The Protein Subcellular Mislocalization in Human Cancers 2 2 Several sets of proteins exist in eukaryotic cells wherein accurately localized in appropriate cellular locations containing plasma membrane, cytoplasm, nucleus and different membrane-enclosed organelles. Each protein is involved in distinct biochemical processes within the normal cell; therefore, the correct subcellular localization of protein is vital; as it provides the physiological context for protein function. However, protein mislocalization described as changing the appropriate subcellular localization of protein, was reported as a key feature of many proteins in a variety of human cancers 1. Mislocalization has important implications in alteration of activation condition, biological function and interaction network of a protein. Particularly,  the aberrant localization of tumor-suppressor proteins and proto-oncoproteins can alter their functions, respectively in either suppressing or supporting the cancer initiation in normal cells whereby cancer development, metastasis and drug resistance are increased 2.  The mechanisms by which subcellular mislocalization is arising in cancer are various and deeply reviewed by Wang and Li 2. They can be described as modification of signals directing proteins into a particular location, dysregulation of sorter and transporter machinery, Endoplasmic Reticulum (ER) retention of misfolded proteins, aberrant endocytosis and vesicular trafficking, dysregulation of signal transduction and protein post-translational modification and so on 2. The aberrant subcellular position of proteins in cancer tissues has been investigated broadly by antibody-based strategies including Immunohistochemistry (IHC). The major localization of sortilin on cell surface of ovarian carcinoma tissues rather than the main resident in ER-Golgi compartment of normal tissues was detected using IHC technique 3. The new method of Dissociable Antibody Microarray (DAMA) combining the power of IHC staining with protein microarray, provides the facility of high-throughput detection of protein subcellular localization 4. The property of malignant cells in differently subcellular localization of proteins might be recruited as an intelligent strategy for detection of malignant but not normal cells in clinical diagnostic and prognostic applications 5.  For instance, fibromodulin, a type of proteoglycan resides in extracellular matrix, has been irregularly located on cell surface of Chronic Lymphocytic Leukemia (CLL) cells but not in normal samples by which a new CLL diagnostic biomarker appropriate for cell surface flow cytometric detection  might be suggested 6. Moreover, directly targeting of locations in where proteins are accumulated in cancer cells plus selecting the structural dissimilarities of proteins by biologic weapons help us to specifically eradicate malignant but not normal cells. In conclusion, the subscellular mislocalization of proteins is a frequent event in cancers defined as an accessory approach for proliferation, survival and invasion of tumors. However, targeting and trapping proteins in specific cellular compartments has been conceptualized as a promising approach for diagnostic, prognostic and therapeutic clinical use. 1 1 Fatemeh Ghaemimanesh Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Editorial 20411.pdf Hung MC, Link W. Protein localization in disease and therapy. J Cell Sci 2011;124(Pt 20):3381-3392.##Wang X, Li S. Protein mislocalization: mechanisms, functions and clinical applications in cancer. Biochim Biophys Acta 2014;1846(1):13-25.##Hemmati S, Zarnani AH, Mahmoudi AR, Sadeghi MR, Soltanghoraee H, Akhondi MM, et al. Ectopic expression of sortilin 1 (NTR-3) in patients with ovarian carcinoma. Avicenna J Med Biotechnol 2009;1(2):125-131.##Wang Y. Immunostaining with dissociable antibody microarrays. Proteomics 2004;4(1):20-26.##Sajic T, Ciuffa R, Lemos V, Xu P, Leone V, Li C, et al. A new class of protein biomarkers based on subcellular distribution: application to a mouse liver cancer model. Scientific Reports 2019;9(1):6913.##Farahi L, Ghaemimanesh F, Milani S, Razavi SM, Hadavi R, Bayat AA, et al. GPI-anchored fibromodulin as a novel target in chronic lymphocytic leukemia: diagnostic and therapeutic implications. Iranian J Immunology 2019;16(2):127-141.##

AS-LAMP: A New and Alternative Method for Genotyping 2 2 In recent decades, different methods have been introduced for the genotyping of Single Nucleotide Polymorphisms (SNPs) and mutations in nucleic acid sequences. These methods have several applications ranging from agriculture to medicine. The Loop-mediated isothermal amplification (LAMP) method was first introduced by Notomi et al. Since then, different methods derived from LAMP have been extensively applied in detecting pathogens. The LAMP method is an isothermal technique that amplifies the target DNA segment using four different primers that have been uniquely designed for recognizing six distinct zones on the objective gene; the process of reaction continues at a constant temperature via a strand displacement reaction. Amplifying and detecting the targeted zone can be accomplished in one stage. Although the LAMP method is mostly used for pathogen detection, several studies have used this method for genotyping. The present article reviewed various studies that used the LAMP method for SNP detection. The outcomes indicated that the LAMP technique could be a reliable and alternative technique for genotyping. Further studies are recommended to use this approach for genotyping. 2 8 Pooria Gill Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences Iran Arash Hadian-Amree Student Research Committee, Thalassemia Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences Student Research Committee, Thalassemia Research Center, Hemoglobinopathy Institute, Mazandaran University of Medical Sciences Iran AS-LAMPGenotypingLAMPSingle nucleotide polymorphisms 20412.pdf Moore P. PCR: replicating success. 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J Biomol Tech 2011;22(2):60-66.##Mori Y, Nagamine K, Tomita N, Notomi T. Detection of loopmediated isothermal amplification by turbidity derived from magnesium pyrophosphate formation. Biochem Biophys Res Commun 2001;289(1):150-154.##Mori Y, Kitao M, Tomita N, Notomi T. Real-time turbidimetry of LAMP reaction for quantifying template DNA. J Biochem Biophys Methods 2004;59(2):145-157.##Zhao X, Lin CW, Wang J, Oh DH. Advances in rapid detection methods for foodborne pathogens. J Microbiol Biotechnol 2014;24(3):297-312.##Law JW, Ab Mutalib NS, Chan KG, Lee LH. Rapid methods for the detection of foodborne bacterial pathogens: principles, applications, advantages and limitations. Front Microbiol 2015;5:770.##Namountougou M, Diabate A, Etang J, Bass C, Sawadogo SP, Gnankinie O, et al. First report of the L1014S kdr mutation in wild populations of anopheles gambiae M and S molecular forms in Burkina Faso (West Africa). Acta Trop 2013;125(2):123-127.##Badolo A, Bando H, Traoré A, Ko-Ketsu M, Guelbeogo WM, Kanuka H, et al. Detection of G119S ace-1 (R) mutation in field-collected Anopheles gambiae mosquitoes using allele-specific loop-mediated isothermal amplification (AS-LAMP) method. Malar J 2015;14:477.##Yongkiettrakul S, Kampeera J, Chareanchim W, Rattanajak R, Pornthanakasem W, Kiatpathomchai W, et al. Simple detection of single nucleotide polymorphism in Plasmodium falciparum by SNP-LAMPassay combined with lateral flow dipstick. Parasitol Int 2017;66(1):964-971.##Ikeda S, Takabe K, Inagaki M, Funakoshi N, Suzuki K. Detection of gene point mutation in paraffin sections using in situ loop-mediated isothermal amplification. Pathol Int 2007;57(9):594-599.##Nakamura N, Ito K, Takahashi M, Hashimoto K, Kawamoto M, Yamanaka M, et al. Detection of six single-nucleotide polymorphisms associated with rheumatoid arthritis by a loop-mediated isothermal amplification method and an electrochemical DNA chip. 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Comparison of single nucleotide polymorphism genotyping of CYP2C19 by loop-mediated isothermal amplification and real-time PCR melting curve analysis. Clin Chim Acta 2018;478:45-50.##Chuang LY, Yang CH, Tsui KH, Cheng YH, Chang PL, Wen CH, et al. Restriction enzyme mining for SNPs in genomes. Anticancer Res 2008;28(4A):2001-2007.##Ota M, Asamura H, Oki T, Sada M. Restriction enzyme analysis of PCR products. Methods Mol Biol 2009;578:405-414.##Chang HW, Yang CH, Chang PL, Cheng YH, Chuang LY. SNP-RFLPing: restriction enzyme mining for SNPs in genomes. BMC Genomics 2006;7:30.##Shao Y, Zhu S, Jin C, Chen F. Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk. Int J Food Microbiol 2011;148(2):75-79.##Yoshida N, Fujino M, Ota Y, Notomi T, Nakayama T. Simple differentiation method of mumps Hoshino vaccine strain from wild strains by reversetranscription loop-mediated isothermal amplification (RT-LAMP). 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Evaluation of Immune Response and Protection Induced by V-ATPase Subunit F as DNA Vaccine Against Leishmania tropica (LCED Syrian 01) After Detection and Sequencing 2 2 Background: Leishmaniasis is one of the major emerging health problems worldwide and Leishmania tropica (L. tropica) is most prevalent in the Middle East due to conflict and environmental factors, and there is no effective prevention strategy available until now. An effective vaccine has not been developed to date. DNA vaccines are considered a promising approach to protect against this infection. In this study, since vacuolar (H+)-ATPase (V-ATPase) enzyme has an essential role in the life cycle of eukaryotes, V-ATPase subunit F gene has been chosen to design DNA vaccine and evaluate its immunogenicity in BALB\c mice.  Methods: Genomic DNA was isolated from promastigote culture, synthesized complementary DNA (cDNA) after standardization of Polymerase Chain Reaction (PCR) conditions. The V-ATPase subunit F gene was placed into plasmid PCI. Then, recombinant plasmids were transformed into competent cells. Cloning was confirmed by PCR, restriction enzyme assays, and finally, DNA sequence analysis, after making miniprep from positive colonies and finally the gene was sequenced. BALB/c mice were immunized subcutaneously three times at an interval of two weeks with designed vaccine. BALB\c mice were challenged with 106 promastigotes of L. tropica 7 days post-immunization. IL-12, IFN-γ and IL-4 were quantified by RT-qPCR.  Results: The present study proved the existence of subunit F gene in Syrian strain of L. tropica (LCED Syrian 01) promastigotes genome. Its expression was also proved in these parasites and the gene length was 414 bp.  Conclusion: This study showed that vaccination of BALB\c mice with this gene induced partial protection against Leishmania by reduction of lesion size by 41.9% and parasite burden reduction by 3-log in the dLNs when compared with control group. IFN-γ\IL-4 was 1.6 after challenge test, so the immune response consisted of both Th1 and Th2. 9 16 Amira Orabi Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Syria Mohammad Maarouf Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Syria Mustafa Alammori Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Department of Biochemistry and Parasitology, Faculty of Pharmacy, Damascus University Syria BALB\c miceDNA vaccineLeishmania tropicaParasite loadRT-PCRV-ATPase subunit F 10397.pdf World Health Organization. Leishmaniasis: worldwide epidemiological. World Health Organization, Geneva, 2017.##World Health Organization. Leishmaniasis: worldwide epidemiological. World Health Organization, 2018.##Hayani k, Dandashli A, Weisshaar, E. Cutaneous leishmaniasis in Syria: clinical features, current status and the effects of war. Acta Derm Venereol 2015;95(1):62-66.##Youssef A, Yaseer S, Harfouch R, Marouf M, Hasan F. Chiclero's ulcer: An unusual presentation of Leishmania tropica in Syria. Avicenna J Med 2018;8(3):117-119.##Masoudzadeh N, Mizbani A, Taslimi Y, Mashayekhi V, Mortazavi H, Sadeghipour P, et al. Leishmania tropica infected human lesions: Whole-genome transcription profiling. Acta Trop 2017;176:236-241.##Croft SL, Olliaro P. Leishmaniasis chemotherapy--challenges and opportunities. Clin Microbiol Infect 2011;17(10):1478-1483.##Seyed N, Taheri T, Rafati S. Post-genomics and vaccine improvement for Leishmania. Front Microbiol 2016;7:467.##Herrera-Najera C, Piña-Aguilar R, Xacur-Garcia F, Ramirez-Sierra MJ, Dumonteil E. Mining the Leishmania genome for novel antigens and vaccine candidates. Proteomics 2009;9(5):1293-1301.##Taheri T, Rafati S. Leishmaniasis: recombinant DNA vaccination and different approaches for vaccine development. Clin Invest 2013;3(11):1023-1044.##Lowe DB, Shearer MH, Kennedy RC. DNA vaccines: Successes and limitations in cancer and infectious disease. J Cell Biochem 2006;98(2):235-242.##Gurunathan S, Wu CY, Freidag BL, Seder RA. DNA vaccines: a key for inducing long-term cellular immunity. Curr Opin Immunol 2000;12(4):442-447.##Marshansky V, Rubinstein JL, Grüber G. Eukaryotic V-ATPase: novel structural findings and functional insights. Biochim Biophys Acta 2014;1837(6):857-879.##Kishikawa J, Yokoyama K. Reconstitution of vacuolar-type rotary H+-ATPase/synthase from Thermus thermophilus. J Biol Chem 2012;287(29):24597-24603.##Castellano LR, Filho DC, Argiro L, Dessein H, Prata A, Dessein A, et al. Th1/Th2 immune responses are associated with active cutaneous leishmaniasis and clinical cure is associated with strong interferon-gamma production. Hum Immunol 2009;70(6):383-390.##Alexander J, Russell DG. The interaction of Leishmania species with macrophages. Adv Parasitol 1992;31:175-254.##Morris L, Aebischer T, Handman E KA. Resistance of BALB/c mice to leishmania major infection is associated with a decrease in the precursor frequency of antigen- specific CD4+ cells secreting interleukin-4. Int Immunol 1993;5:761-769.##Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25(4):402-408.##Makyio H, Iino R, Ikeda C, Imamura H, Tamakoshi M, Iwata M, et al. Structure of a central stalk subunit F of prokaryotic V-type ATPase/synthase from Thermus thermophilus. EMBO J 2005;24:3974-3983.##Selvapandiyan A, Dey R, Gannavaram S, Lakhal-Naouar I, Duncan R, Salotra P, et al. Immunity to visceral leishmaniasis using genetically defined live-attenuated parasite. J Trop Med 2001:1-12.##

Optimization of In vitro Expansion and Activation of Human Natural Killer Cells against a Breast Cancer Cell Line 2 2 Background: Regarding to the increase of cancer deaths in recent years and disability of common therapies to eradicate cancers, as well as expansion of Natural Killer (NK) cell therapy, it seems so vital to find new useful therapies against cancers. Breast cancer is the second main cause of cancer death among women. As it is impossible for a majority of patients to receive NK cell therapy, an attempt was made to establish a low-cost and efficient method for expanding and activating NK cells against breast cancer cell line (MCF7). Methods: NK cells were isolated from Peripheral Blood Mononuclear Cells (PBMCs) applying either MACS based NK cell enrichment kit or antibodies and complement as cytotoxic method. Then, the NK cells were cultured in Stem Cell Growth Medium (SCGM) with feeder layer (irradiated PBMCs) along with PHA or OKT3. IL-2, IL-15 and IL-21 were used to expand NK cells and finally their cytotoxic activity was investigated by flow cytometry. Results: Highly pure NK cells were obtained and no significant difference between the two isolation methods was found. Using IL-2 plus IL-15, the number of NK cells increased up to100 fold after 16 days. No significant effect was observed after IL-21 treatment. Conclusion: Our data indicated that cytotoxicity method can be considered a low-cost alternative for NK cell isolation kits. It seems that culturing NK cells for 14 days in either PHA or OKT3 supplemented SCGM medium would be more effective than culturing for 16 days in the presence of IL-21. 17 23 Farzaneh Peighambarzadeh Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Iran Anahita Najafalizadeh Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Iran Nafiseh Esmaeil Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Iran Abbas Rezaei Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Iran Farzaneh Ashrafi Hematology Division, Department of Internal Medicine, Isfahan University of Medical Sciences Hematology Division, Department of Internal Medicine, Isfahan University of Medical Sciences Iran Mazdak Ganjalikhani-Hakemi Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences Iran InterleukinsImmunotherapyNatural killer cells 10400.pdf Smith AJ, Oertle J, Prato D. Immunotherapy in cancer treatment. Open J Med Microbiol 2014;4(3):178-191.##Vinay DS, Ryan EP, Pawelec G, Talib WH, Stagg J, Elkord E, et al. Immune evasion in cancer: mechanistic basis and therapeutic strategies. Semin Cancer Biol 2015;35 Suppl:S185-S198.##Yu LY, Tang J, Zhang CM, Zeng WJ, Yan H, Li MP, et al. New immunotherapy strategies in breast cancer. Int J Environ Res Public Health 2017;14(1). pii: E68.##Shen J, Pan J, Du C, Si W, Yao M, Xu L, et al. Silencing NKG2D ligand-targeting miRNAs enhances natural killer cell-mediated cytotoxicity in breast cancer. Cell Death Dis 2017;8(4):e2740.##Shenouda MM, Gillgrass A, Nham T, Hogg R, Lee AJ, Chew MV, et al. Ex vivo expanded natural killer cells from breast cancer patients and healthy donors are highly cytotoxic against breast cancer cell lines and patient-derived tumours. Breast Cancer Res 2017;19(1):76.##Lee AV, Oesterreich S, Davidson NE. MCF-7 cells--changing the course of breast cancer research and care for 45 years. J Natl Cancer Inst 2015;107(7). pii: djv073.##Spanholtz J, Preijers F, Tordoir M, Trilsbeek C, Paardekooper J, De Witte T, et al. Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process. PLoS One 2011;6(6):e20740.##Zhou J. Advances and prospects in cancer immunotherapy. New J Sci 2014;2014.##Lim O, Jung MY, Hwang YK, Shin EC. Present and future of allogeneic natural killer cell therapy. Front Immunol 2015;6:286.##Bodduluru LN, Kasala ER, Madhana RM, Sriram CS. Natural killer cells: the journey from puzzles in biology to treatment of cancer. Cancer Lett 2015;357(2):454-467.##Levy EM, Roberti MP, Mordoh J. Natural killer cells in human cancer: from biological functions to clinical applications. J Biomed Biotechnol 2011;2011:676198.##Mandal A, Viswanathan C. Natural killer cells: in health and disease. Hematol Oncol Stem Cell Ther 2015;8(2):47-55.##Childs RW, Berg M. Bringing natural killer cells to the clinic: ex vivo manipulation. Hematology Am Soc Hematol Educ Program 2013;2013:234-246.##Selvan SR, Dowling JP. “Adherent” versus other isolation strategies for expanding purified, potent, and activated human NK cells for cancer immunotherapy. Biomed Res Int 2015;2015:869547.##Shook DR, Campana D. Natural killer cell engineering for cellular therapy of cancer. Tissue Antigens 2011;78(6):409-415.##Granzin M, Soltenborn S, Müller S, Kollet J, Berg M, Cerwenka A, et al. Fully automated expansion and activation of clinical-grade natural killer cells for adoptive immunotherapy. Cytotherapy 2015;17(5):621-632.##Dahlberg CI, Sarhan D, Chrobok M, Duru AD, Alici E. Natural killer cell-based therapies targeting cancer: possible strategies to gain and sustain anti-tumor activity. Front Immunol 2015;6:605.##Yang Y, Lim O, Kim TM, Ahn Y-O, Choi H, Chung H, et al. Phase I study of random healthy donor-derived allogeneic natural killer cell therapy in patients with malignant lymphoma or advanced solid tumors. Cancer Immunol Res 2016;4(3):215-224.##Somanchi SS, McCulley KJ, Somanchi A, Chan LL, Lee DA. A novel method for assessment of natural killer cell cytotoxicity using image cytometry. PLoS One 2015;10(10):e0141074.##Ahn YO, Kim S, Kim TM, Song EY, Park MH, Heo DS. Irradiated and activated autologous PBMCs induce expansion of highly cytotoxic human NK cells in vitro. J Immunother 2013;36(7):373-381.##Alici E, Sutlu T, Björkstrand B, Gilljam M, Stellan B, Nahi H, et al. Autologous antitumor activity by NK cells expanded from myeloma patients using GMP-compliant components. Blood 2008;111(6):3155-3162.##South AM, Grimm PC. Transplant immuno-diagnostics: crossmatch and antigen detection. Pediatr Nephrol 2016;31(6):897-905.##Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T, et al. Expansion of highly cytotoxic human natural killer cells for cancer cell therapy. Cancer Res 2009;69(9):4010-4017.##Alnabhan R, Madrigal A, Saudemont A. Differential activation of cord blood and peripheral blood natural killer cells by cytokines. Cytotherapy 2015;17(1):73-85.##Domogala A, Madrigal JA, Saudemont A. Natural killer cell immunotherapy: from bench to bedside. Front Immunol 2015;6:264.##Delirezh N, Shojaeefar E, Parvin P, Asadi B. Comparison the effects of two monocyte isolation methods, plastic adherence and magnetic activated cell sorting methods, on phagocytic activity of generated dendritic cells. Cell J 2013;15(3):218-223.##Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Front Immunol 2015;6:578.##Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. 2005;106(1):376-383.##Siegler U, Meyer-Monard S, Jörger S, Stern M, Tichelli A, Gratwohl A, et al. Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients. Cytotherapy 2010;12(6):750-763.##Skak K, Frederiksen KS, Lundsgaard D. Interleukin‐21 activates human natural killer cells and modulates their surface receptor expression. Immunology 2008;123(4):575-583.##Granzin M, Stojanovic A, Miller M, Childs R, Huppert V, Cerwenka AJO. Highly efficient IL-21 and feeder cell-driven ex vivo expansion of human NK cells with therapeutic activity in a xenograft mouse model of melanoma. Oncoimmunology 2016;5(9):e1219007.##Duarte RF, Chen FE, Lowdell MW, Potter MN, Lamana ML, Prentice HG, et al. Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy. Gene Ther 2002;9(20):1359-1368.##Klöß S, Oberschmidt O, Morgan M, Dahlke J, Arseniev L, Huppert V, et al. Optimization of human NK cell manufacturing: Fully automated separation, improved ex vivo expansion using IL-21 with autologous feeder cells, and generation of anti-CD123-CAR-expressing effector cells. Hum Gene Ther 2017;28(10):897-913.##Park KH, Park H, Kim M, Kim Y, Han K, Oh EJ. Evaluation of NK cell function by flowcytometric measurement and impedance based assay using real-time cell electronic sensing system. Biomed Res Int 2013;2013:210726.##

Expression of Human Placenta-specific 1 (PLAC1) in CHO-K1 Cells 2 2 Background: Placenta-specific 1 (PLAC1), as a new Cancer/Testis Antigen (CTA), is frequently expressed in a variety of cancers and localized to cytoplasm and plasma membrane. Surface expression of cancer target antigens is of great importance that enables antibody-mediated cancer immunotherapy. The aim of the current study was to express the intact human PLAC1 protein on plasma membrane of a eukaryotic cell as a model for future anti-PLAC1-based cancer immunotherapy. Methods: In the first approach, entire human PLAC1 gene including its own Signal Peptide (SP) was cloned into pIRES2-EGFP and LeGO-iG2 vectors and expressed in CHO-K1 cells. In the second approach, cytosolic and Signal-Anchor (SA) sequence of Transferrin Receptor Protein 1 (TFR1) were fused to extracellular portion of PLAC1 and expressed as above. Expression of PLAC1 was then assessed using Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western Blot (WB), Immunocytochemistry (ICC), Immunofluorescence (IF) and Flow Cytometry (FC). Results: The first approach resulted in the expression of PLAC1 in submembranous but not in the surface of transfected CHO-K1 cells. Using the chimeric human PLAC1 construct, the same intracellular expression pattern was observed. Conclusion: These results indicated that there are some yet unknown PLAC1 localization signals employed by cancer cells for surface expression of PLAC1. 24 31 Jafar Mahmoudian Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS)Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) IranIran Mahboobeh Nazari Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Roya Ghods Oncopathology Research Center, Iran University of Medical Sciences (IUMS)Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS) Oncopathology Research Center, Iran University of Medical Sciences (IUMS)Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences (IUMS) IranIran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Seyed Naser Ostad Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS)Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS)Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) IranIran Mohammad Hossein Ghahremani Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS)Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS)Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences (TUMS) IranIran Sedigheh Vafaei Reproductive Immunology Research Center, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Amiri Department of Immunology, School of Public Health, Tehran University of Medical Sciences (TUMS) Department of Immunology, School of Public Health, Tehran University of Medical Sciences (TUMS) Iran Amir-Hassan Zarnani Immunology Research Center (IRC), Iran University of Medical Sciences (IUMS) Immunology Research Center (IRC), Iran University of Medical Sciences (IUMS) Iran Eukaryotic cellsPLAC1 protein expressionProtein transport 10402.pdf Gubin MM, Artyomov MN, Mardis ER, Schreiber RD. 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Effect of Poly-Histidine Tag Position toward Inhibition Activity of Secretory Leukocyte Protease Inhibitor as Candidate for Material Wound Healing 2 2 Background: The Secretory Leukocyte Protease Inhibitors (SLPI) has many biological functions including anti-bacterial, anti-fungal, anti-viral, anti-inflammatory, and immuno-modulatory. Previous studies have shown that gene-encoding human SLPI have successfully been expressed in Escherichia coli (E. coli) with a C-terminal polyhistidine tag (His-tag). The aim of this research was to investigate the inhibition activity of N-terminal His-tag position (NSLPI) and C-terminal His-tag position (CSLPI). We hypothesized that a His-tag close to an active site SLPI domain may interfere with the inhibition activity of SLPIs.  Methods: A NSLPI and CSLPI were constructed with polymerase chain reaction (PCR) amplification. The PCR products were then ligated into pET-30a plasmid and transformed into E. coli TOP10. Recombinant plasmids were verified by using restriction analysis and nucleotide sequence analysis. pET-NSLPI and pET-CSLPI were then subcloned in E. coli BL21(DE3) for its expression. The SLPI protein was expressed using Isopropyl β-D-1-thiogalactopyranoside induction (IPTG). The inhibition effect of both SLPI against Porcine Pancreatic Elastase (PPE) enzyme was tested using the N-succinyil-alanyl-L-alanyl-L-prolyl-L-phenylalanyl-4-nitroanalide (NPN) substrate.  Results: The SLPI gene was successfully cloned and expressed in E. coli BL21. Fusion proteins of NSLPI and CSLPI were generated with His-tag in the N-terminal and C-terminal position, respectively. The inhibition effect of NSLPI and CSLPI on PPE indicated that both SLPI were active. The inhibition activity of NSLPI was 66.7%, higher than CSLPI by 44.4%.  Conclusion: The His-tag position on the C-terminal of SLPI reduced the inhibition activity of SLPI. 32 36 Elly Munadziroh Department of Dental Material and Technology, Faculty of Dentistry, Universitas Airlangga Department of Dental Material and Technology, Faculty of Dentistry, Universitas Airlangga Indonesia Evi Ulfa Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas JemberDepartment of Chemistry, Faculty of Science and Technology, Universitas Airlangga Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas JemberDepartment of Chemistry, Faculty of Science and Technology, Universitas Airlangga IndonesiaIndonesia Amaliah Labiqah Department of Health Analysis, Stikes Kesetiakawanan Sosial Indonesia Department of Health Analysis, Stikes Kesetiakawanan Sosial Indonesia Indonesia One Asmarani Proteomic Study Group, Institute of Tropical Disease, Universitas Airlangga Proteomic Study Group, Institute of Tropical Disease, Universitas Airlangga Indonesia Ni Nyoman Puspaningsih Department of Chemistry, Faculty of Science and Technology, Universitas AirlanggaProteomic Study Group, Institute of Tropical Disease, Universitas Airlangga Department of Chemistry, Faculty of Science and Technology, Universitas AirlanggaProteomic Study Group, Institute of Tropical Disease, Universitas Airlangga IndonesiaIndonesia Escherichia coli (E. coli)Gene expressionPoly histidineSecretory leukocyte protease inhibitor 20413.pdf Nukiwa T, Suzuki T, Fukuhara T, Kikuchi T. Secretory leukocyte peptidase inhibitor and lung cancer. Cancer Sci 2008;99(5):849-855.##Moreau T, Baranger K, Dadé S, Dallet-Choisy S, Guyot N, Zani ML. Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family. Biochimie 2008;90(2):284-295.##Ashcroft GS, Lei K, Jin W, Longenecker G, Kulkarni AB, Greenwell-wild T, et al. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat Med 2000;6(10):1147-1153.##Ma G, Greenwell-Wild T, Lei K, Jin W, Swisher J, Hardegen N, et al. Secretory leukocyte protease inhibitor binds to annexin II , a cofactor for macrophage HIV-1 infection. J Exp Med 2004;200(10):1337-1346.##Fitriani D, Cahyati M, Prasetyaningrum N, Budhy TI, Munadziroh E. Acceleration of wound healing with use of secretory leukocyte protease inhibitor could be seen by osteopontin expression in Rattus norvegicus post tooth extraction acceleration of wound healing with use of secretory leukocyte protease inhibitor could be s. J Physic 2018;1073.##Majchrzak-Gorecka M, Majewski P, Grygier B, Murzyn K, Cichy J. Secretory leukocyte protease inhibitor (SLPI), a multifunctional protein in the host defense response. Cytokine Growth Factor Rev 2016;28:79-93.##Scott A, Weldon S, Taggart CC. SLPI and elafin : multifunctional antiproteases of the WFDC family. Biochem Soc Trans 2011;39(5):1437-1440.##Mikami Y, Iwase T, Komiyama Y, Matsumoto N. Secretory leukocyte protease inhibitor inhibits expression of polymeric immunoglobulin receptor via the NF- κB signaling pathway. Mol Immunol 2015;67(2 pt):568-574.##Paiyabhroma N, Nernpermpisooth N, Kumphune S. The recombinant human secretory leukocyte protease inhibitor (SLPI) protects cardiac fibroblasts injury against an in vitro ischemia/reperfusion injury. J Appl Pharm Sci 2018;8(06):156-162.##Koizumi M, Fujino A, Fukushima K, Kamimura T, Takimoto-kamimura M. Complex of human neutrophil elastase with 1/2SLPI. J Synchrotron Radiat 2008;15(Pt 3):308-311.##Fukushima K, Kamimura T, Takimoto-kamimura M. Structure basis 1/2SLPI and porcine pancreas trypsin interaction. J Synchrotron Radiat 2013;20(Pt 6):943-947.##Ying QL, Kemme M, Simon SR. Functions of the N-Terminal domain of secretory leukoprotease inhibitor. Biochemistry 1994;33(18):5445-5450.##Munadziroh E, Purnamasari S, Puspaningsih NN., Soetjipto, Rubianto M, Ismaya W. Generation of a soluble and active recombinant human secretory leukocyte protease inhibitor. Biotechnol Apl 2017;34(2):2231-2234.##Arnau J, Lauritzen C, Petersen GE, Pedersen J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 2006;48(1):1-13.##Majorek KA, Kuhn ML, Chruszcz M, Anderson WF, Minor W. Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci 2014;23(10):1359-1368.##Freydank AC, Brandt W, Dräger B. Protein structure modeling indicates hexahistidine-tag interference with enzyme activity. Proteins 2008;72(1):173-183.##Dickson JM, Lee W, Shepherd PR, Buchanan CM. Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase. Biosci Rep 2013;33(6). pii: e00079.##Thielges MC, Chung JK, Axup JY, Fayer MD. Influence of histidine tag attachment on picosecond protein dynamics. Biochemistry 2011;50(25):5799-5805.##Purnamasari S. Inhibitory analysis of recombinant Secretory leukocyte protease inhibitor (SLPI) from human amniotic membrane on Porcine Pancreatic Elastase (PPE) activity. Master Thesis. Faculty of Science and Technology: Universitas Airlangga, Surabaya; 2011.##Yeon YJ, Park HJ, Park HY, Yoo YJ. Effect of His-tag location on the catalytic activity of 3-hydroxybutyrate dehydrogenase. Biotechnol Bioprocess Eng 2014;19(5):798-802.##

A Single Tube Overlap Extension PCR Method for Splicing of Multiple DNA Fragments 2 2 Background: Despite the ease of conventional splicing by overlap-extension (SOEing) PCR technique in theory, when splicing more than two fragments, and especially if one of the complementary sequences is A-T rich, the attachment of the fragments would be challenging. A new rapid and highly efficient SOEing PCR assay was developed for simultaneous splicing of multiple DNA fragments and induction of site-directed mutagenesis in a single tube. Methods: The method was adapted for splicing human beta-globin UTRs to OCT4, SOX2, KLF4, C-MYC, LIN28A, and destabilized GFP for the construction of chimeric DNA fragments for in vitro transcription. In addition, the native Kozak sequence of beta-globin (K1) was replaced by the strongest Kozak sequence (K2) using site-directed mutagenesis to enhance the expression of target genes. Results: ChimericGFPd2/K1, GFPd2/K2, OCT4, and KLF4 were created by the optimized conventional SOEing PCR. The single tube method was able to create the chimeric SOX2, C-MYC, and LIN28A in high quality and quantity in comparison with the conventional SOEing PCR. Moreover, using single tube SOEing PCR, the reaction time and materials that are required in the conventional SOEing PCR were significantly reduced. Fluorescent microscopy and flow cytometry examinations indicated highly efficient translation of K2 sequence in comparison with the K1sequence. Conclusion: Single tube SOEing PCR is a valuable method to construct more multiple fragments with high yield. The method can successfully be applied for construction of various kinds of complex chimeric genes. 37 43 Farzaneh Zarghampoor Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz Iran Abbas Behzad-Behbahani Negar Azarpira Saeed Reza Khatami Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz Iran Maryam Fanian Transplant Research Centre, Shiraz University of Medical Science Transplant Research Centre, Shiraz University of Medical Science Iran Mahdokht Hossein-Aghdaie Transplant Research Centre, Shiraz University of Medical Science Transplant Research Centre, Shiraz University of Medical Science Iran Gholam Reza Rafiei Dehbidi Diagnostic Laboratory Sciences and Technology Research Centre, Faculty of Paramedical Sciences, Shiraz University of Medical Science Diagnostic Laboratory Sciences and Technology Research Centre, Faculty of Paramedical Sciences, Shiraz University of Medical Science Iran Beta-globinsMutagenesisPolymerase chain reaction Site-directedUntranslated regions 20415.pdf Higuchi R, Krummel B, Saiki R. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 1988;16(15):7351-7367.##Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 1989;77(1):51-59.##Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 1989;77(1):61-68.##Mullis KB, Faloona FA. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 1987;155:335-350.##Mullis K, Faloona F, Scharf S, Saiki R, Horn G, editors. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harbor symposia on quantitative biology; 1986: Cold Spring Harbor Laboratory Press.##Charlier N, Molenkamp R, Leyssen P, Vandamme AM, De Clercq E, Bredenbeek P, et al. A rapid and convenient variant of fusion-PCR to construct chimeric flaviviruses. J Virol Methods 2003;108(1):67-74.##Goh KM, Liew KJ, Chai KP, Illias RM. Use of megaprimer and overlapping extension PCR (OE-PCR) to mutagenize and enhance cyclodextrin glucosyltransferase (CGTase) function. Methods Mol Biol 2017;498:385-396.##You BJ, Lee MH, Chung KR. Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach. Arch Microbiol 2009;191(7):615-622.##Nakamura M, Suzuki A, Hoshida H, Akada R. Minimum GC-rich sequences for overlap extension PCR and primer annealing. Methods Mol Biol 2014;1116:165-181.##Shevchuk NA, Bryksin AV, Nusinovich YA, Cabello FC, Sutherland M, Ladisch S. Construction of long DNA molecules using long PCR‐based fusion of several fragments simultaneously. Nucleic Acids Res 2004;32(2):e19.##Luo WG, Liu HZ, Lin WH, Kabir MH, Su Y. Simultaneous splicing of multiple DNA fragments in one PCR reaction. 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HR9: An Important Cell Penetrating Peptide for Delivery of HCV NS3 DNA into HEK-293T Cells 2 2 Background: The delivery of exogenous genes into cells for functional expression is required for development of DNA vaccine and gene therapy in medicine and pharmacology. Cell Penetrating Peptides (CPPs) were considered to mediate gene and drug delivery into living cells. In this study, an attempt was made to evaluate the efficiency of an arginine-rich CPP, HR9, in HCV NS3 gene delivery compared to TurboFect cationic polymer and supercharged +36 GFP into HEK-293T cells. Methods: The recombinant pEGFP-NS3 was constructed and their accuracy was confirmed by digestion and sequencing. Then, the recombinant plasmid was transfected into HEK-293T cells by TurboFect, +36 GFP and HR9 gene delivery systems. The expression of NS3 protein was assessed by fluorescent microscopy, flow cytometry and western blotting. Results: Our data indicated that HR9 peptide was able to form stable complexes with plasmid DNA and increased its delivery into HEK-293T cells in a non-covalent manner. Furthermore, treatment of cells with HR9 and HR9/DNA complexes resulted in a viability of 90-95% indicating this CPP was not cytotoxic. The analysis of zeta potential and size showed the importance of interactions between positively-charged HR9/pEGFP-NS3 complexes and negatively-charged plasma membranes.   Conclusion: The non-toxic HR9 CPP can be considered an effective carrier for delivering plasmid DNA harboring Hepatitis C virus (HCV) gene in therapeutic vaccine design. 44 51 Sina Alizadeh Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University Iran Shiva Irani Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University Department of Biology, Faculty of Basic Sciences, Science and Research Branch, Islamic Azad University Iran Azam Bolhassani Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Seyed Mehdi Sadat Department of Hepatitis and AIDS, Pasteur Institute of Iran Iran Cell penetrating peptideDNAGene delivery systemHepatitic C virusVaccines 20416.pdf Amer AF. Progress in developing hepatitis C virus prophylactic and therapeutic vaccines. Int J Curr Microbiol App Sci 2014;3(7):891-906.##Quadeer AA, Louie RH, Shekhar K, Chakraborty AK, Hsing IM, McKay MR. Statistical linkage analysis of substitutions in patient-derived sequences of genotype 1a hepatitis C virus nonstructural protein 3 exposes targets for immunogen design. J Virol 2014;88(13):7628-7644.##Naeem A, Waheed Y. Sequence analysis of hepatitis C virus nonstructural protein 3-4A serine protease and prediction of conserved B and T cell epitopes. Biomed Rep 2017;7(6):563-566.##Alaee M, Rajabi P, Sharifi Z, Farajollahi MM. Immunoreactivity assessment of hepatitis C virus NS3 protease and NS5A proteins expressed in TOPO cloning system. J Microbiol Immunol Infect 2014;47(4):282-291.##Kim AY. Expression of the non-structural proteins NS3/4A of the hepatitis C virus using a genetically modified vesicular stomatitis virus vector system. 2016 Undergraduate Awards 2016;1-26.##Anticoli S, Falcone E, Ruggieri A, Federico M. Engineered exosomes boost the HCV NS3-specific CD8+ T lymphocyte immunity in humans. Trials Vaccinol 2016;5:105-110.##Vajdy M, Selby M, Medina-Selby A, Coit D, Hall J, Tandeske L, et al. Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses. J Gen Virol 2006;87(Pt 8):2253-2262.##Jafari S, Maleki Dizaj S, Adibkia K. Cell-penetrating peptides and their analogues as novel nanocarriers for drug delivery. Bioimpacts 2015;5(2):103-111.##Liu MJ, Chou JC, Lee HJ. A gene delivery method mediated by three arginine-rich cell-penetrating peptides in plant cells. Adv Stud Biol 2013;5(2):71-88.##Durzynska J, Przysiecka L, Nawrot R, Barylski J, Nowicki G, Warowicka A, et al. Viral and other cell-penetrating peptides as vectors of therapeutic agents in medicine. J Pharmacol Exp Ther 2015;354(1):32-42.##Ahlén G, Holmström F, Gibbs A, Alheim M, Frelin L. Long-term functional duration of immune responses to HCV NS3/4A induced by DNA vaccination. Gene Ther 2014;21(8):739-750.##McNaughton BR, Cronican JJ, Thompson DB, Liu DR. Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins. Proc Natl Acad Sci USA 2009;106(15):6111-6116.##Motevalli F, Bolhassani A, Hesami S, Shahbazi S. Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo. Immunol Lett 2018;194:29-39.##Lee SH, Kim SH, Park TG. Intracellular siRNA delivery system using poly-electrolyte complex micelles prepared from VEGF siRNA-PEG conjugate andcationic fusogenic peptide. Biochem Biophys Res Commun 2007;357(2):511-516.##Tang Q, Cao B, Wu H, Cheng G. Cholesterol-peptide hybrids to form liposome-like vesicles for gene delivery. PLoS One 2013;8(1):e54460.##Moret I, Esteban Peris J, Guillem VM, Benet M, Revert F, Dası´F, et al. Stability of PEI-DNA and DOTAP-DNA complexes: Effect of alkaline pH, heparin and serum. J Control Release 2001;76(1):169-181.##Sadeghian F, Hosseinkhani S, Alizadeh A, Hatefi A. Design, engineering and preparation of a multi-domain fusion vector for gene delivery. Int J Pharm 2012;427(2):393-399.##Krishnadas DK, Ahn JS, Han J, Kumar R, Agrawal B. Immunomodulation by hepatitis C virus-derived proteins: targeting human dendritic cells by multiple mechanisms. Int Immunol 2010;22(6):491-502.##Krishnadas DK, Li W, Kumar R, Tyrrell LJ, Agrawal B. In vitro activation and differentiation of naive CD4+ and CD8+ T cells into HCV core- and NS3-specific armed effector cells: a new role for CD4+ T cells. Cell Immunol 2009;259(2):141-149.##Haller AA, Lauer GM, King TH, Kemmler C, Fiolkoski V, Lu Y, et al. Whole recombinant yeast-based immunotherapy induces po¬tent T cell responses targeting HCV NS3 and Core proteins. Vaccine 2007;25(8):1452-1463.##Sarobe P, Lasarte JJ, Zabaleta A, Arribillaga L, Arina A, Melero I, et al. Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune re¬sponses. J Virol 2003;77(20):10862-10871.##Liu BR, Chan MH, Chen HH, Lo SY, Huang YW, Lee HJ. Effects of surface charge and particle size of cell-penetrating peptide/nanoparticle complexes on cellular internalization. In: Mandraccia L, Slavin G, editors. Cell membrane. USA: Nova Science Publishers, Inc; 2013. p. 43-57.##Chen YJ, Liu BR, Dai YH, Lee CY, Chan MH, Chen HH, et al. A gene delivery system for insect cells mediated by arginine-rich cell-penetrating peptides. Gene 2012;493(2):201-210.##Liu BR, Chen HH, Chan MH, Huang YW, Aronstam RS, Lee HJ. Three arginine-rich cell-penetrating peptides facilitate cellular internalization of red-emitting quantum dots. J Nanosci Nanotechnol 2015;15(3):2067-2078.##Revon Liu BR, Chiang HJ, Huang YW, Chan MH, Chen HH, Lee HJ. Cellular internalization of quantum dots mediated by cell-penetrating peptides. Pharm Nanotechnol 2013;1(2):151-161.##Liu BR, Huang YW, Winiarz JG, Chiang HJ, Lee HJ. Intracellular delivery of quantum dots mediated by a histidine- and arginine-rich HR9 cell-penetrating peptide through the direct membrane translocation mechanism. Biomaterials 2011;32(13):3520-353##Grubor-Bauk B, Yu W, Wijesundara D, Gummow J, Garrod T, Brennan AJ, et al. Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs. Gene Ther 2016;23(1):26-37.##Chen CP, Chou JC, Liu BR, Chang M, Lee HJ. Transfection and expression of plasmid DNA in plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation. 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Investigation of the Correlation between Androgen Receptor and ZEB1 and its Value in Progression of Gastric Cancer 2 2 Background: Zinc-finger Enhancer Binding protein (ZEB1) acts as a transcription factor to promote cancer progression through regulating Epithelial to Mesenchymal Transition (EMT). It is well-known that ZEB1 mRNA expression is directly induced by both Estrogen Receptor (ER) and Progesterone Receptor (PR). Moreover, Androgen Receptor (AR) and PR could bind to the same regulatory element. Since it has been shown that AR overexpresses in Gastric Cancer (GC) as a male-predominant tumor, the goal of this study was to evaluate whether AR could regulate ZEB1 expression in GC. Methods: The expression profile of ZEB1 in 60 fresh GC and adjacent non-tumor tissues and 50 normal gastric specimens was assessed by qRT-PCR, and the association of ZEB1 expression with clinicopathological features was investigated. Furthermore, possible correlation between ZEB1 and AR was evaluated to elucidate a novel prognostic marker using Kaplan-Meier method and Cox regression model. Finally, molecular interaction of ZEB1 and AR was assessed using a potent AR antagonist in GC cells. Results: Among GC patients, 70.2% (40/57) overexpressed ZEB1 and 64.91% (37/57) overexpressed AR relative to normal gastric tissues. ZEB1 overexpression was significantly correlated with the AR overexpression in GC patients. Moreover, ZEB1 overexpression was remarkably associated with lower overall survival; however, it was not an independent prognostic factor. Evidence shows that simultaneous evaluation of ZEB1 and AR expression could independently predict survival of GC patients (HR=2.193, p=0.047). Conclusion: These findings have clinical importance suggesting simultaneous evaluation of ZEB1 and AR expression as a potential prognostic marker. Moreover, AR may regulate ZEB1 expression in GC cells proposing a possible promising targeted therapy for GC patients. 52 60 Shahrzad Soleymani Fard Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Masoud Sotoudeh Digestive Oncology Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences Digestive Oncology Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences Iran Kioomars Saliminejad Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mansour Yazdanbod Department of Surgery, Madaen Hospital Department of Surgery, Madaen Hospital Iran Habibollah Mahmoodzadeh Department of Surgical Oncology, Cancer Institute, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences Department of Surgical Oncology, Cancer Institute, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences Iran Shaghayegh Kouchaki Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Marjan Yaghmaei Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Seyed Asadollah Mousavi Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Reza Malekzadeh Digestive Oncology Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences Digestive Oncology Research Center, Digestive Diseases Research Institute, Tehran University of Medical Sciences Iran Kamran Alimoghaddam Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Seyed Hamidollah Ghaffari Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Hematology, Oncology and Stem Cell Transplantation Research Institute, Faculty of Medicine, Tehran University of Medical Sciences Iran Androgen receptorEnzalutamideGastric cancerPrognostic markerTargeted therapyZEB1 20419.pdf Catalano V, Labianca R, Beretta GD, Gatta G, de Braud F, Van Cutsem E. Gastric cancer. Crit Rev Oncol Hematol 2009;71(2):127-164.##Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, et al. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial. Lancet 2010;376(9742):687-697.##Huang L, Wu RL, Xu AM. Epithelial-mesenchymal transition in gastric cancer. Am J Translational Res 2015;7(11):2141-2158.##Klymkowsky MW, Savagner P. Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe. Am J Pathol 2009;174(5):1588-1593.##Christiansen JJ, Rajasekaran AK. Reassessing epithelial to mesenchymal transition as a prerequisite for carcinoma invasion and metastasis. Cancer Res 2006;66(17):8319-8326.##Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M, et al. DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 2005;24(14):2375-2385.##Spaderna S, Schmalhofer O, Hlubek F, Berx G, Eger A, Merkel S, et al. A transient, EMT-linked loss of basement membranes indicates metastasis and poor survival in colorectal cancer. Gastroenterology 2006;131(3):830-840.##Graham TR, Zhau HE, Odero-Marah VA, Osunkoya AO, Kimbro KS, Tighiouart M, et al. Insulin-like growth factor-I-dependent up-regulation of ZEB1 drives epithelial-to-mesenchymal transition in human prostate cancer cells. Cancer Res 2008;68(7):2479-2488.##Graham TR, Yacoub R, Taliaferro-Smith L, Osunkoya AO, Odero-Marah VA, Liu T, et al. Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells. Breast Cancer Res Treat 2010;123(1):139-147.##Romero S, Musleh M, Bustamante M, Stambuk J, Pisano R, Lanzarini E, et al. Polymorphisms in TWIST1 and ZEB1 are associated with prognosis of gastric cancer patients. Anticancer Res 2018;38(7):3871-3877.##Murai T, Yamada S, Fuchs BC, Fujii T, Nakayama G, Sugimoto H, et al. Epithelial-to-mesenchymal transition predicts prognosis in clinical gastric cancer. J Surg Oncol 2014;109(7):684-689.##Xue Y, Zhang L, Zhu Y, Ke X, Wang Q, Min H. Regulation of proliferation and epithelial-to-mesenchymal transition (EMT) of gastric cancer by ZEB1 via modulating Wnt5a and related mechanisms. Med Sci Monit 2019;25:1663-1670.##Ma WL, Hsu CL, Wu MH, Wu CT, Wu CC, Lai JJ, et al. Androgen receptor is a new potential therapeutic target for the treatment of hepatocellular carcinoma. Gastroenterology 2008;135(3):947-955, 955.e1-5.##Li Y, Izumi K, Miyamoto H. The role of the androgen receptor in the development and progression of bladder cancer. Jpn J Clin Oncol 2012;42(7):569-577.##Konduri S, Schwarz MA, Cafasso D, Schwarz RE. Androgen receptor blockade in experimental combination therapy of pancreatic cancer. J Surg Res 2007;142(2):378-386.##Feng H, Cheng AS, Tsang DP, Li MS, Go MY, Cheung YS, et al. Cell cycle-related kinase is a direct androgen receptor-regulated gene that drives beta-catenin/T cell factor-dependent hepatocarcinogenesis. J Clin Invest 2011;121(8):3159-3175.##Tang W, Liu R, Yan Y, Pan X, Wang M, Han X, et al. Expression of estrogen receptors and androgen receptor and their clinical significance in gastric cancer. Oncotarget 2017;8(25):40765-40777.##Tian Y, Wan H, Lin Y, Xie X, Li Z, Tan G. Androgen receptor may be responsible for gender disparity in gastric cancer. Med Hypotheses 2013;80(5):672-674.##Miao L, Yang L, Li R, Rodrigues DN, Crespo M, Hsieh JT, et al. Disrupting androgen receptor signaling induces snail-mediated epithelial-mesenchymal plasticity in prostate cancer. Cancer Res 2017;77(11):3101-3112.##Cottard F, Asmane I, Erdmann E, Bergerat JP, Kurtz JE, Ceraline J. Constitutively active androgen receptor variants upregulate expression of mesenchymal markers in prostate cancer cells. PLoS One 2013;8(5):e63466.##Anose BM, Sanders MM. Androgen receptor regulates transcription of the ZEB1 transcription factor. Int J Endocrinol 2011;2011:903918.##Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008;3(6):1101-1108.##Mateo J, Smith A, Ong M, de Bono JS. Novel drugs targeting the androgen receptor pathway in prostate cancer. Cancer Metastasis Rev 2014;33(2-3):567-579.##Jia B, Liu H, Kong Q, Li B. Overexpression of ZEB1 associated with metastasis and invasion in patients with gastric carcinoma. Mol Cell Biochem 2012;366(1-2):223-229.##Zhang GJ, Zhou T, Tian HP, Liu ZL, Xia SS. High expression of ZEB1 correlates with liver metastasis and poor prognosis in colorectal cancer. Oncol Lett 2013;5(2):564-568.##Yabusaki N, Yamada S, Murai T, Kanda M, Kobayashi D, Tanaka C, et al. Clinical significance of zinc-finger E-box binding homeobox 1 mRNA levels in peritoneal washing for gastric cancer. Mol Clin Oncol 2015;3(2):435-441.##Zhou YM, Cao L, Li B, Zhang RX, Sui CJ, Yin ZF, et al. Clinicopathological significance of ZEB1 protein in patients with hepatocellular carcinoma. Ann Surg Oncol 2012;19(5):1700-1706.##Okugawa Y, Toiyama Y, Tanaka K, Matsusita K, Fujikawa H, Saigusa S, et al. Clinical significance of zinc finger E-box binding homeobox 1 (ZEB1) in human gastric cancer. J Surg Oncol 2012;106(3):280-285.##Zhang BG, Du T, Zang MD, Chang Q, Fan ZY, Li JF, et al. Androgen receptor promotes gastric cancer cell migration and invasion via AKT-phosphorylation dependent upregulation of matrix metalloproteinase 9. Oncotarget 2014;5(21):10584-10595.##Chen H, Lu W, Huang C, Ding K, Xia D, Wu Y, et al. Prognostic significance of ZEB1 and ZEB2 in digestive cancers: a cohort-based analysis and secondary analysis. Oncotarget 2017;8(19):31435-31448.##Wong TS, Gao W, Chan JY. Transcription regulation of E-cadherin by zinc finger E-box binding homeobox proteins in solid tumors. Biomed Res Int 2014;2014:921564.##Zhou L, Hu YL, Wu SW, Yu L, Cheng ZN, Zhu B. [Expressions of Slug, ZEB1 and KISS-1 in gastric adenocarcinoma and their clinical significance]. Nan Fang Yi Ke Da Xue Xue Bao 2016;36(4):532-537. Chinese.##Pena C, Garcia JM, Silva J, Garcia V, Rodriguez R, Alonso I, et al. E-cadherin and vitamin D receptor regulation by SNAIL and ZEB1 in colon cancer: clinicopathological correlations. Hum Mol Genet 2005;14(22):3361-3370.##Han J, Xie C, Pei T, Wang J, Lan Y, Huang K, et al. Deregulated AJAP1/beta-catenin/ZEB1 signaling promotes hepatocellular carcinoma carcinogenesis and metastasis. Cell Death Dis 2017;8(4):e2736.##Liu YN, Liu Y, Lee HJ, Hsu YH, Chen JH. Activated androgen receptor downregulates E-cadherin gene expression and promotes tumor metastasis. Mol Cell Biol 2008;28(23):7096-7108.##Chiurillo MA. Role of the Wnt/beta-catenin pathway in gastric cancer: An in-depth literature review. World J Exp Med 2015;5(2):84-102.##Ware KE, Somarelli JA, Schaeffer D, Li J, Zhang T, Park S, et al. Snail promotes resistance to enzalutamide through regulation of androgen receptor activity in prostate cancer. Oncotarget 2016;7(31):50507-50521.##Aghdassi A, Sendler M, Guenther A, Mayerle J, Behn CO, Heidecke CD, et al. Recruitment of histone deacetylases HDAC1 and HDAC2 by the transcriptional repressor ZEB1 downregulates E-cadherin expression in pancreatic cancer. Gut 2012;61(3):439-448.##

Evaluation of the Bioactive Potential of Secondary Metabolites Produced by a New Marine Micrococcus Species Isolated from the Persian Gulf 2 2 Background: In the present work, a newly isolated marine bacterium, Micrococcus sp. MP76, from coastal area of Persian Gulf around Bushehr province, Iran, was identified with the ability to produce bioactive compounds. Methods: The pigment production was optimized by changing carbon and nitrogen sources in bacterial growth media at 28°C and 220 rpm for 5 days. Partial purification of the pigment was carried out using suitable solvents. Results: Maximum pigment extract was achieved (1.4 g/l) when cultured in the medium containing 0.5% (v/v) molasses, 0.5% (w/v) peptone, 1% (w/v) sea salt, 0.01% (w/v) potassium phosphate, and 0.05% (w/v) yeast extract, pH=7.0. Antibacterial effect assessment of the extract against pathogenic bacteria revealed the MIC values in the range of 4.2-7.5 mg/ml depending on different pathogens. The pigment extracted from medium supplemented by molasses and ammonium sulfate had 81% radical scavenging activity, and its IC50 value was 0.28 mg/ml. Conclusion: The newly isolated strain of Micrococcus genus from the Persian Gulf revealed a valuable source to access worth medicinal ingredients when cultured under optimized conditions. 61 65 Hamid Reza Karbalaei-Heidari Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz UniversityInstitute of Biotechnology, Shiraz University Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz UniversityInstitute of Biotechnology, Shiraz University IranIran Mozhdeh Partovifar Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz University Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz University Iran Mina Memarpoor-Yazdi Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz University Molecular Biotechnology Lab., Department of Biology, Faculty of Science, Shiraz University Iran Antibacterial effectAntioxidant activityBioactive compoundsMarine bacteriaPersian Gulf 20420.pdf Schinke C, Martins T, Queiroz SC, Melo IS, Reyes FG. 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Importance of Argan Oil in Human Health According to the Dosage of Antioxidants in the Algerian Argan Fruits (Argania spinosa) 2 2 No Abstract 66 66 Zohra Benaouf Research Laboratory in Geo Environment and Spatial Development LGEDE, University of Mustapha Stambouli Research Laboratory in Geo Environment and Spatial Development LGEDE, University of Mustapha Stambouli Algeria Imen Benbahi Laboratory of Plant Ecology and Environment, Faculty of Biological Sciences, USTHB University, Bab Zouar Laboratory of Plant Ecology and Environment, Faculty of Biological Sciences, USTHB University, Bab Zouar Algeria Oussama Djorf Laboratory of Biochemistry, Faculty of Chemistry, USTHB University, Bab Zouar Laboratory of Biochemistry, Faculty of Chemistry, USTHB University, Bab Zouar Algeria Zahira Souidi Research Laboratory on Biological Systems and Geomancy (L.R.S.B.G), University of Mustapha Stambouli Research Laboratory on Biological Systems and Geomancy (L.R.S.B.G), University of Mustapha Stambouli Algeria Reda Kechairi Laboratory of Plant Ecology and Environment, Faculty of Biological Sciences, Telemcen University Laboratory of Plant Ecology and Environment, Faculty of Biological Sciences, Telemcen University Algeria AntioxidantsArgan oilHealthHumanMorocco 20421.pdf Hilali M, Charrouf Z, Soulhi Ael A, Hachimi L, Guillaume D. Influence of origin and extraction method on argan oil physico-chemical characteristics and composition. J Agric Food Chem 2005;53(6):2081-2087. ##Harhar H, Gharby S, Kartah B, El Monfalouti H, Guillaume D, Charrouf Z. Influence of argan kernel roasting-time on virgin argan oil composition and oxidative stability. Plant Food Hum Nutr 2011;66(2):163-168.##Derouiche A, Cherki M, Drissi A, Bamou Y, El Messal AM, Idrissi-Oudghiri A, et al. Nutritional intervention study with argan oil in man: effects on lipids and apolipoproteins. Ann Nutr Metab 2005;49(3):196-201.##Khallouki F, Younos C, Soulimani R, Oster T, Charrouf Z, Spiegelhalder B, et al. Consumption of argan oil (Morocco) with its unique profile of fatty acids, tocopherols, squalene, sterols and phenolic compounds should confer valuable cancer chemopreventive effects. Eur J Cancer Prev 2003;12(1):67-75.##Emonard H, Marcq V, Mirand C, Hornebeck W. Inhibition of gelatinase A by oleic acid. Ann N Y Acad Sci 1999;878:647-649.##Berrougui M, Alvarez de Sotomayor M, Perz-Guerrero C, Ettaib A, Hmamouchi E, Marhuenda E, et al. Argan (Argania spinosa) oil lowers blood pressure and improves endothelial dysfunction in spontaneously hypertensive rats. Br J Nutr 2004;92(6):921-929.##