Protection against the Omicron and Subsequent Coronavirus Variants: Medical-grade Masking, Third Dose Vaccination, Updating Vaccines, and Pursuing Universal Vaccine 2 2 No Abstract 1 2 Ahmad Shamabadi School of Medicine, Tehran University of Medical Sciences School of Medicine, Tehran University of Medical Sciences Iran Shahin Akhondzadeh COVID-19 PandemicMaskMass active immunizationSARS-CoV-2 60489.pdf ####

CRISPR-Cas System: A Promising Diagnostic Tool for Covid-19 2 2 More than a year has passed since the beginning of the 2019 novel coronavirus diseases (COVID-19) pandemic which has created massive problems globally affecting all aspects of people's life. Due to the emergence of new strains of the SARS-CoV-2, pandemic risk still remains, despite the start of vaccination. Therefore, rapid diagnostic tests are essential to control infection, improve clinical care and stop the spread of the disease. Recently CRISPR-based diagnostic tools have facilitated rapid diagnostic. Here, we review the diagnostic applications of CRISPR-Cas system in COVID-19. 3 9 Hashem Khanbabaei Department of Radiologic Sciences and Medical Physics, Faculty of Allied Medicine, Kerman University of Medical Sciences Department of Radiologic Sciences and Medical Physics, Faculty of Allied Medicine, Kerman University of Medical Sciences Iran Samaneh Abbasi Abadan University of Medical Sciences Abadan University of Medical Sciences Iran Mona Fani Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd Department of Pathobiology and Laboratory Sciences, School of Medicine, North Khorasan University of Medical Sciences, Bojnurd Iran Saber Soltani Department of Virology, School of Public Health, Tehran University of Medical Sciences Department of Virology, School of Public Health, Tehran University of Medical Sciences Iran Milad Zandi Department of Virology, School of Public Health, Tehran University of Medical Sciences Department of Virology, School of Public Health, Tehran University of Medical Sciences Iran Zahra Najafimemar Infectious Diseases Research Center, Golestan University of Medical Sciences Infectious Diseases Research Center, Golestan University of Medical Sciences Iran Saeedeh Ebrahimi COVID-19CRISPR-Cas systemsSARS-CoV-2 40489.pdf 1. 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Non-gynaecological Applications of Menstrual-derived Stem Cells: A Systematic Review 2 2 Menstrual-derived Stem Cells (MenSC) are a potential novel source of mesenchymal stem cells. There is an increased interest in investigating the therapeutic potential of MenSC due to the various advantages they exhibit, when compared to other types of stem cells. MenSC are obtained non-invasively from menstrual blood. Thus, collection of MenSC is simple, reproducible and can be carried out periodically, with minimal complications. MenSC are present in abundance, are highly proliferative, exhibit a low immunogenicity and lack ethical issues. MenSC have shown the ability to differentiate into several lineages. The therapeutic potential of MenSC in non-gynaecological applications has been investigated in wound healing, neurological, musculo-skeletal,  cardiovascular, respiratory, and liver disorders, as well as in diabetes and cancer. Human clinical trials are limited. To date, therapeutic efficacy and safety have been reported in patients with Avian influenza A subtype H7N9, COVID-19, congestive heart failure, multiple sclerosis and Duchene muscular dystrophy. However, further clinical trials in humans should be conducted, to study the long-term therapeutic effects of these stem cells in various diseases and to further explore their mechanism of action. This systematic review focuses on the application of MenSC in non-gynaecological diseases. 10 29 Claire Galea Nicoletta Riva Department of Anatomy, Faculty of Medicine and Surgery, University of MaltaDepartment of Pathology, Faculty of Medicine and Surgery, University of Malta Department of Anatomy, Faculty of Medicine and Surgery, University of MaltaDepartment of Pathology, Faculty of Medicine and Surgery, University of Malta MaltaMalta Jean Calleja-Agius Department of Anatomy, Faculty of Medicine and Surgery, University of Malta Department of Anatomy, Faculty of Medicine and Surgery, University of Malta Malta Cell therapyMenstruationMesenchymal stem cellsRegenerative medicineStem cells 40490.pdf 1. Meng X, Ichim TE, Zhong J, Rogers A, Yin Z, Jackson J, et al. Endometrial regenerative cells: A novel stem cell population. J Transl Med 2007;5:57.##2. Patel AN, Silva F. Menstrual blood stromal cells: The potential for regenerative medicine. Regen Med 2008;3(4):443-4.##3. Khoury M, Alcayaga-Miranda F, Illanes SE, Figueroa FE. The promising potential of menstrual stem cells for antenatal diagnosis and cell therapy. Front Immunol 2014;5:205.##4. Lv H, Hu Y, Cui Z, Jia H. Human menstrual blood: a renewable and sustainable source of stem cells for regenerative medicine. Stem Cell Res Ther 2018;9(1):325.##5. Liu Y, Niu R, Li W, Lin J, Stamm C, Steinhoff G, et al. Therapeutic potential of menstrual blood-derived endometrial stem cells in cardiac diseases. Cell Mol Life Sci 2019;76(9):1681-95.##6. Chen L, Qu J, Cheng T, Chen X, Xiang C. Menstrual blood-derived stem cells: Toward therapeutic mechanisms, novel strategies, and future perspectives in the treatment of diseases. Stem Cell Res Ther 2019;10(1):406.##7. Chen S, Dong C, Zhang J, Tang B, Xi Z, Cai F, et al. Human menstrual blood-derived stem cells protect H9c2 cells against hydrogen peroxide-associated apoptosis. In Vitro Cell Dev Biol Anim 2019;55(2):104-12.##8. Alcayaga-Miranda F, Cuenca J, Luz-Crawford P, Aguila-Díaz C, Fernandez A, Figueroa FE, et al. Characterization of menstrual stem cells: Angiogenic effect, migration and hematopoietic stem cell support in comparison with bone marrow mesenchymal stem cells. Stem Cell Res Ther 2015;6(1):32.##9. Azedi F, Kazemnejad S, Zarnani AH, Soleimani M, Shojaei A, Arasteh S. Comparative capability of menstrual blood versus bone marrow derived stem cells in neural differentiation. Mol Biol Rep 2017;44(1):169-82.##10. Khanjani S, Khanmohammadi M, Zarnani AH, Akhondi M, Ahani A, Ghaempanah Z, et al. Comparative evaluation of differentiation potential of menstrual blood- versus bone marrow-derived stem cells into hepatocyte-like cells. PloS One 2014;9(2):e86075.##11. Farzamfar S, Salehi M, Ehterami A, Naseri-Nosar M, Vaez A, Zarnani AH, et al. Promotion of excisional wound repair by a menstrual blood-derived stem cell-seeded decellularized human amniotic membrane. Biotechnol Lett 2018;8(4):393-8.##12. Akhavan-Tavakoli M, Fard M, Khanjani S, Zare S, Edalatkhah H, Mehrabani D, et al. In vitro differentiation of menstrual blood stem cells into keratinocytes: A potential approach for management of wound healing. Biologicals 2017;48:66-73.##13. Dalirfardouei R, Jamialahmadi K, Mahdipour E. A feasible method for the isolation of mesenchymal stem cells from menstrual blood and their exosomes. Tissue Cell 2018;55:53-62.##14. Arasteh S, Katebifar S, Shirazi R, Kazemnejad S. Differentiation of menstrual blood stem cells into keratinocyte-like cells on bilayer nanofibrous scaffold. Methods Mol Bio 2020;2125:129-56.##15. Zhao Y, Chen X, Wu Y, Wang Y, Li Y, Xiang C. Transplantation of human menstrual blood-derived mesenchymal stem cells alleviates alzheimer’s disease-like pathology in app/ps1 transgenic mice. Front Mol Neurosci 2018;11:140.##16. Zhong Z, Patel AN, Ichim TE, Riordan N, Wang H, Min WP, et al. Feasibility investigation of allogeneic endometrial regenerative cells. J Transl Med 2009;7:15.##17. Sanberg PR, Eve DJ, Willing AE, Garbuzova-Davis S, Tan J, Sanberg CD, et al. The treatment of neurodegenerative disorders using umbilical cord blood and menstrual blood-derived stem cells. Cell Transplant 2011;20(1):85-94.##18. Cui CH, Uyama T, Miyado K, Terai M, Kyo S, Kiyono T, et al. Menstrual blood-derived cells confer human dystrophin expression in the murine model of duchenne muscular dystrophy via cell fusion and myogenic transdifferentiation. Mol Biol Cell 2007;18(5):1586-94.##19. Vu NB, Trinh VN, Phi LT, Phan NK, Van Pham P. Human menstrual blood-derived stem cell transplantation for acute hind limb ischemia treatment in mouse models. Regen Med 2014.##20. Zhang Z, Wang JA, Xu Y, Jiang Z, Wu R, Wang L, et al. Menstrual blood derived mesenchymal cells ameliorate cardiac fibrosis via inhibition of endothelial to mesenchymal transition in myocardial infarction. Int J Cardiol 2013;168(2):1711-4.##21. Rahimi M, Zarnani A-H, Mohseni-Kouchesfehani H, Soltanghoraei H, Akhondi M-M, Kazemnejad S. Comparative evaluation of cardiac markers in differentiated cells from menstrual blood and bone marrow-derived stem cells in vitro. Mol Biotechnol 2014;56(12):1151-62.##22. Lu J, Xie ZY, Zhu DH, Li LJ. Human menstrual blood-derived stem cells as immunoregulatory therapy in COVID-19: A case report and review of the literature. World J Clin cases 2021;9(7):1705-13.##23. Chen L, Zhang C, Chen L, Wang X, Xiang V, Wu X, et al. Human menstrual blood-derived stem cells ameliorate liver fibrosis in mice by targeting hepatic stellate cells via paracrine mediators. Stem Cells Transl Med 2017;6(1):272-84.##24. Cen PP, Fan LX, Wang J, Chen JJ, Li LJ. Therapeutic potential of menstrual blood stem cells in treating acute liver failure. World J Gastroenterol 2019;25(41):6190-204.##25. Wu X, Luo Y, Chen J, Pan R, Xiang B, Du X, et al. Transplantation of human menstrual blood progenitor cells improves hyperglycemia by promoting endogenous progenitor differentiation in type 1 diabetic mice. Stem Cells Dev 2014;23(11):1245-57.##26. Dalirfardouei R, Jamialahmadi K, Jafarian AH, Mahdipour E. Promising effects of exosomes isolated from menstrual blood-derived mesenchymal stem cell on wound-healing process in diabetic mouse model. Tissue Eng Regen Med 2019;13(4):555-68.##27. Moreno R, Fajardo CA, Farrera-Sal M, Perisé-Barrios AJ, Morales-Molina A, Al-Zaher AA, et al. Enhanced antitumor efficacy of oncolytic adenovirus-loaded menstrual blood-derived mesenchymal stem cells in combination with peripheral blood mononuclear cells. Mol Cancer Ther 2019;18(1):127-38.##28. Rosenberger L, Ezquer M, Lillo-Vera F, Pedraza PL, Ortúzar MI, González PL, et al. Stem cell exosomes inhibit angiogenesis and tumor growth of oral squamous cell carcinoma. Sci Rep 2019;9(1):663.##29. Alcayaga-Miranda F, González PL, Lopez-Verrilli A, Varas-Godoy M, Aguila-Díaz C, Contreras L, et al. Prostate tumor-induced angiogenesis is blocked by exosomes derived from menstrual stem cells through the inhibition of reactive oxygen species. Oncotarget 2016;7(28):44462-77.##30. Wang XJ, Xiang BY, Ding YH, Chen L, Zou H, Mou XZ, et al. Human menstrual blood-derived mesenchymal stem cells as a cellular vehicle for malignant glioma gene therapy. Oncotarget 2017;8(35):58309-21.##31. Guo Y, Zhang Z, Xu X, Xu Z, Wang S, Huang D, et al. Menstrual blood-derived stem cells as delivery vehicles for oncolytic adenovirus virotherapy for colorectal cancer. Stem Cells Dev 2019;28(13):882-96.##32. Feng P, Li P, Tan J. Human menstrual blood-derived stromal cells promote recovery of premature ovarian insufficiency via regulating the ECM-dependent FAK/AKT signaling. Stem Cell Rev Rep 2019;15(2):241-55.##33. Noory P, Navid S, Zanganeh BM, Talebi A, Borhani-Haghighi M, Gholami K, et al. Human menstrual blood stem cell-derived granulosa cells participate in ovarian follicle formation in a rat model of premature ovarian failure in vivo. Cell Reprogram 2019;21(5):249-59.##34. Wang X, Wang C, Cong J, Bao H, Liu X, Hao C. Regenerative potential of menstrual blood-derived stem cells and platelet-derived growth factor in endometrial injury. Med Sci Monit 2020;26:e919251.##35. Hu J, Song K, Zhang J, Zhang Y, Tan BZ. Effects of menstrual blood-derived stem cells on endometrial injury repair. Mol Med Rep 2019;19(2):813-20.##36. Tan J, Li P, Wang Q, Li Y, Li X, Zhao D, et al. Autologous menstrual blood-derived stromal cells transplantation for severe Asherman's syndrome. Hum Reprod 2016;31(12):2723-9.##37. Zhang S, Li P, Yuan Z, Tan J. Platelet-rich plasma improves therapeutic effects of menstrual blood-derived stromal cells in rat model of intrauterine adhesion. Stem Cell Res Ther 2019;10(1):61.##38. Meng X, Ichim TE, Zhong J, Rogers A, Yin Z, Jackson J, et al. Endometrial regenerative cells: A novel stem cell population. J Transl Med 2007;5:57.##39. Chen L, Qu J, Xiang C. The multi-functional roles of menstrual blood-derived stem cells in regenerative medicine. Stem Cell Res Ther 2019;10(1):1.##40. Zhong Z, Patel AN, Ichim TE, Riordan NH, Wang H, Min W, et al. Feasibility investigation of allogeneic endometrial regenerative cells. J Transl Med 2009;7:15.##41. Jiang Z, Hu X, Yu H, Xu Y, Wang L, Chen H, et al. Human endometrial stem cells confer enhanced myocardial salvage and regeneration by paracrine mechanisms. J Cell Mol Med 2013;17(10):1247-60.##42. Lai D, Wang F, Yao X, Zhang Q, Wu X, Xiang C. Human endometrial mesenchymal stem cells restore ovarian function through improving the renewal of germline stem cells in a mouse model of premature ovarian failure. J Transl Med 2015;13(1):155.##43. Wang K, Jiang Z, Webster KA, Chen J, Hu H, Zhou Y, et al. Enhanced cardioprotection by human endometrium mesenchymal stem cells driven by exosomal microRNA-21. Stem Cells Transl Med 2017;6(1):209-22.##44. Liu Y, Niu R, Yang F, Yan Y, Liang S, Sun Y, et al. Biological characteristics of human menstrual blood-derived endometrial stem cells. J Cell Mol Med 2018;22(3):1627-39.##45. Liu Y, Yang F, Liang S, Liu Q, Fu S, Wang Z, et al. N-cadherin upregulation promotes the neurogenic differentiation of menstrual blood-derived endometrial stem cells. Stem Cells Intl 2018;2018:3250379-10.##46. Zhao Y, Chen X, Wu Y, Wang Y, Li Y, Xiang C. Transplantation of human menstrual blood-derived mesenchymal stem cells alleviates alzheimer’s disease-like pathology in app/ps1 transgenic mice. Front Mol Neurosci 2018;11:140.##47. Moreno R, Rojas LA, Villellas FP, Soriano VC, Garcia-Castro J, Fajardo CA, et al. Human menstrual blood-derived mesenchymal stem cells as potential cell carriers for oncolytic adenovirus. Stem Cells Int 2017;2017:3615729-10.##48. Mou XZ, Lin J, Chen JY, Li YF, Wu XX, Xiang BY, et al. Menstrual blood-derived mesenchymal stem cells differentiate into functional hepatocyte-like cells. J Zhejiang Uni Sci B 2013;14(11):961-72.##49. Chen L, Xiang B, Wang X, Xiang C. Exosomes derived from human menstrual blood-derived stem cells alleviate fulminant hepatic failure. Stem Cell Res Ther 2017;8(1):9.##50. Khanjani S, Khanmohammadi M, Zarnani AH, Talebi S, Edalatkhah H, Eghtesad S, et al. Efficient generation of functional hepatocyte-like cells from menstrual blood-derived stem cells. J Tissue Eng Regen Med 2015;9(11):E124-E134.##51. Khanmohammadi M, Khanjani S, Edalatkhah H, Zarnani AH, Heidari-Vala H, Soleimani M, et al. Modified protocol for improvement of differentiation potential of menstrual blood-derived stem cells into adipogenic lineage. Cell Prolif 2014;47(6):615-23.##52. Zheng SX, Wang J, Wang XL, Ali A, Wu LM, Liu YS. Feasibility analysis of treating severe intrauterine adhesions by transplanting menstrual blood-derived stem cells. Int J Mol Med 2018;41(4):2201-2212.##53. Chen L, Zhang C, Chen L, Wang X, Xiang B, Wu X, et al. Human menstrual blood-derived stem cells ameliorate liver fibrosis in mice by targeting hepatic stellate cells via paracrine mediators. Stem CellsTransl Med 2017;6(1):272-84.##54. Liu T, Huang Y, Zhang J, Qin W, Chi H, Chen J, et al. Transplantation of human menstrual blood stem cells to treat premature ovarian failure in mouse model. Stem Cells Dev 2014;23(13):1548-57.##55. Wang Z, Wang Y, Yang T, Li J, Yang X. Study of the reparative effects of menstrual-derived stem cells on premature ovarian failure in mice. Stem Cell Res Ther 2017;8(1):11.##56. Alcayaga-Miranda F, Cuenca J, Luz-Crawford P, Aguila-Díaz C, Fernandez A, Figueroa FE, et al. Characterization of menstrual stem cells: Angiogenic effect, migration and hematopoietic stem cell support in comparison with bone marrow mesenchymal stem cells. Stem Cell Res Ther 2015;6(1):32.##57. Wu X, Luo Y, Chen J, Pan R, Xiang B, Du X, et al. Transplantation of human menstrual blood progenitor cells improves hyperglycemia by promoting endogenous progenitor differentiation in type 1 diabetic mice. Stem Cells Dev 2014;23(11):1245-57.##58. Patel AN, Park E, Kuzman M, Benetti F, Silva FJ, Allickson JG. Multipotent menstrual blood stromal stem cells: isolation, characterization, and differentiation. Cell Transplant 2008;17(3):303-11.##59. Nikoo S, Ebtekar M, Jeddi-Tehrani M, Shervin A, Bozorgmehr M, Kazemnejad S, et al. Effect of menstrual blood-derived stromal stem cells on proliferative capacity of peripheral blood mononuclear cells in allogeneic mixed lymphocyte reaction. J Obstet Gynaecol Res 2012;38(5):804-9.##60. 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New Generation Vaccines for COVID-19 Based on Peptide, Viral Vector, Artificial Antigen Presenting Cell, DNA or mRNA 2 2 At present, effective vaccines have been developed as the most successful approaches for preventing widespread infectious disease. The global efforts are focusing with the aim of eliminating and overcoming the Coronavirus Disease 2019 (COVID-19) and are developing vaccines from the date it was announced as a pandemic disease. In this study, PubMed, Embase, Cochrane Library, Clinicaltrial.gov, WHO reports, Science Direct, Scopus, Google Scholar, and Springer databases were searched for finding the relevant studies about the COVID-19 vaccines. This article provides an overview of multiple vaccines that have been manufactured from December 2020 up to April 2021 and also offers a perspective on their efficacy, safety, advantages, and limitations. Currently, there are several categories of COVID-19 vaccines based on Protein Subunit (PS), Inactivated Virus (IV), Virus Like Particle (VLP), Live Attenuated Virus (LAV), Viral Vector (replicating) (VVr) and Viral Vector (non-replicating) (VVnr) in progress or finalized as indicated by the WHO reporting of April 1, 2020. 30 36 Marzieh Rezaei Department of Cell & Molecular Biology and Microbiology, Faculty of Science and Biotechnology, University of Isfahan Department of Cell & Molecular Biology and Microbiology, Faculty of Science and Biotechnology, University of Isfahan Iran Mahboobeh Nazari COVID-19PandemicsSARS-CoV-2Vaccines 40491.pdf 1. Drosten C, Günther S, Preiser W, Van Der Werf S, Brodt H-R, Becker S, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. New Eng J Med 2003;348(20):1967-76.##2. Zaki AM, Van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. 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Monoclonal Antibody Against Sortilin Induces Apoptosis in Human Breast Cancer Cells 2 2 Background: Sortilin has an important role in various malignances and can be used as a promising target to eradicate cancer cells. Methods: In this study, the expression of sortilin in 4T1 and MDA-MB231 cell lines was evaluated by flow cytometry and immunocytochemistry. Apoptosis assay was also applied to evaluate apoptosis induction in 4T1 and MDA-MB231 cell lines. Results: Based on cell surface flow cytometry results, anti-sortilin (2D8-E3) mAb could recognize sortilin molecules in 79.2% and 90.3% of 4T1 and MDA-MB231 cell-lines, respectively. The immunocytochemistry staining results confirmed sortilin surface expression. Apoptosis assay indicated that anti-sortilin mAb could induce apoptosis in 4T1 and MDA-MB231 cell lines. Conclusion: Our study revealed the important role of surface sortilin in breast carcinoma cell survival and its possible application as a therapeutic agent in cancer targeted therapies. 37 45 Miganoosh Simonian Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences Iran Mozhan Haji Ghaffari Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences Iran Ali Salimi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Ebrahim Mirzadegan Nanobiotechnology Research Center, Avicenna Research Institute, ACECR Iran Niloufar Sadeghi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Nasim Ebrahimnezhad Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Ghazaleh Fazli Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Ramina Fatemi Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Ali Ahmad Bayat Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Saeideh Milani Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine Iran Babak Negahdari Hodjattallah Rabbani Breast cancerFlow cytometryMonoclonal antibodySortilin 40492.pdf 1. 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The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein. EMBO J 2001;20(9):2180-90.##16. Nykjaer A, Lee R, Teng KK, Jansen P, Madsen P, Nielsen MS, et al. Sortilin is essential for proNGF-induced neuronal cell death. Nature 2004;427(6977):843-8.##17. Patel KM, Strong A, Tohyama J, Jin X, Morales CR, Billheimer J, et al. Macrophage sortilin promotes LDL uptake, foam cell formation, and atherosclerosis. Circ Res 2015;116(5):789-96.##18. Mazella J, Vincent J-P. Functional roles of the NTS2 and NTS3 receptors. Peptides 2006;27(10):2469-75.##19. Roselli S, Pundavela J, Demont Y, Faulkner S, Keene S, Attia J, et al. Sortilin is associated with breast cancer aggressiveness and contributes to tumor cell adhesion and invasion. Oncotarget 2015;6(12):10473.##20. Akil H, Perraud A, Mélin C, Jauberteau M-O, Mathonnet M. Fine-tuning roles of endogenous brain-derived neurotrophic factor, TrkB and sortilin in colorectal cancer cell survival. PloS One 2011;6(9):e25097.##21. Wilson CM, Naves T, Vincent F, Melloni B, Bonnaud F, Lalloué F, et al. Sortilin mediates the release and transfer of exosomes in concert with two tyrosine kinase receptors. J Cell Sci 2014;127(18):3983-97.##22. Tanimoto R, Morcavallo A, Terracciano M, Xu S-Q, Stefanello M, Buraschi S, et al. Sortilin regulates progranulin action in castration-resistant prostate cancer cells. Endocrinology 2015;156(1):58-70.##23. Truzzi F, Marconi A, Lotti R, Dallaglio K, French LE, Hempstead BL, et al. Neurotrophins and their receptors stimulate melanoma cell proliferation and migration. J Invest Dermatol 2008;128(8):2031-40.##24. Tan S, Li D, Zhu X. Cancer immunotherapy: Pros, cons and beyond. Biomed Pharmacother 2020;124:109821.##25. Dal Farra C, Sarret P, Navarro V, Botto JM, Mazella J, Vincent JP. Involvement of the neurotensin receptor subtype NTR3 in the growth effect of neurotensin on cancer cell lines. Int J Cancer 2001;92(4):503-9.##26. Miguel A, Berger J, Sánchez-Prieto R, Saada S, Naves T, Guillaudeau A, et al. p75 neurotrophin receptor and pro-BDNF promote cell survival and migration in clear cell renal cell carcinoma. Oncotarget 2016;7(23):34480.##27. Béraud-Dufour S, Devader C, Massa F, Roulot M, Coppola T, Mazella J. Focal adhesion kinase-dependent role of the soluble form of neurotensin receptor-3/sortilin in colorectal cancer cell dissociation. Int J Mol Sci 2016;17(11):1860.##28. Yang W, Wu PF, Ma JX, Liao MJ, Wang XH, Xu LS, et al. Sortilin promotes glioblastoma invasion and mesenchymal transition through GSK-3β/β-catenin/twist pathway. Cell Death Dis 2019;10(3):1-15.##29. Demeule M, Charfi C , Currie JC, Larocque A, Zgheib A , Kozelko S, et al. TH1901, a novel curcumin-peptide conjugate for the treatment of Sortilin-positive (SORT1+) cancer. Cancer Sci 2021 Oct;112(10):4317-34.##30. Marsolais C, Charfi C, Demeule M, Currie J-C, Larocque A, Zgheib A, et al. 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Fauchais AL, Lalloué F, Lise MC, Boumediene A, Preud'Homme JL, Vidal E, et al. Role of endogenous brain-derived neurotrophic factor and sortilin in B cell survival. J Immunol 2008;181(5):3027-38.##36. Farahi L, Ghaemimanesh F, Milani S, Razavi SM, Akhondi MM, Rabbani H. Sortilin as a novel diagnostic and therapeutic biomarker in chronic lymphocytic leukemia. Avicenna J Med Biotechnol 2019;11(4):270.##37. Ludwig DL, Pereira DS, Zhu Z, Hicklin DJ, Bohlen P. Monoclonal antibody therapeutics and apoptosis. Oncogene 2003;22(56):9097-106.##38. Ghaemimanesh F, Ahmadian G, Talebi S, Zarnani A-H, Behmanesh M, Hemmati S, et al. The effect of sortilin silencing on ovarian carcinoma cells. Avicenna J Med Biotechnol 2014;6(3):169-77.##39. Faulkner S, Jobling P, Rowe CW, Oliveira SR, Roselli S, Thorne RF, et al. Neurotrophin receptors TrkA, p75NTR, and sortilin are increased and targetable in thyroid cancer. Am J Pathol 2018;188(1):229-41.##40. Tsai YC, Tsai TH, Chang CP, Chen SF, Lee YM, Shyue SK. 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Exogenous Production of N-acetylmuramyl-L Alanine Amidase (LysM2) from Siphoviridae Phage Affecting Anti-Gram-Negative Bacteria: Evaluation of Its Structure and Function 2 2 Background: To obtain endolysin with impact(s) on gram-negative bacteria as well as gram-positive bacteria, N-acetylmuramyl L-alanine-amidase (MurNAc-LAA) from a Bacillus subtilis-hosted Siphoviridae phage (SPP1 phage, Subtilis Phage Pavia 1) was exogenously expressed in Escherichia coli (E. coli).  Methods: The sequences of MurNAc-LAA genes encoding peptidoglycan hydrolases were obtained from the Virus-Host database. The sequence of MurNAc-LAA was optimized by GenScript software to generate MurNAc-LAA-MMI (LysM2) for optimal expression in E. coli. Furthermore, the structure and function of LysM2 was evaluated in silico. The optimized gene was synthesized, subcloned in the pET28a, and expressed in E. coli BL21(DE3). The antibacterial effects of the protein on the peptidoglycan substrates were studied. Results: LysM2, on 816 bp gene encoding a 33 kDa protein was confirmed as specific SPP1 phage enzyme. The enzyme is composed of 271 amino acids, with a half-life of 10 hr in E. coli. In silico analyses showed 34.2% alpha-helix in the secondary structure, hydrophobic N-terminal, and lysine-rich C-terminal, and no antigenic properties in LysM2 protein. This optimized endolysin revealed impacts against Proteus (sp) by turbidity, and an antibacterial activity against Klebsiella pneumoniae, Salmonella typhimurium, and Proteus vulgaris in agar diffusion assays. Conclusion: Taken together, our results confirmed that LysM2 is an inhibiting agent for gram-negative bacteria. 46 53 Morteza Miri Department of Biotechnology, Faculty of Biotechnology, Semnan University Department of Biotechnology, Faculty of Biotechnology, Semnan University Iran Sepideh Yazdianpour Department of Biotechnology, Faculty of Biotechnology, Semnan University Department of Biotechnology, Faculty of Biotechnology, Semnan University Iran Shamsozoha Abolmaali Shakiba Darvish Alipour Astaneh Department of Biotechnology, Faculty of Biotechnology, Semnan University Department of Biotechnology, Faculty of Biotechnology, Semnan University Iran Antibacterial activityBacteriophage SPP1EndolysinSiphoviridae 50488.pdf 1. Scheurwater EM, Pfeffer JM, Clarke AJ. Production and purification of the bacterial autolysin N-acetylmuramoyl-L-alanine amidase B from Pseudomonas aeruginosa. Protein Expr Purif 2007;56(1):128-37.##2. Kashani HH, Schmelcher M, Sabzalipoor H, Hosseini ES, Moniri R. Recombinant endolysins as potential therapeutics against antibiotic-resistant Staphylococcus aureus: current status of research and novel delivery strategies. Clin Microbiol Rev 2018;31(1):e00071-17.##3. Grishin AV, Karyagina AS, Vasina DV, Vasina IV, Gushchin VA, Lunin VG. Resistance to peptidoglycan-degrading enzymes. Crit Rev Microbiol 2020;46(6):703-26.##4. Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials. Future Microbiol 2012;7(10):1147-71.##5. Santos SB, Oliveira A, Melo LD, Azeredo J. Identification of the first endolysin Cell Binding Domain (CBD) targeting Paenibacillus larvae. Sci Rep 2019;9(1):2568.##6. Morita M, Tanji Y, Orito Y, Mizoguchi K, Soejima A, Unno H. Functional analysis of antibacterial activity of Bacillus amyloliquefaciens phage endolysin against Gram‐negative bacteria. FEBS Lett 2001;500(1-2):56-9.##7. Roach DR, Donovan DM. Antimicrobial bacteriophage-derived proteins and therapeutic applications. Bacteriophage 2015;5(3):e1062590.##8. Fernández-Ruiz I, Coutinho FH, Rodriguez-Valera F. Thousands of novel endolysins discovered in uncultured phage genomes. Front Microbiol 2018;9:1033.##9. São-José C. Correction: São-José, C.Engineering of phage-derived lytic enzymes: improving their potential as antimicrobials. Antibiotics 2018, 7, 29. Antibiotics (Basel) 2018;7(3):56.##10. Love MJ, Abeysekera GS, Muscroft-Taylor AC, Billington C, Dobson RC. On the catalytic mechanism of bacteriophage endolysins: Opportunities for engineering. Biochim Biophys Acta (BBA) Proteins Proteom 2020;1868(1):140302.##11. Guo M, Feng C, Ren J, Zhuang X, Zhang Y, Zhu Y, et al. A novel antimicrobial endolysin, LysPA26, against Pseudomonas aeruginosa. Front Microbiol 2017;8:293.##12. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, et al. CDD: NCBI's conserved domain database. Nucleic acids Res 2015;43(Database issue):D222-D6.##13. Quintavalla S, Vicini L. Antimicrobial food packaging in meat industry. Meat Sci 2002;62(3):373-80.##14. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids Res 2004;32(5):1792-7.##15. Reuter JS, Mathews DH. RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 2010;11(1):129.##16. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, et al. Protein identification and analysis tools on the ExPASy server. Methods Mol Biol 1999;112:531-52.##17. Geourjon C, Deleage G. SOPMA: significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments. Comput Appl Biosci 1995;11(6):681-4.##18. Krogh A, Larsson B, Von Heijne G, Sonnhammer EL. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 2001;305(3):567-80.##19. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015;10(6):845.##20. Wiederstein M, Sippl MJ. ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic acids Res 2007;35(Web server issue):W407-W10.##21. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 1993;26(2):283-91.##22. Doytchinova IA, Flower DR. VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics 2007;8(1):4.##23. Noormohammadi H, Abolmaali S, Astaneh SDA. Identification and characterization of an endolysin–like from Bacillus subtilis. Microb Pathog 2018;119:221-4.##24. Sarjoughian MR, Rahmani F, Abolmaali Sh, Darvish Alipour Sh. Bacillus phage endolysin, lys46, bactericidal properties against gram-negative bacteria. Iran J Microbiol 2020;12(6):505-12.##25. Dong H, Zhu C, Chen J, Ye X, Huang YP. Antibacterial Activity of Stenotrophomonas maltophilia Endolysin P28 against both gram-positive and gram-negative bacteria. Front Microbiol 2015;6:1299.##26. Tossi A, Sandri L, Giangaspero A. Amphipathic, α‐helical antimicrobial peptides. Biopolymers 2000;55(1):4-30.##27. Regamey A, Karamata D. The N-acetylmuramoyl-l-alanine amidase encoded by the Bacillus subtilis 168 prophage SPβ. Microbiology 1998;144(Pt 4):885-93.##28. Lai MJ, Lin NT, Hu A, Soo PC, Chen LK, Chen LH, et al. Antibacterial activity of Acinetobacter baumannii phage ϕAB2 endolysin (LysAB2) against both gram-positive and gram-negative bacteria. Appl Microbiol Biotechnol 2011;90(2):529-39.##29. Ghose C, Euler CW. Gram-negative bacterial lysins. Antibiotics 2020;9(2):74.##30. Oliveira H, Thiagarajan V, Walmagh M, Sillankorva S, Lavigne R, Neves-Petersen MT, et al. A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids. PLoS One 2014;9(10):e108376.##31. Yakhnina AA, Bernhardt TG. The Tol-Pal system is required for peptidoglycan-cleaving enzymes to complete bacterial cell division. Proc Natl Acad Sci 2020;117(12):6777-83.##32. Heidrich C, Templin MF, Ursinus A, Merdanovic M, Berger J, Schwarz H, et al. Involvement of N‐acetylmuramyl‐L‐alanine amidases in cell separation and antibiotic‐induced autolysis of Escherichia coli. Mol Microbiol 2001;41(1):167-78.##33. Plotka M, Sancho-Vaello E, Dorawa S, Kaczorowska AK, Kozlowski LP, Kaczorowski T, et al. Structure and function of the Ts2631 endolysin of Thermus scotoductus phage vB_Tsc2631 with unique N-terminal extension used for peptidoglycan binding. Sci Rep 2019;9(1):1261.##34. Plotka M, Kapista M, Dorawa S, Kaczorowska AK, Kaczorowski T. Ts2631 Endolysin from the extremophilic Thermus scotoductus bacteriophage vB_Tsc2631 as an antimicrobial agent against gram-negative multidrug-resistant bacteria. Viruses 2019;11(7):657.##35. Plotka M, Szadkowska M, Håkansson M, Kovacic R, Al-Karadaghi S, Walse B, et al. Molecular characterization of a novel lytic enzyme LysC from Clostridium intestinale URNW and its antibacterial activity mediated by positively charged N-terminal extension. Int J Mol Sci 2020;21(14):4894.##

Genome Analysis of the Enterococcus faecium Entfac.YE Prophage 2 2 Background: Bacteriophages are viruses that infect bacteria. Bacteriophages are widely distributed in various environments. The prevalence of bacteriophages in water sources, especially wastewaters, is naturally high. These viruses affect evolution of most bacterial species. Bacteriophages are able to integrate their genomes into the chromosomes of their hosts as prophages and hence transfer resistance genes to the bacterial genomes. Enterococci are commensal bacteria that show high resistance to common antibiotics. For example, prevalence of vancomycin-resistant enterococci has increased within the last decades. Methods: Enterococcal isolates were isolated from clinical samples and morphological, phenotypical, biochemical, and molecular methods were used to identify and confirm their identity. Bacteriophages extracted from water sources were then applied to isolated Enterococcus faecium (E. faecium). In the next step, the bacterial genome was completely sequenced and the existing prophage genome in the bacterial genome was analyzed. Results: In this study, E. faecium EntfacYE was isolated from a clinical sample. The EntfacYE genome was analyzed and 88 prophage genes were identified. The prophage content included four housekeeping genes, 29 genes in the group of genes related to replication and regulation, 25 genes in the group of genes related to structure and packaging, and four genes belonging to the group of genes associated with lysis. Moreover, 26 genes were identified with unknown functions. Conclusion: In conclusion, genome analysis of prophages can lead to a better understanding of their roles in the rapid evolution of bacteria. 54 60 Yara Elahi Department of Genetics, Faculty of Life Sciences, Islamic Azad University Tehran North Branch Department of Genetics, Faculty of Life Sciences, Islamic Azad University Tehran North Branch Iran Ramin Mazaheri Nezhad Fard Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences Iran Arash Seifi Department of Infectious Diseases, School of Medicine, Tehran University of Medical Sciences Department of Infectious Diseases, School of Medicine, Tehran University of Medical Sciences Iran Saeideh Mahfouzi Department of Virology, School of Public Health, Tehran University of Medical Sciences, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Iran Ali Akbar Saboor Yaraghi Antibacterial agentsBacteriophagesEnterococcus faeciumGenome analysisProphages 50489.pdf 1. Monstein H-J, Quednau M, Samuelsson A, Ahrne S, Isaksson B, Jonasson J. Division of the genus Enterococcus into species groups using PCR-based molecular typing methods. Microbiology 1998;144:1171-9.##2. Tsakris A, Woodford N, Pournaras S, Kaufmann M, Douboyas J. Apparent increased prevalence of high-level aminoglycoside-resistant Enterococcus durans resulting from false identification by a semiautomatic software system. J Clin Microbiol 1998;36:1419-21.##3. Tünger A, Aydemir S, Uluer S, Cilli F. In vitro activity of linezolid & quinupristin/dalfopristin against Gram-positive cocci. Indian J Med Res 2004;120(6):546-52.##4. Alavidze Z, Aminov R, Betts A, Bardiau M, Bretaudeau L, Caplin J, et al. Silk route to the acceptance and re-implementation of bacteriophage therapy - Expert round table on acceptance and re-implementation of bacteriophage therapy. Biotechnol. J 2016;11(5):595-600.##5. Sime-Ngando T. Environmental bacteriophages: viruses of microbes in aquatic ecosystems. Front Microbiol 2014;24;5:355.##6. Nobrega FL, Costa AR, Kluskens LD, Azeredo J. Revisiting phage therapy: new applications for old resources. Trends Microbiol 2015;23(4):185-91.##7. Pirnay JP, Blasdel BG, Bretaudeau L, Buckling A, Chanishvili N, Clark JR, et al. Quality and safety requirements for sustainable phage therapy products. Pharm Res 2015;32(7):2173-9.##8. Al-Rammahi SA, Hussein JM, Kadhem EJ, Al-Hadad AS. Bacteriological study of Enterococcus bacteria isolated from different diseases in the female cows. Biomed Pharmacol J 2020;13(2):989-98.##9. Karna A, Baral R, Khanal B. Characterization of clinical isolates of enterococci with special reference to glycopeptide susceptibility at a tertiary care center of Eastern Nepal. Int J Microbiol 2019;2019.##10. Chatterjee A, Willett JL, Nguyen UT, Monogue B, Palmer KL, Dunny GM, et al. Parallel genomics uncover novel enterococcal-bacteriophage interactions. MBio 2020;11(2):e03120-19.##11. Arbabi L, Boustanshenas M, Adabi M, Fathizadeh S, Rasouli Koohi S, Afshar M, et al. Isolation and antibiotic susceptibility pattern among vancomycin-resistant enter-ococci isolated from clinical samples of different parts of Rasoul-E-Akram Hospital. J Ardabil University of Medi-cal Sciences (JAUMS) 2016;15(4):404-13.##12. Samadi H, Pirhajati Mahabadi R, Pournajaf A, Omidi S, Moghimyan S, Alyasin N. An investigation of the vanA and vanB genes in Enterococcus faecalis and Enterococcus faecium strains isolated from the hospitalized patients in Shariati Hospital and evaluation of their antibiotic susceptibility. Qom Univ Med Sci J 2015;9(3):32-8.##13. Bhardwaj SB, Mehta M, Sood S, Sharma J. Isolation of a novel phage and targeting biofilms of drug-resistant oral enterococci. J Glob Infect Dis 2020;12(1):11.##14. Lopes A, Amarir-Bouhram J, Faure G, Petit MA, Guerois R. Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2. 5 remote homologs. 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Optimization of Expression and Purification of Recombinant Mouse plac1 2 2 Background: Placenta-specific 1 (PLAC1) is one of the recently-discovered Cancer-Testis-Placenta (CTP) antigen with restricted normal tissue and ectopic expression in a wide range of cancer cells from different histological origins. The production of recombinant human PLAC1 has already been optimized; however, no study has been reported so far on the production and purification of mouse plac1. In this study, mouse plac1 expression and purification was optimized in a prokaryotic system and the effects of the generated proteins on inducing humoral responses in mice were investigated. Methods: A fusion protein containing full extracellular domain of mouse plac1, immunostimulatory peptides, tetanus toxin P2P30 and PADRE and KDEL3 signal (main plac1), and the same fragment without immunostimulatory peptides (control plac1) was produced. To optimize production and purification steps, different parameters including bacterial strain, cultivation temperature, cultivation time, IPTG concentration, culture medium, and also different buffers for purification of the recombinant proteins were tested. After confirming the identity of recombinant plac1 proteins with Western Blotting (WB) and ELISA assays, these proteins were subcutaneously injected in mice with Freund's adjuvant and the anti-plac1 antibody response was detected by ELISA. Results: The optimal expression level of main and control plac1 was obtained in BL21 (DE3) and TB culture medium in the presence of 0.25 mM IPTG after 24 hr of induction at 15°C. The buffer containing 2% sarkosyl produced higher yield and purity. Our results showed specific reactivity of anti-human recombinant plac1 polyclonal antibody with both main and control plac1 recombinant proteins in WB and ELISA analysis. Both proteins induced humoral responses in mice; however, anti-plac1  antibody titer was significantly higher in sera of mice immunized with main compared to control plac1. Conclusion: In this study, an optimized protocol for production and purification of mouse plac1 was reported and it was shown that insertion of immunostimulatory peptides in gene construct could efficiently enhance humoral immune responses against mouse plac1, which could potentially augment cellular immune responses against plac1 leading to more effective anti-cancer responses. 61 69 Shaghayegh Rahdan Department of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical Sciences Iran Mahboobeh Nazari Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Sorour Shojaeian Department of Biochemistry, School of Medical Sciences, Alborz University of Medical Sciences Department of Biochemistry, School of Medical Sciences, Alborz University of Medical Sciences Iran Fazel Shokri Department of Immunology, School of Public Health, Tehran University of Medical SciencesMonoclonal Antibody Research Center, Avicenna Research Institute, ACECR Department of Immunology, School of Public Health, Tehran University of Medical Sciences IranIran Mohammad Mehdi Amiri Department of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical Sciences Iran Amin Ramezani Institute for Cancer Research, Shiraz University of Medical Sciences Institute for Cancer Research, Shiraz University of Medical Sciences Iran Amir-Hassan Zarnani Seyed Alireza Razavi Department of Immunology, School of Public Health, Tehran University of Medical Sciences Department of Immunology, School of Public Health, Tehran University of Medical Sciences Iran ExpressionMouse plac1OptimizationPurificationRecombinant proteins 50490.pdf 1. Mahmoudian J, Ghods R, Nazari M, Jeddi-Tehrani M, Ghahremani MH, Ghaffari-Tabrizi-Wizsy N, et al. PLAC1: biology and potential application in cancer immunotherapy. Cancer Immunol Immunother 2019;68(7):1039-58.##2. Fant M, Farina A, Nagaraja R, Schlessinger D. PLAC1 (Placenta-specific 1): a novel, X-linked gene with roles in reproductive and cancer biology. Prenat Diagn 2010;30(6):497-502.##3. Koslowski M, Sahin U, Mitnacht-Kraus R, Seitz G, Huber C, Tureci O. A placenta-specific gene ectopically activated in many human cancers is essentially involved in malignant cell processes. Cancer Res 2007;67(19):9528-34.##4. Devor EJ, Leslie KK. The oncoplacental gene placenta-specific protein 1 is highly expressed in endometrial tumors and cell lines. Obstet Gynecol Int 2013;2013:807849.##5. Yang L, Zha TQ, He X, Chen L, Zhu Q, Wu WB, et al. Placenta-specific protein 1 promotes cell proliferation and invasion in non-small cell lung cancer. Oncol Rep 2018;39(1):53-60.##6. Wu Y, Lin X, Di X, Chen Y, Zhao H, Wang X. Oncogenic function of Plac1 on the proliferation and metastasis in hepatocellular carcinoma cells. Oncol Rep 2017;37(1):465-73.##7. Liu FF, Dong XY, Pang XW, Xing Q, Wang HC, Zhang HG, et al. The specific immune response to tumor antigen CP1 and its correlation with improved survival in colon cancer patients. Gastroenterology 2008;134(4):998-1006.##8. Liu F, Shen D, Kang X, Zhang C, Song Q. New tumour antigen PLAC1/CP1, a potentially useful prognostic marker and immunotherapy target for gastric adenocarcinoma. J Clin Pathol 2015;68(11):913-6.##9. Ghods R, Ghahremani MH, Madjd Z, Asgari M, Abolhasani M, Tavasoli S, et al. High placenta-specific 1/low prostate-specific antigen expression pattern in high-grade prostate adenocarcinoma. Cancer Immunol Immunother. 2014;63(12):1319-27.##10. Nejadmoghaddam MR, Zarnani AH, Ghahremanzadeh R, Ghods R, Mahmoudian J, Yousefi M, et al. Placenta-specific1 (PLAC1) is a potential target for antibody-drug conjugate-based prostate cancer immunotherapy. Sci Rep 2017;7(1):13373.##11. Mahmoudi AR, Ghods R, Rakhshan A, Madjd Z, Bolouri MR, Mahmoudian J, et al. Discovery of a potential biomarker for immunotherapy of melanoma: PLAC1 as an emerging target. Immunopharmacol Immunotoxicol 2020;42(6):604-13.##12. Nazari M, Zarnani AH, Ghods R, Emamzadeh R, Najafzadeh S, Minai-Tehrani A, et al. Optimized protocol for soluble prokaryotic expression, purification and structural analysis of human placenta specific-1(PLAC1). Protein Expr Purif 2017;133:139-51.##13. Alexander J, Sidney J, Southwood S, Ruppert J, Oseroff C, Maewal A, et al. Development of high potency universal DR-restricted helper epitopes by modification of high affinity DR-blocking peptides. Immunity 1994;1(9):751-61.##14. Percival-Alwyn JL, England E, Kemp B, Rapley L, Davis NH, McCarthy GR, et al. Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags". MAbs 2015;7(1):129-37.##15. Liao CW, Chen CA, Lee CN, Su YN, Chang MC, Syu MH, et al. Fusion protein vaccine by domains of bacterial exotoxin linked with a tumor antigen generates potent immunologic responses and antitumor effects. Cancer Res 2005;65(19):9089-98.##16. Mahmoudian J, Ghods R, Nazari M, Jeddi-Tehrani M, Ghahremani MH, Ostad SN, et al. Expression profiling of plac1 in murine cancer cell lines. Exp Oncol 2019;41(1):7-13.##17. Tao H, Liu W, Simmons BN, Harris HK, Cox TC, Massiah MA. Purifying natively folded proteins from inclusion bodies using sarkosyl, Triton X-100, and CHAPS. Biotechniques 2010;48(1):61-4.##18. Silva WA, Jr., Gnjatic S, Ritter E, Chua R, Cohen T, Hsu M, et al. PLAC1, a trophoblast-specific cell surface protein, is expressed in a range of human tumors and elicits spontaneous antibody responses. Cancer Immun 2007;7:18.##19. Murphy K, Weaver C. Janeway's Immunobiology. 19th ed. Garland Science. 2016. 924 p.##20. Rice J, Ottensmeier CH, Stevenson FK. DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer 2008;8(2):108-20.##21. Nielsen FS, Sauer J, Backlund J, Voldborg B, Gregorius K, Mouritsen S, et al. Insertion of foreign T cell epitopes in human tumor necrosis factor alpha with minimal effect on protein structure and biological activity. J Biol Chem 2004;279(32):33593-600.##22. Nezafat N, Sadraeian M, Rahbar MR, Khoshnoud MJ, Mohkam M, Gholami A, et al. Production of a novel multi-epitope peptide vaccine for cancer immunotherapy in TC-1 tumor-bearing mice. Biologicals 2015;43(1):11-7.##

Modern Paradigm Towards Potential Target Identification for Antiviral (SARS-nCoV-2) and Anticancer Lipopeptides: A Pharmacophore-Based Approach 2 2 Background: Lipopeptides are potential microbial metabolites that are abandoned with broad spectrum biopharmaceutical properties ranging from antimicrobial, antiviral and anticancer, etc. Clinical studies are not much explored beyond the experimental methods to understand drug mechanisms on target proteins at the molecular level for large molecules. Due to the less available studies on potential target proteins of lipopeptide based drugs, their potential inhibitory role for more obvious treatment on disease have not been explored in the direction of lead optimization. However, Computational approaches need to be utilized to explore drug discovery aspects on lipopeptide based drugs, which are time saving and cost-effective techniques. Methods: Here a ligand-based drug discovery approach is coupled with reverse pharmacophore-mapping for the prediction of potential targets for antiviral (SARS-nCoV-2) and anticancer lipopeptides. Web-based servers PharmMapper and SwissTargetPrediction are used for the identification of target proteins for lipopeptides surfactin and iturin produced by Bacillus subtilis. Results: The studies have given the insight to treat the diseases with next-generation large molecule therapeutics. Results also indicate the affinity for Angiotensin-Converting Enzymes (ACE) and proteases as the potential viral targets for these categories of peptide therapeutics. A target protein for the Human Papilloma Virus (HPV) has also been mapped. Conclusion: The work will further help in exploring computer-aided drug designing of novel compounds with greater efficiency where the structure of the target proteins and lead compounds are known. 70 78 Manisha Yadav Department of Biotechnology, National Institute of Technology Raipur Department of Biotechnology, National Institute of Technology Raipur India J. Satya Eswari Antiviral AgentsBacillus subtillisDrug discoveryLigandsLipopeptidesPeptide hydrolases 50492.pdf 1. Bruno A, Costantino G, Sartori L, Radi M. The in silico drug discovery toolbox: applications in lead discovery and optimization. Curr Med Chem 2019;26(21):3838-73.##2. Sauna ZE, Lagassé HAD, Alexaki A, Simhadri VL, Katagiri NH, Jankowski W, et al. Recent advances in (therapeutic protein) drug development. F1000Res 2017;6:113.##3. Neu HC. The crisis in antibiotic resistance. Science 1992;257(5073):1064-73.##4. Meena KR, Kanwar SS. Lipopeptides as the Antifungal and antibacterial agents: applications in food safety and therapeutics. Biomed Res Int 2015;2015:473050.##5. Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol 2019;10:302.##6. Cochrane SA, Vederas JC. Lipopeptides from Bacillus and Paenibacillus spp.: A gold mine of antibiotic candidates. Med Res Rev 2016;36(1):4-31.##7. Jujjavarapu SE, Dhagat S, Yadav M. Computer-Aided Design of Antimicrobial Lipopeptides As Prospective Drug Candidates. CRC Press LLC; 2019. 146 p.##8. Pham JV, Yilma MA, Feliz A, Majid MT, Maffetone N, Walker JR, et al. A review of the microbial production of bioactive natural products and biologics. Front Microbiol 2019;10:1404.##9. Xia S, Liu M, Wang C, Xu W, Lan Q, Feng S, et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res 2020;30(4):343-55.##10. Lau JL, Dunn MK. Therapeutic peptides: Historical perspectives, current development trends, and future directions. Bioorganic Med Chem 2018;26(10):2700-7.##11. Craik DJ, Fairlie DP, Liras S, Price D. The future of peptide-based drugs. Chem Biol Drug Des 2013;81:136-47.##12. Yadav M, Dhagat S, Eswari JS. Emerging strategies on in silico drug development against COVID-19: challenges and opportunities. Eur J Pharm Sci 2020;155:105522.##13. Chowdhury T, Baindara P, Mandal SM. LPD-12: a promising lipopeptide to control COVID-19. Int J Antimicrob Agents 2021;57(1):106218.##14. de Vries RD, Schmitz KS, Bovier FT, Predella C, Khao J, Noack D, et al. Intranasal fusion inhibitory lipopeptide prevents direct-contact SARS-CoV-2 transmission in ferrets. Science 2021;371(6536):1379-82.##15. Chartier M, Najmanovich R. Detection of binding site molecular interaction field similarities. J Chem Inf Model 2015;55(8):1600-15.##16. Ciemny M, Kurcinski M, Kamel K, Kolinski A, Alam N, Schueler-Furman O, et al. Protein–peptide docking: opportunities and challenges. Drug Discov Today 2018;23(8):1530-7.##17. Liu X, Ouyang S, Yu B, Liu Y, Huang K, Gong J, et al. PharmMapper server: a web server for potential drug target identification using pharmacophore mapping approach. Nucleic Acids Res 2010;38(Web Server issue):W609-14.##18. Meshram RJ, Baladhye VB, Gacche RN, Karale BK, Gaikar RB. Pharmacophore mapping approach for drug target identification: a chemical synthesis and in silico study on novel thiadiazole compounds. J Clin Diagn Res 2017;11(5):KF01-KF08.##19. Mojsoska B, Jenssen H. Peptides and peptidomimetics for antimicrobial drug design. Pharmaceuticals. Pharmaceuticals (Basel) 2015;8(3):366-415.##20. Sable R, Parajuli P, Jois S. Peptides, peptidomimetics, and polypeptides from marine sources: A wealth of natural sources for pharmaceutical applications. Mar Drugs 2017;15(4):124.##21. Kumar S, Singh J, Narasimhan B, Shah SAA, Lim SM, Ramasamy K, et al. Reverse pharmacophore mapping and molecular docking studies for discovery of GTPase HRas as promising drug target for bis-pyrimidine derivatives. Chem Cent J 2018;12(1):106.##22. Bhattacharjee B, Chatterjee J. Identification of proapoptopic, anti-inflammatory, anti- proliferative, anti-invasive and anti-angiogenic targets of essential oils in cardamom by dual reverse virtual screening and binding pose analysis. Asian Pac J Cancer Prev 2013;14(6):3735-42.##23. Meshram RJ, Baladhye VB, Gacche RN, Karale BK, Gaikar RB. Pharmacophore mapping approach for drug target identification: A chemical synthesis and in silico study on novel thiadiazole compounds. J Clin Diagn Res 2017;11(5):KF01-KF08.##24. Seydlová G, Svobodová J. Review of surfactin chemical properties and the potential biomedical applications. Cent Eur J Med 2008;123-33.##25. SwissTargetPrediction: A Web Server for Target Prediction of Bioactive Small Molecules - PubMed [Internet]. [cited 2020 Jul 10]. Available from: https://pubmed.ncbi.nlm.nih.gov/24792161/##26. Gfeller D, Grosdidier A, Wirth M, Daina A, Michielin O, Zoete V. SwissTargetPrediction: A web server for target prediction of bioactive small molecules. Nucleic Acids Res 2014;42(Web Server issue):W32-8.##27. Daina A, Michielin O, Zoete V. SwissTargetPrediction: updated data and new features for efficient prediction of protein targets of small molecules. Nucleic Acids Res 2019;47(W1):W357-364.##28. Inès M, Dhouha G. Lipopeptide surfactants: Production, recovery and pore forming capacity. Peptides 2015 Sep;71:100-12.##29. Kracht M, Rokos H, Özel M, Kowall M, Pauli G, Vater J. Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot (Tokyo) 1999;52(7):613-9.##30. Kopp F, Marahiel MA. Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Nat Prod Rep 2007;24(4):735-49.##31. Lavecchia A, Giovanni C. Virtual screening strategies in drug discovery: A critical review. Curr Med Chem 2013;20(23):2839-60.##32. Rishton GM. Nonleadlikeness and leadlikeness in biochemical screening. Drug Discov Today 2003;8(2):86-96.##33. Wu YS, Ngai SC, Goh BH, Chan KG, Lee LH, Chuah LH. Anticancer activities of surfactin potential application of nanotechnology assisted surfactin delivery. Front Pharmacol 2017;8:761.##34. Katsila T, Spyroulias GA, Patrinos GP, Matsoukas MT. Computational approaches in target identification and drug discovery. Comput Struct Biotechnol J 2016;14:177-84.##35. Maget-Dana R, Thimon L, Peypoux F, Ptak M. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie 1992;74(12):1047-51.##36. Maget-Dana R, Peypoux F. Iturins, a special class of pore-forming lipopeptides: biological and physicochemical properties. Toxicology 1994;87(1-3):151-74.##37. Clark A, MacKenzie S. Targeting cell death in tumors by activating caspases. Curr Cancer Drug Targets 2008;8(2):98-109.##38. Yu S, Sun L, Jiao Y, Lee LTO. The role of G protein-coupled receptor kinases in cancer. Int J Biol Sci 2018;14(2):189-203.##39. Demirbaş A, Eker H, Elmas ÖF, Ulutaş Demirbaş G, Atasoy M, Türsen Ü, et al. COVID-19 and human papillomavirus: Paradoxical immunity. J Cosmet Dermatol 2021;20(7):2001-3.##40. Wang J. New strategy for COVID-19 vaccination: targeting the receptor-binding domain of the SARS-CoV-2 spike protein Cell Mol Immunol 2021 Feb;18(2):243-44.##

Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum Dots 2 2 Background: Methicillin Resistance Staphylococcus aureus (MRSA) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices. Methods: Using molecular diagnostic methods are important for identification of MRSA from the Methicillin Sensitive Staphylococcus aureus (MSSA). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of MRSA. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of MRSA. The mecA gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with MRSA positive results were tested with QD-LAMP, the conventional LAMP, GV-LAMP, and Polymerase Chain Reaction (PCR). Results: Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of mecA positive MRSA samples; however, the Limit of Detection (LoD) of QD-LAMP was 1.5×104 Colony Forming Unit (CFU). Conclusion: The results suggested that the QD-LAMP assay was easy to operate and could be used for detection of MRSA in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of MRSA. 79 88 Aily Aliasgharian Research Center for Thalassemia, Mazandaran University of Medical SciencesDepartment of Medical Microbiology, Faculty of Medicine, Mazandaran University of Medical Sciences Research Center for Thalassemia, Mazandaran University of Medical SciencesDepartment of Medical Microbiology, Faculty of Medicine, Mazandaran University of Medical Sciences IranIran Pooria Gill Mohammad Ahanjan Department of Medical Microbiology, Faculty of Medicine, Mazandaran University of Medical SciencesResearch Center for Pediatric Infectious Diseases, Mazandaran University of Medical Sciences Department of Medical Microbiology, Faculty of Medicine, Mazandaran University of Medical SciencesResearch Center for Pediatric Infectious Diseases, Mazandaran University of Medical Sciences IranIran Adele Rafati Immunogenetics Research Center, Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences Immunogenetics Research Center, Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences Iran LAMPMRSAMSSAQuantum dotsmecA gene 60491.pdf 1.Wang XR, Wu LF,Wang Y, Ma YY, Chen FH, Ou HL. Rapid detection of Staphylococcus aureus by loop-mediated isothermal amplification. Appl Biochem Biotechnol 2015;175(2):882-91.##2. Sudhaharan S, Vanjari L, Mamidi N, Ede N, Vemu L. Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of nuc and mecA genes among clinical isolates of Staphylococcus spp. J Clin Diag Res 2015;9(8):DC06- 9.##3. Rahimzadeh G, Gill P, Rezai MS. Characterization of methicillin-resistant Staphylococcus aureus (MRSA) phages from sewage at a tertiary pediatric hospital. Arch Pediatr Infect Dis 2015;5: e39615.##4. Su J, Liu X, Cui H, Li Y, Chen D, Li Y, et al. Rapid and simple detection of methicillin-resistance Staphylococcus aureus by orfX loop-mediated isothermal amplification assay. BMC Biotechnol 2014;14:8.##5. You R, Gui Z, Xu Z, Shirtliff ME, Yu G, Zhao X, et al. Methicillin-resistance Staphylococcus aureus detection by an improved rapid polymerase chain reaction (PCR) assay. Afr J Microbiol 2012;Res 6:7131-3.##6. Ghindilis AL, Smith MW, Simon HM, Seoudi IA, Yazvenko NS, Murray IA, et al. Restriction cascade exponential amplification (RCEA) assay with an attomolar detection limit: a novel, highly specific, isothermal alternative to qPCR. Sci Rep 2015;5:7737.##7. Metwally L, Gomaa N, Hassan R. Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests. East Mediterr Health J 2014;20(4):273-9.##8. Lim KT, Teh CSJ, Thong KL. Loop-mediated isothermal amplification assay for the rapid detection of Staphylococcus aureus. Biomed Res Int 2013;2013:895816.##9. Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother 2007;13(3):134-40.##10. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 2000;28(12):E63.##11. Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Cli. Chem 2001;47(9):1742-3.##12. Notomi T. [Loop-mediated isothermal amplification]. Nihon Rinsho 2007;65(5):957-61. Japanese.##13. Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S, Kumar A, et al. Loop-mediated isothermal amplification of DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak J Biol Sci 2014;17(2):151-66.##14. Gill P, Ghaemi A. Nucleic acid isothermal amplification technologies - a review. Nucleosides Nucleotides Nucleic Acids 2008;27(3):224-43.##15. Song T, Toma C, Nakasone N, Iwanaga M. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop‐mediated isothermal amplification method. FEMS Microbiol Lett 2005;243(1):259-63.##16. Horisaka T, Fujita K, Iwata T, Nakadai A, Okatani A.T, Horikita T, et al. Sensitive and specific detection of Yersinia pseudotuberculosis by loop-mediated isothermal amplification. J Clin Microbiol 2004;42(11):5349-52.##17. Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett 2005;253(1):155-61.##18. Yamazaki W, Ishibashi M, Kawahara R, Inoue K. Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus. BMC Microbiol 2008;8:163.##19. Chen S, Ge B. Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus. BMC Microbiol 2010;10:41.##20. EI-Jakee J, Marouf S, Ata NS, Abdel-Rahman EH, El-Moez SIA, Samy AA, et al. Rapid method for detection of Staphylococcus aureus enterotoxins in food. Glob Vet 2013;11:335-41.##21. Xia Y, Guo XG, Zhou S. Rapid detection of Streptococcus pneumoniae by real-time fluorescence loop-mediated isothermal amplification. J Thorac Dis 2014;6:1193-9.##22. Goto M, Honda E, Ogura A, Nomoto A, Hanaki KI. Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques 2009;46(2009):167-72.##23. Nikbakht H, Gill P, Tabarraei A, Niazi A. Nanomolecular detection of human influenza virus type A using reverse transcription loop-mediated isothermal amplification assisted with rod-shaped gold nanoparticles. RSC Adv 2014;4:13575-80.##24. Zanoli LM, Spoto G. Isothermal amplification methods for the detection of nucleic acids in microfluidic devices. Biosensors (Basel) 2012;(1)3:18-43.##25. Sharafdarkolaei SH, Motovali-Bashi M, Gill P. Fluorescent detection of point mutation via ligase reaction assisted by quantum dots and magnetic nanoparticle-based probes. RSC Adv 2017;7:25665-72.##26. Yeh HC, Ho YP, Shih IM, Wang TH. Homogeneous point mutation detection by quantum dot-mediated two-color fluorescence coincidence analysis. Nucleic Acids Res 2006;34(5):e35.##27. Weinstein MP. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. 11th ed. PA, USA: Clinical and Laboratory Standards Institute; 2018. p.112.##28. Bauer A, Kirby W, Sherris J.C, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45(4):493-6.##29. Brown DF, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, et al. Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother 2005;56(6):1000-18.##30. QIAamp DNA Mini Blood Mini Handbook. Qiagen. 2012.##31. Goldmeyer J, Li H, McCormac M, Cook S, Stratton C, Lemieux B, et al. Identification of Staphylococcus aureus and determination of methicillin resistance directly from positive blood cultures by isothermal amplification and a disposable detection device. J Clin Microbiol 2008;46(4):1534-6.##32. Lucchi NW, Demas A, Narayanan J, Sumari D, Kabanywanyi A, Kachur SP, et al. Real-time fluorescence loop mediated isothermal amplification for the diagnosis of malaria. PLoS One 2010;5(10):e13733.##33. Mori Y, Notomi T. Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother 2009;15(2):62-9.##34. Zhao XH, Li Y, Park MS, Wang J, Zhang Y, He X, et al. Loop-mediated isothermal amplification assay targeting the femA gene for rapid detection of Staphylococcus aureus from clinical and food samples. J Microbiol Biotechnol 2013;23(2):246-50.##35. Nie K, Zhao X, Ding X, Li X, Zou S, Guo J, et al. Visual detection of human infection with influenza A (H7N9) virus by subtype‐specific reverse transcription loop‐mediated isothermal amplification with hydroxynaphthol blue dye. Clin Microbiol Infect 2013;19(8):E372-5.##36. Sowmya N, Thakur M, Manonmani H. Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods. J Appl Microbiol 2012;113(1):106-13.##37. Xu H, Zhang L, Shen G, Feng C, Wang X, Yan J, et al. Establishment of a novel one-step reverse transcription loop-mediated isothermal amplification assay for rapid identification of RNA from the severe fever with thrombocytopenia syndrome virus. J Virol Methods 2013;194(1-2):21-5.##38. Soleimani M, Shams S, Majidzadeh-A K. Developing a real‐time quantitative loop‐mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis. J Appl Microbiol 2013;115(3):828-34.##

Antitumor Activities of Green Tea by Up-regulation of miR -181a Expression in LNCaP Cells Using 3D Cell Culture Model 2 2 Background: Prostate Cancer (PCa) is the major reason for the high mortality rates among men worldwide. In fact, current therapeutic approaches are not successful. It appears that discovering more effective methods considering several parameters such as availability, low cost, and no toxicity to normal cells is one of the biggest challenges for interested researchers. Green tea (extracted from the plant Camellia sinensis) with high level of polyphenolic compounds and as the most globally consumed beverage has attracted considerable interest. MicroRNAs (or miRNAs) were considered as novel tools in cancer therapy which modulate various biological events in cell by regulation of gene expression. The aim of the current study was to evaluate the antitumor activity of green tea in LNCaP cells through up-regulation of miR-181a expression. Methods: First, LNCaP cells were cultured and by using quantitative real time PCR (qRT-PCR) and western blot methods, the expression levels of Bax and BCL2 were analyzed. Next, a 3D cell culture model was applied to evaluate the expression of miRNA-181a in LNCaP cells. Results: It was shown that green tea induced cellular apoptosis. The high number of apoptotic nuclei was also shown by using DAPI staining. The inhibition of tumor growth was revealed by analyzing the size and number of spheroids. Also, up-regulation of miR-181a expression in LNCaP cells was revealed after treatment with green tea. Conclusion: Our results are helpful to design antitumor regimens based on consumption of green tea through up-regulation of miRNA-181a expression and induction of apoptosis. 89 94 Fatemeh Safari Narjes Rayat Azad Department of Biology, Faculty of Science, University of Guilan Department of Biology, Faculty of Science, University of Guilan Iran Ali Alizadeh Ezdiny Department of Biology, Faculty of Basic Sciences, Rasht Branch, Islamic Azad University Department of Biology, Faculty of Basic Sciences, Rasht Branch, Islamic Azad University Iran Safoora Pakizehkar Cellular and Molecular Endocrine Research Center (CMERC), Research Institute for Endocrine Science, Shahid Beheshti University of Medical Sciences Cellular and Molecular Endocrine Research Center (CMERC), Research Institute for Endocrine Science, Shahid Beheshti University of Medical Sciences Iran Zeinab Khazaei Koohpar Department of Cell and Molecular Biology, Faculty of Biological Sciences, Tonekabon Branch, Islamic Azad University Department of Cell and Molecular Biology, Faculty of Biological Sciences, Tonekabon Branch, Islamic Azad University Iran Najmeh Ranji Department of Biology, Faculty of Basic Sciences, Rasht Branch, Islamic Azad University Department of Biology, Faculty of Basic Sciences, Rasht Branch, Islamic Azad University Iran ApoptosisCell culture techniquesGreen teaProto-oncogene proteins c-bcl-2Up-regulation 50494.pdf 1. 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Pulse Voltage Electrical Stimulation for Bacterial Inactivation and Wound Healing in Mice with Diabetes 2 2 Background: Treatment of wounds in diabetes often gets less than perfect healing. One of the reasons for the difficulty in treating wounds in diabetes is the growth of aerobic and anaerobic bacteria. This study aims to determine the pulse voltage and treatment time that can optimally inactivate bacteria, and their effect on wound healing in mice suffering from diabetes. Methods: The study used electrical stimulation with a direct voltage of 10 volts given a pulse voltage of 50-80 volts, a width of 50 µs, and the number of pulses of 65 per second. The research samples were Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) bacteria that grew on beef and mice (Mus musculus) with diabetes. The treatment for S. aureus and P. aeruginosa bacteria was carried out using a pulse voltage of 50-80 volts for 5-15 min/day and repeated for 3 days. Meanwhile, treatment of mice wounds was carried out with a pulse voltage of 80 volts for 15 min/day and repeated for 7 days. Results: The results showed that treatment with a pulse voltage of 50-80 volts and a treatment time of 5-15 min significantly reduced the number of S. aureus and P. aeruginosa bacteria in beef (p£0.05). Treatment with a pulse voltage of 80 volts for 15 min made beef free from bacteria. Meanwhile, treatment with a pulse voltage of 80 volts for 15 min per day for seven days resulted in the wound state of three mice in the maturation phase and two mice in the proliferation phase on day 8 with an average wound area of 0.108 cm 2. Conclusion: The treatment with a pulse voltage of 80 volts for 15 min made the beef sterile, the mice wounds healed quickly, and the mice not stressed. 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