Forge D, Port M, Roch A, Robic C, Vander Elst L, et al. Magnetic iron oxide nanoparticles: synthesis, stabilization, vectorization, physicochemical characterizations, and biological applications. Chem Rev 2008;108(6):2064-2110.##Zeng C, Shi X, Wu B, Zhang D, Zhang W. Colloids containing gadolinium-capped gold nanoparticles as high relaxivity dual-modality contrast agents for CT and MRI. Colloids Surf B Biointerfaces 2014;123:130-135.##Shahbazi-Gahrouei D, Abdolahi M. Detection of MUC1-expressing ovarian cancer by C595 monoclonal antibody-conjugated SPIONs using MR imaging. Scientific World Journal 2013;2013:609151.##Barcena C, Sra AK, Chaubey GS, Khemtong C, Liu JP, Gao J. Zinc ferrite nanoparticles as MRI contrast agents. Chem Commun (Camb) 2008;(19):2224-2226.##Hoque SM, Srivastava C, Srivastava N, Venkateshan N, Chattopadhyay K. Synthesis and characterization of Fe-and Co-based ferrite nanoparticles and study of the T1 and T2 relaxivity of chitosan-coated particles. J Mater Sci 2013;48(2):812-818.##Arsalani N, Fattahi H, Nazarpoor M. Synthesis and characterization of PVP-functionalized superparamagnetic Fe3O4 nanoparticles as an MRI contrast agent. Exp Polym Lett 2010;4(6):329-338.##Tong S, Hou S, Zheng Z, Zhou J, Bao G. Coating optimization of superparamagnetic iron oxide nanoparticles for high T2 relaxivity. Nano Lett 2010;10(11):4607-4613.##Jun YW, Huh YM, Choi JS, Lee JH, Song HT, Kim S, et al. Nanoscale size effect of magnetic nanocrystals and their utilization for cancer diagnosis via magnetic resonance imaging. J Am Chem Soc 2005;127(16):5732-5733.##Seo WS, Lee JH, Sun X, Suzuki Y, Mann D, Liu Z, et al. FeCo/graphitic-shell nanocrystals as advanced magnetic-resonance-imaging and near-infrared agents. Nat Mater 2006;5(12):971-976.##Ge G, Wu H, Xiong F, Zhang Y, Guo Z, Bian Z, et al. The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells. Nanoscale Res Lett 2013;8(1):215.##Manouchehri S, Ghasemian Z, Shahbazi-Gahrouei D, Abdolahi M. Synthesis and characterization of cobalt-zinc ferrite nanoparticles coated with DMSA. Chem Xpress 2013;2(3):147-152.##Cabrera LI, Somoza A, Marco JF, Serna CJ, Morales MP. Synthesis and surface modification of uniform MFe2O4 (M=Fe, Mn, and Co) nanoparticles with tunable sizes and functionalities. J Nanopar Res 2012;14(6):1-14.##Shahbazi-Gahrouei D, Ghasemian Z, Abdolahi M, Manouchehri S, Javanmard SH, Dana N. In vitro evaluation of cobalt-zinc ferrite nanoparticles coated with DMSA on human prostate cancer cells. J Mol Biomark Diagn 2013;4(3):154.##

Release Studies on Ciprofloxacin Loaded Non-ionic Surfactant Vesicles 2 2 Background: Development of new drug carriers would be an interesting approach if it allowed increased efficacy of antibiotics and a reduction in doses, thus reducing the risk of developing resistance. As with most drug carriers, niosomes have been used to improve the selective delivery and the therapeutic index of antimicrobial agents. Methods: In this study, different formulation of niosomes containing ciprofloxacin (CPFX), Span (20, 60 or 80), Tween (20, 60 or 80) and cholesterol were prepared by film hydration method. The release of the drug from different formulations was studied by using Franz diffusion cell. The niosomes were further characterized by optical microscopy and particle size analysis, and their antimicrobial activity was evaluated. Results: Size of the niosomes was significantly dependent on the amount of cholesterol and surfactant type and varied from 8.56 to 61.3 μm. The entrapment efficiency of CPFX niosomes prepared by remote loading was more than 74%. Niosomes composed of Span/Tween 60 provided a higher CPFX release rate than other formulations. The obtained results indicated a diffusion-based mechanism for drug leakage through bilayers. All formulations presented more antibacterial activity as compared to free CPFX solution. Conclusion: Niosomal CPFX appears to be a promising approach in the management of bacterial infections, especially ophthalmic ones, and should be further evaluated by in vivo experiments. 69 75 Vajihe Akbari Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran Daryoush Abedi Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran Abbas Pardakhty Kerman Pharmaceutics Research Center and Department of Pharmaceutics, Kerman University of Medical Sciences Kerman Pharmaceutics Research Center and Department of Pharmaceutics, Kerman University of Medical Sciences Iran Hojjat Sadeghi-Aliabadi Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran CiprofloxacinNiosomesRelease 205.pdf Oliphant CM, Green GM. 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Efficacy of ciprofloxacin-releasing bioabsorbable osteoconductive bone defect filler for treatment of experimental osteomyelitis due to Staphylococcus aureus. Antimicrob Agents Chemother 2005;49(4):1502-1508.##Drulis-Kawa Z, Dorotkiewicz-Jach A. Liposomes as delivery systems for antibiotics. Int J Pharm 2010;387(1):187-198.##Kumar R, Kumar S, Jha SS, Jha AK. Vesicular system-carrier for drug delivery. Der Pharmacia Sinica 2011;2(4):192-202.##Chen C-S, Yao J, Durst RA. Liposome encapsulation of fluorescent nanoparticles: Quantum dots and silica nanoparticles. J Nanopart Res 2006;8(6):1033-1038.##Mahale NB, Thakkar PD, Mali RG, Walunj DR, Chaudhari SR. Niosomes: novel sustained release nonionic stable vesicular systems--an overview. Adv Colloid Interface Sci 2012;183-184:46-54.##Pardakhty A, Moazeni E. Nano-niosomes in drug, vaccine and gene delivery: a rapid overview. Nanomedicine J 2013;1(1):1-12.##Abdelkader H, Ismail S, Kamal A, Alany RG. Design and evaluation of controlled-release niosomes and discomes for naltrexone hydrochloride ocular delivery. J Pharm Sci 2011;100(5):1833-1846.##Hasan AA, Madkor H, Wageh S. Formulation and evaluation of metformin hydrochloride-loaded niosomes as controlled release drug delivery system. Drug Deliv 2013;20(3-4):120-126.##Mokhtar M, Sammour OA, Hammad MA, Megrab NA. Effect of some formulation parameters on flurbiprofen encapsulation and release rates of niosomes prepared from proniosomes. Int J Pharm 2008;361(1-2):104-111.##Guinedi AS, Mortada ND, Mansour S, Hathout RM. Preparation and evaluation of reverse-phase evaporation and multilamellar niosomes as ophthalmic carriers of acetazolamide. Int J Pharm 2005;306(1-2):71-82.##Akbari V, Abedi D, Pardakhty A, Sadeghi-Aliabadi H. Ciprofloxacin nano-niosomes for targeting intracellular infections: an in vitro evaluation. J Nanopart Res 2013;15(4):1-14.##Pardakhty A, Varshosaz J, Rouholamini A. 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TP53 Binding to BRCA1 and RAD51 in MCF7 and MDA-MB-468 Breast Cancer Cell Lines In vivo and In vitro 2 2 Background: Tumour suppressor genes such as TP53, BRCA1 and RAD51 are involved in DNA repair and their malfunctions result in genomic instability and cancer. Wild type (WT) TP53 binds to BRCA1and RAD51 in vivo and in vitro. However, mutated TP53 in tumours can interfere with WT TP53 function. We studied how mutation of TP53 in MDA-MB-468 cell line could affect its binding capacity and interfere with WT TP53 interaction with these DNA repair proteins. Methods: Binding capacity of mutated TP53 in MDA-MB-468 breast cancer cell line to BRCA1 and RAD51 proteins in comparison to WT TP53 in MCF7 cell line was studied by Immunoprecipitation. In vitro studies were performed by GST-WT p53 pull-down assays in these cell lines to assess the interaction of GST-WT p53 with BRCA1 and RAD51 proteins. Results: The results showed that mutated TP53 in MDA-MB-468 cells interacted with BRCA1 protein in vivo and did not effect WT TP53 binding to this protein in vitro. The Immunoprecipitation assays revealed that the mutated TP53 did not bind to RAD51 in comparison to WT TP53. However, this mutated protein could not interfere with binding of RAD51 to GST-WT p53 in MDA-MB-468 cell line by in vitro experiment. Conclusion: It was found that WT TP53 interactions with BRCA1 and RAD51 did not interfere with mutated TP53 in MDA-MB-468 cell line. In addition, RAD51 did not bind to TP53 with R273C mutation in vivo. 76 79 Mozhgan Rasti Recombinant Lab, Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Recombinant Lab, Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Iran Tayebeh Azimi Recombinant Lab, Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Recombinant Lab, Department of Biochemistry, Faculty of Medicine, Shiraz University of Medical Sciences Iran BRCA1 proteinImmunoprecipitationRAD51 proteinTP53 206.pdf Mousavi SM, Montazeri A, Mohagheghi MA, Jarrahi AM, Harirchi I, Najafi M, et al. Breast cancer in Iran: an epidemiological review. Breast J 2007;13(4):383-391.##Lee EY, Muller WJ. Oncogenes and tumor suppressor genes. Cold Spring Harb Perspect Biol 2010;2(10): a003236.##Xu Y. Induction of genetic instability by gain-of-function p53 cancer mutants. Oncogene 2008;27(25):3501-3507.##Song H, Hollstein M, Xu Y. p53 gain-of-function cancer mutants induce genetic instability by inactivating ATM. Nat Cell Biol 2007;9(5):573-580.##Brosh R, Rotter V. When mutants gain new powers: news from the mutant p53 field. Nat Rev Cancer 2009;9(10):701-713.##Menendez D, Inga A, Resnick MA. The expanding universe of p53 targets. Nat Rev Cancer 2009;9(10):724-737.##Zheng L, Pan H, Li S, Flesken-Nikitin A, Chen PL, Boyer TG, et al. Sequence-specific transcriptional corepressor function for BRCA1 through a novel zinc finger protein, ZBRK1. Mol Cell 2000;6(4):757-768.##Yu X, Chini CC, He M, Mer G, Chen J. The BRCT domain is a phospho-protein binding domain. Science 2003;302(5645):639-642.##Davies AA, Masson JY, McIlwraith MJ, Stasiak AZ, Stasiak A, Venkitaraman AR, et al. Role of BRCA2 in control of the RAD51 recombination and DNA repair protein. Mol Cell 2001;7(2):273-282.##Galkin VE, Wu Y, Zhang XP, Qian X, He Y, Yu X, et al. The Rad51/RadA N-terminal domain activates nucleo- protein filament ATPase activity. Structure 2006;14(6):983-992.##Somyajit K, Subramanya S, Nagaraju G. RAD51C: a novel cancer susceptibility gene is linked to Fanconi anemia and breast cancer. Carcinogenesis 2010;31(12):2031-2038.##Zhang H, Somasundaram K, Peng Y, Tian H, Zhang H, Bi D, et al. BRCA1 physically associates with p53 and stimulates its transcriptional activity. Oncogene 1998;16(13):1713-1721.##Buchhop S, Gibson MK, Wang XW, Wagner P, Sturzbecher HW, Harris CC. Interaction of p53 with the human Rad51 protein. Nucleic Acids Res 1997;25(19):3868-3874.##Goh AM, Coffill CR, Lane DP. The role of mutant p53 in human cancer. J Pathol 2011;223(2):116-126.##Rasti M, Khatooni Z, Mostafavi- pour Z. Production and evaluation of polyclonal rabbit anti-human p53 antibody using bacterially expressed glutathione S-transferase-p53 fusion protein. Iran J Biotechnol 2012;10(4):240-248.##Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-254.##Arabsolghar R, Azimi T, Rasti M. Mutant p53 binds to estrogen receptor negative promoter via DNMT1 and HDAC1 in MDA-MB-468 breast cancer cells. Mol Biol Rep 2013;40(3):2617-2625.##Casey G, Lo-Hsueh M, Lopez ME, Vogelstein B, Stanbridge EJ. Growth suppression of human breast cancer cells by the introduction of a wild-type p53 gene. Oncogene 1991;6(10):1791-1797.##Muller PA, Caswell PT, Doyle B, Iwanicki MP, Tan EH, Karim S, et al. Mutant p53 drives invasion by promoting integrin recycling. Cell 2009;139(7):1327-1341.##Rodriguez MC, Songyang Z. BRCT domains: phosphopeptide binding and signaling modules. Front Biosci 2008;13:5905-5915.##Johnson RD, Jasin M. Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells. 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Maternal Supplementary Folate Intake, Methylenetetrahydrofolate Reductase (MTHFR) C677T and A1298C Polymorphisms and the Risk of Orofacial Cleft in Iranian Children 2 2 Background: The purpose of this study was to describe the association of MTHFR gene single nucleotide polymorphisms (C677T and A1298C) and maternal supplementary folate intake with orofacial clefts in the Iranian population. Methods: In this case-control study, peripheral venous blood was taken from 65 patients with orofacial clefts and 215 unaffected controls for DNA extraction and kept in EDTA for further analysis. The genotyping was carried out using Polymerase Chain Reaction (PCR) followed by Restriction Fragment Length Polymorphism (RFLP) and gel electrophoresis. Data were analyzed using Chi square test and logistic regression tests. Results: Genotype frequencies of 677TT were reported to be 13.5 and 36.1% in controls and CL/P patients, respectively, which showed a significant difference compared to CC as reference (OR=4.118; 95% CI=1.997-8.492; p=0.001). Conversely, 1298CC with frequencies of 10.8 and 12.7% in controls and patients, respectively, showed no significant difference compared to AA (OR=2.359; 95% CI=0.792-7.023; p=0.123). Comparing patients whose mothers did not report the folate supplement intake during pregnancy, to controls, it was observed that lack of folate intake was a predisposing factor for having a child with oral clefts (OR=5/718, p=0.000). Conclusion: Children carrying the 677TT variant of the MTHFR gene may have an increased risk of CL/P. In addition, the finding that the risk associated with this allele was obviously higher when the mothers didn't use folic acid, supports the hypothesis that folic acid may play a role in the etiology of CL/P. 80 84 Asghar Ebadifar Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Dentofacial Deformities Research Center, Research Institute of Dental Sciences, Shahid Beheshti University of Medical Sciences Iran Hamid Reza Khorram Khorshid Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Genetic Research Center, University of Social Welfare and Rehabilitation Sciences Iran Koorosh Kamali Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mehdi Salehi Zeinabadi Pediatric Department, Dental School, Semnan University of Medical Sciences Pediatric Department, Dental School, Semnan University of Medical Sciences Iran Tayyebeh Khoshbakht Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Genetic Research Centre, University of Social Welfare and Rehabilitation Sciences Iran Nazila Ameli Orthodontic Department, Dental school, Semnan University of Medical Sciences Orthodontic Department, Dental school, Semnan University of Medical Sciences Iran Cleft lipCleft palateGenesPolymorphism 207.pdf Mirfazeli A, Kaviany N, Hosseinpour KR, Golalipour MJ. Incidence of cleft lip and palate in Gorgan-Northern Iran: an epidemiological study. Oman Med J 2012;27(6):461-464.##Leck I. The geographical distribution of neural tube defects and oral clefts. Br Med Bull 1984;40(4):390-395.##Farhud DD, Walizadeh GR, Kamali MS. Congenital malformations and genetic diseases in Iranian infants. Hum Genet 1986;74(4):382-385.##Das SK, Runnels RS Jr, Smith JC, Cohly HH. Epidemiology of cleft lip and cleft palate in Mississippi. South Med J 1995;88(4):437-442.##Derijcke A, Eerens A, Carels C. The incidence of oral clefts: a review. Br J Oral Maxillofac Surg 1996;34(6): 488-494.##Rajabian MH, Sherkat M. An epidemiologic study of oral clefts in Iran: analysis of 1,669 cases. Cleft Palate Craniofac J 2000;37(2):191-196.##Robert E, KallEn B, Harris J. The epidemiology of orofacial clefts. Some general epidemiological characteristics. J Craniofac Genet Dev Biol 1996;16(4):234-241.##Taher AA. Cleft lip and palate in Tehran. Cleft Palate Craniofac J 1992;29(1):15-16.##Semic-Jusufagic A, Bircan R, Celebiler O, Erdim M, Akarsu N, Elcioglu NH. Association between C677T and A1298C MTHFR gene polymorphism and nonsyndromic orofacial clefts in the Turkish population: a case-parent study. Turk J Pediatr 2012;54(6):617-625.##Jagomagi T, Nikopensius T, Krjutskov K, Tammekivi V, Viltrop T, Saag M, et al. MTHFR and MSX1 contribute to the risk of nonsyndromic cleft lip/palate. Eur J Oral Sci 2010;118(3):213-220.##Aslar D, Ozdiler E, Altug AT, Tastan H. Determination of Methylenetetrahydrofolate Reductase (MTHFR) gene polymorphism in Turkish patients with nonsyndromic cleft lip and palate. Int J Pediatr Otorhinolaryngol 2013;77(7):1143-1146.##Pan Y, Zhang W, Ma J, Du Y, Li D, Cai Q, et al. Infants' MTHFR polymorphisms and nonsyndromic orofacial clefts susceptibility: a meta-analysis based on 17 case-control studies. Am J Med Genet A 2012;158A(9):2162-2169.##Jugessur A, Wilcox AJ, Lie RT, Murray JC, Taylor JA, Ulvik A, et al. Exploring the effects of methylenetetrahydrofolate reductase gene variants C677T and A1298C on the risk of orofacial clefts in 261 Norwegian case-parent triads. Am J Epidemiol 2003;157(12):1083-1091.##Mills JL, Molloy AM, Parle-McDermott A, Troendle JF, Brody LC, Conley MR, et al. Folate-related gene polymorphisms as risk factors for cleft lip and cleft palate. Birth Defects Res A Clin Mol Teratol 2008;82(9):636-643.##Limpach A, Dalton M, Miles R, Gadson P. Homocysteine inhibits retinoic acid synthesis: a mechanism for homocysteine-induced congenital defects. Exp Cell Res 2000;260(1):166-174.##Greene ND, Dunlevy LE, Copp AJ. Homocysteine is embryotoxic but does not cause neural tube defects in mouse embryos. Anat Embryol (Berl) 2003;206(3):185-191.##Reutter H, Birnbaum S, Lacava AD, Mende M, Henschke H, Berge S, et al. Family-based association study of the MTHFR polymorphism C677T in patients with nonsyndromic cleft lip and palate from central Europe. Cleft Palate Craniofac J 2008;45(3):267-271.##Han Y, Pan Y, Du Y, Tong N, Wang M, Zhang Z, et al. Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and nonsyndromic orofacial clefts susceptibility in a southern Chinese population. DNA Cell Biol 2011;30(12):1063-1068.##van der Put NM, Steegers-Theunissen RP, Frosst P, Trijbels FJ, Eskes TK, van den Heuvel LP, et al. Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet 1995;346(8982):1070-1071.##van der Put NM, Gabreels F, Stevens EM, Smeitink JA, Trijbels FJ, Eskes TK, et al. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am J Hum Genet 1998;62(5):1044-1051.##Gudnason V, Stansbie D, Scott J, Bowron A, Nicaud V, Humphries S. C677T (thermolabile alanine/valine) polymorphism in methylenetetrahydrofolate reductase (MTHFR): its frequency and impact on plasma homocysteine concentration in different European populations. EARS group. Atherosclerosis 1998;136(2):347-354.##Yin G, Ming H, Zheng X, Xuan Y, Liang J, Jin X. Methylenetetrahydrofolate reductase C677T gene polymorphism and colorectal cancer risk: A case-control study. Oncol Lett 2012;4(2):365-369.##Wang SM, Wang JH, Yu JC, Wei B, Wang KH, Liu JY, et al. [Association between parental MTHFR gene polymorphism 677C/T and nonsyndromic cleft lip and palate in offspring]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 2012;29(4):464-467. Chinese.##van Rooij IA, Vermeij-Keers C, Kluijtmans LA, Ocke MC, Zielhuis GA, Goorhuis-Brouwer SM, et al. Does the interaction between maternal folate intake and the methylenetetrahydrofolate reductase polymorphisms affect the risk of cleft lip with or without cleft palate? Am J Epidemiol 2003;157(7):583-591.##Wan WD, Wang LJ, Zhou XP, Zhou DL, Zhang QG, Huang JL, et al. [Relationship between nonsyndromic cleft lip with or without cleft palate (NSCL/P) and genetic polymorphisms of MTHFR C677T and A1298C]. Zhonghua Zheng Xing Wai Ke Za Zhi 2006;22(1):8-11. Chinese.##Kelly D, O'Dowd T, Reulbach U. Use of folic acid supplements and risk of cleft lip and palate in infants: a population-based cohort study. Br J Gen Pract 2012;62 (600):e466-472.##Butali A, Little J, Chevrier C, Cordier S, Steegers-Theunissen R, Jugessur A, et al. Folic acid supplementation use and the MTHFR C677T polymorphism in orofacial clefts etiology: An individual participant data pooled-analysis. Birth Defects Res A Clin Mol Teratol 2013;97(8):509-514.##Wehby GL, Murray JC. Folic acid and orofacial clefts: a review of the evidence. Oral Dis 2010;16(1):11-19.##Little J, Gilmour M, Mossey PA, Fitzpatrick D, Cardy A, Clayton-Smith J, et al. Folate and clefts of the lip and palate--a U.K.-based case-control study: Part II: Biochemical and genetic analysis. Cleft Palate Craniofac J 2008;45(4):428-438.##Munger RG, Sauberlich HE, Corcoran C, Nepomuceno B, Daack-Hirsch S, Solon FS. Maternal vitamin B-6 and folate status and risk of oral cleft birth defects in the Philippines. Birth Defects Res A Clin Mol Teratol 2004;70 (7):464-471.##

Evaluation of a Modified DNA Extraction Method for Isolation of Cell-Free Fetal DNA from Maternal Serum 2 2 Background: Discovery of short cell free fetal DNA (cffDNA) fragments in maternal plasma has created major changes in the field of prenatal diagnosis. The use of cffDNA to set up noninvasive prenatal test is limited due to the low concentration of fetal DNA in maternal plasma therefore, employing a high efficiency extraction method leads to more accurate results. The aim of this study was to evaluate the efficiency of Triton/Heat/Phenol (THP) protocol in comparison with the QIAamp DNA Blood mini Kit for cffDNA purification. Methods: In order to evaluate the efficiency of THP protocol, DNA of Rhesus D (RhD) negative pregnant women's plasma was collected, then real-time PCR for RHD exon 7 was performed. The Ct value data of real time PCR obtained by two different methods were compared and after delivery serology test on cord blood was done to validate the real time PCR results. Results: The results indicated significant differences between two extraction methods (p=0.001). The mean±SD of Ct-value using THP protocol was 33.8±1.6 and 36.1±2.47 using QIAamp DNA Blood mini Kit. Conclusion: our finding demonstrated that THP protocol was more effective than the QIAamp DNA Blood mini Kits for cffDNA extraction and lead to decrease the false negative results. 85 88 Zeinab Keshavarz Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical SciencesStudent Research Committee, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical SciencesStudent Research Committee, Shiraz University of Medical Sciences IranIran Leili Moezzi 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 Reza Ranjbaran 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 Farzaneh Aboualizadeh 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 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 Masooma Abdullahi 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 Sedigheh Sharifzadeh 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 FetusPrenatal diagnosisReal time polymerase chain reactionTHP protocol 208.pdf Costa JM, Benachi A, Gautier E. New strategy for prenatal diagnosis of X-linked disorders. N Engl J Med 2002;346(19):1502.##Zhong XY, Holzgreve W, Hahn S. Circulatory fetal and maternal DNA in pregnancies at risk and those affected by preeclampsia. Ann N Y Acad Sci 2001;945(1):138-140.##Schmidt B, Weickmann S, Witt C, Fleischhacker M. Improved method for isolating cell-free DNA. Clin Chem 2005;51(8):1561-1563.##Chan KA, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50(1):88-92.##Bianchi DW. Circulating fetal DNA: its origin and diagnostic potential-a review. Placenta 2004;25(Suppl A):S93-S101.##Edlow AG, Bianchi DW. Tracking fetal development through molecular analysis of maternal biofluids. Biochim Biophys Acta 2012;1822(12):1970-1980.##Anker P, Stroun M. Immunological aspects of circulating DNA. Ann N Y Acad Sci 2006;1075(1):34-39.##Bianchi DW, LeShane ES, Cowan JM. Large amounts of cell-free fetal DNA are present in amniotic fluid. Clin Chem 2001;47(10):1867-1869.##Hui L, Bianchi DW. Cell-free fetal nucleic acids in amniotic fluid. Hum Reprod Update 2011;17(3):362-371.##Swanson A, Sehnert AJ, Bhatt S. Non-invasive prenatal testing: technologies, clinical assays and implementation strategies for women’s healthcare practitioners. Curr Genet Med Rep 2013;1(2):113-121.##Yang YH, Han SH, Lee KR, Kim SY, Ryu HM. Noninvasive prenatal diagnosis using cell-free fetal DNA in maternal plasma: clinical applications. J Genet Med 2011;8(1):10.##Clausen FB, Krog GR, Rieneck K, Dziegiel MH. Improvement in fetal DNA extraction from maternal plasma. Evaluation of the NucliSens Magnetic Extraction system and the QIAamp DSP virus kit in comparison with the QIAamp DNA blood mini kit. Prenatal Diag 2007;27(1):6-10.##Lo Y, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350(9076):485-487.##Xue X, Teare MD, Holen I, Zhu YM, Woll PJ. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta 2009;404(2):100-104.##Wang XD, Wang BL, Ye SL, Liao YQ, Wang LF, He ZM. Non‐invasive foetal RHD genotyping via real‐time PCR of foetal DNA from Chinese RhD‐negative maternal plasma. Eur J Clin Invest 2009;39(7):607-617.##Kimura M, Hara M, Itakura A, Sato C, Ikebuchi K, Ishihara O. Fragment size analysis of free fetal DNA in maternal plasma using Y-STR loci and SRY gene amplification. Nagoya J Med Sci 2011;73(3-4):129-135.##Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn 2007;27(9):824-829.##Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal D status from maternal plasma: introduction of a new noninvasive fetal RHD genotyping service. Transfusion 2002;42(8):1079-1085.##Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Kulovany E, et al. Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma. Fetal Diagn Ther 2005;20(4):275-280.##Hromadnikova I, Vechetova L, Vesela K, Benesova B, Doucha J, Vlk R. Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies. J Histochem Cytochem 2005;53(3):301-305.##Johnson KL, Dukes KA, Vidaver J, LeShane ES, Ramirez I, Weber WD, et al. Interlaboratory comparison of fetal male DNA detection from common maternal plasma samples by real-time PCR. Clin Chem 2004;50(3):516-521.##