The Need to Continue the Education of Medical Doctors in Medical Basic Science: Existing Obstacles 2 2 In recent years, many prominent professors of basic sciences in Iranian medical schools had entered basic sciences from the field of medicine. This would facilitate the connection between basic and clinical sciences, providing a more attractive educational field for medical students in basic sciences 1,2. Of course, this does not mean that all professors of basic sciences must have a medical background. Maybe during my studies in the pharmacy field of Tehran University, Dr. Adibfar, Dr. Maleknia, Dr. Jahanghiri, Dr. Abdulwahabi and Dr. Shadan were all examples of that period. Also, many professors of basic sciences came from pharmacy, the best examples of which were Dr. Zarrindast and Dr. Dehpour. In those years, there was no empty place for Dr. Dehapour’ classes even to sit on the corridors of the salon. In the past two decades, when the salary of an assistant professor with a medicine or pharmacy background became half the average salary of a general practitioner or pharmacist, only the love of the principle of science was no longer enough to attract doctors and pharmacists in basic sciences. Maybe if a student of medicine saw Dr. Farrokh Shadan's old age and retirement period and never be interested in continuing his/her education in basic sciences. I hope that the new Minister of Health and Medical Education, Dr. Zafarqandi, who is a graduate of Tehran Medical School and has seen all these professors, and most importantly, was a biology teacher in high school, will urgently think about reviving basic medical sciences. In the scientific and career history of Dr. Zafarghandi, especially in 8 year-imposed war, the term of triage is very repeated. Mr. Minister, based on the triage, one of the first problems that your Excellency and the vice minister of education should urgently address, are the revival of basic medical science, the maintenance of the living conditions of basic science professors, and the renovation of teaching and research laboratories of basic science departments.   136 136 Shahin Akhondzadeh Editorial 60587.pdf Akhondzadeh S. The Need for Serious Support for Basic Medical Science in Iran. Avicenna J Med Biotechnol 2024;16(3):136. ##Sanjari Moghaddan H, Akhondzadeh S. The Clinician Scientist Training Program in Iran: Catalyzing Clinical Science Advancements. Avicenna J Med Biotechnol 2023;15(3):128. ##

Overview on Immunopathology of Chronic Lymphocytic Leukemia and Tumor-Associated Antigens with Therapeutic Applications 2 2 Chronic Lymphocytic Leukemia (CLL) is a clinically and biologically heterogeneous disease with a variable clinical course. The induction of a generalized state of immunosuppression, leading to susceptibility to infections and the failure of anti-tumor immune responses, is a key feature of the clinical course of CLL. In addition to B-cell receptor (BCR) signaling in CLL, several receptor tyrosine kinases (RTKs) have been reported to be constitutively active in leukemic B cells, resulting in promoted survival and resistance to apoptosis induced by chemotherapy. Several treatment options are available for CLL, including a watch-and-wait strategy, chemotherapy, targeted therapies, immunotherapies such as adoptive cellular therapy (CAR T-Cell Therapy), stem cell transplantation (allogeneic transplantation), radiation therapy and surgery. The identification of Tumor-Associated Antigens (TAAs) is the bottleneck of tumor immunology and immunotherapy, serving as promising targets for precise diagnosis, monitoring, or therapeutic approaches. Numerous TAAs have been identified, and their application in immunotherapy holds promise for the treatment of CLL. Furthermore, extensive ongoing research aims to identify new cancer TAAs. In this review, our objective is to provide a comprehensive overview of CLL immunology and recent findings regarding advances in TAAs with therapeutic applications in CLL. 201 222 Mahdi Shabani Davoud Rostamzadeh Medicinal Plants Research Center, Yasuj University of Medical Sciences Medicinal Plants Research Center, Yasuj University of Medical Sciences Iran Mansoure Mansouri Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR) Iran Cell therapyChronic lymphocytic leukemiaHematologic malignanciesImmunotherapyTumor antigens 60588.pdf Hus I, Rolinski J. Current concepts in diagnosis and treatment of chronic lymphocytic leukemia. Contemp Oncol (Pozn) 2015;19(5):361-7.##Hallek M. Chronic lymphocytic leukemia: 2017 update on diagnosis, risk stratification, and treatment. Am J Hematol 2017;92(9):946-65.##Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med 1995;333(16):1052-7.##Jovanovic D, Djurdjevic P, Andjelkovic N, Zivic L. Possible role of CD22, CD79b and CD20 expression in distinguishing small lymphocytic lymphoma from chronic lymphocytic leukemia. Contemp Oncol (Pozn) 2014;18(1):29-33.##Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. Guidelines for the diagnosis and treatment of chronic lymphocytic leukemia: a report from the International Workshop on Chronic Lymphocytic Leukemia updating the National Cancer Institute-Working Group 1996 guidelines. Blood 2008;111(12):5446-56.##Hallek M, Cheson BD, Catovsky D, Caligaris-Cappio F, Dighiero G, Dohner H, et al. iwCLL guidelines for diagnosis, indications for treatment, response assessment, and supportive management of CLL. Blood 2018;131(25):2745-60.##Chemotherapeutic options in chronic lymphocytic leukemia: a meta-analysis of the randomized trials. CLL Trialists' Collaborative Group. J Natl Cancer Inst 1999;91(10):861-8.##Brown J, Hillmen P, O’Brien S, Barrientos J, Reddy N, Coutre S, et al. Extended follow-up and impact of high-risk prognostic factors from the phase 3 RESONATE study in patients with previously treated CLL/SLL. Leukemia 2018;32(1):83-91.##Molica S, Tam C, Allsup D, Polliack A. Advancements in the treatment of CLL: The rise of zanubrutinib as a preferred therapeutic option. Cancers 2023;15(14):3737.##Andreescu M, Andreescu B. Immune Evasion Through Human Leukocyte Antigen Implications and Its Impact on Targeted Therapy. Cureus 2024;16(1):e52737.##Ghosh AK, Kay NE. Critical signal transduction pathways in CLL. Adv Exp Med Biol 2013;792:215-39.##Mouawad N, Ruggeri E, Capasso G, Martinello L, Visentin A, Frezzato F, et al. How receptor tyrosine kinase‐like orphan receptor 1 meets its partners in chronic lymphocytic leukemia. Hematol Oncol 2024;42(2):e3250.##Riches JC, Ramsay AG, Gribben JG. Immune dysfunction in chronic lymphocytic leukemia: the role for immunotherapy. Curr Pharm Des 2012;18(23):3389-98. ##Platsoucas CD, Galinski M, Kempin S, Reich L, Clarkson B, Good RA. Abnormal T lymphocyte subpopulations in patients with B cell chronic lymphocytic leukemia: an analysis by monoclonal antibodies. J Immunol 1982;129(5):2305-12.##Mu X, Kay NE, Gosland MP, Jennings CD. Analysis of blood T-cell cytokine expression in B-chronic lymphocytic leukaemia: evidence for increased levels of cytoplasmic IL-4 in resting and activated CD8 T cells. Br J Haematol 1997;96(4):733-5.##de Totero D, Reato G, Mauro F, Cignetti A, Ferrini S, Guarini A, et al. IL4 production and increased CD30 expression by a unique CD8+ T-cell subset in B-cell chronic lymphocytic leukaemia. Br J Haematol 1999;104(3):589-99.##Dancescu M, Rubio-Trujillo M, Biron G, Bron D, Delespesse G, Sarfati M. Interleukin 4 protects chronic lymphocytic leukemic B cells from death by apoptosis and upregulates Bcl-2 expression. J Exp Med 1992;176(5):1319-26.##Cerutti A, Kim EC, Shah S, Schattner EJ, Zan H, Schaffer A, et al. Dysregulation of CD30+ T cells by leukemia impairs isotype switching in normal B cells. Nat Immunol 2001;2(2):150-6.##Boncompagni G, Tatangelo V, Lopresti L, Ulivieri C, Capitani N, Tangredi C, et al. Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia. Cell Death Dis 2024;15(2):144.##Buggins AG, Patten PE, Richards J, Thomas NS, Mufti GJ, Devereux S. Tumor-derived IL-6 may contribute to the immunological defect in CLL. Leukemia 2008;22(5):1084-7.##Fayad L, Keating MJ, Reuben JM, O'Brien S, Lee BN, Lerner S, et al. Interleukin-6 and interleukin-10 levels in chronic lymphocytic leukemia: correlation with phenotypic characteristics and outcome. Blood 2001;97(1):256-63.##Gadi D, Griffith A, Tyekucheva S, Wang Z, Rai V, Vartanov A, et al. AT cell inflammatory phenotype is associated with autoimmune toxicity of the PI3K inhibitor duvelisib in chronic lymphocytic leukemia. Leukemia 2022;36(3):723-32.##Gorgun G, Holderried TA, Zahrieh D, Neuberg D, Gribben JG. Chronic lymphocytic leukemia cells induce changes in gene expression of CD4 and CD8 T cells. J Clin Invest 2005;115(7):1797-805.##D'Arena G, Laurenti L, Minervini MM, Deaglio S, Bonello L, De Martino L, et al. Regulatory T-cell number is increased in chronic lymphocytic leukemia patients and correlates with progressive disease. Leuk Res. 2011;35(3):363-8.##Giannopoulos K, Schmitt M, Kowal M, Wlasiuk P, Bojarska-Junak A, Chen J, et al. Characterization of regulatory T cells in patients with B-cell chronic lymphocytic leukemia. Oncol Rep 2008;20(3):677-82.##Lindqvist CA, Christiansson LH, Simonsson B, Enblad G, Olsson-Stromberg U, Loskog AS. T regulatory cells control T-cell proliferation partly by the release of soluble CD25 in patients with B-cell malignancies. Immunology 2010;131(3):371-6.##Lindqvist CA, Christiansson LH, Thorn I, Mangsbo S, Paul-Wetterberg G, Sundstrom C, et al. Both CD4+ FoxP3+ and CD4+ FoxP3- T cells from patients with B-cell malignancy express cytolytic markers and kill autologous leukaemic B cells in vitro. Immunology 2011;133(3):296-306.##Catakovic K, Gassner FJ, Ratswohl C, Zaborsky N, Rebhandl S, Schubert M, et al. TIGIT expressing CD4+ T cells represent a tumor-supportive T cell subset in chronic lymphocytic leukemia. Oncoimmunology 2018;7(1):e1371399.##Van den Hove LE, Vandenberghe P, Van Gool SW, Ceuppens JL, Demuynck H, Verhoef GE, et al. Peripheral blood lymphocyte subset shifts in patients with untreated hematological tumors: evidence for systemic activation of the T cell compartment. Leuk Res 1998;22(2):175-84.##Kay NE, Zarling JM. Impaired natural killer activity in patients with chronic lymphocytic leukemia is associated with a deficiency of azurophilic cytoplasmic granules in putative NK cells. Blood 1984;63(2):305-9.##Kay NE, Zarling J. Restoration of impaired natural killer cell activity of B-chronic lymphocytic leukemia patients by recombinant interleukin-2. Am J Hematol 1987;24(2):161-7.##Burton JD, Weitz CH, Kay NE. Malignant chronic lymphocytic leukemia B cells elaborate soluble factors that down-regulate T cell and NK function. Am J Hematol 1989;30(2):61-7.##Acebes-Huerta A, Huergo-Zapico L, Gonzalez-Rodriguez AP, Fernandez-Guizan A, Payer AR, Lopez-Soto A, et al. Lenalidomide induces immunomodulation in chronic lymphocytic leukemia and enhances antitumor immune responses mediated by NK and CD4 T cells. Biomed Res Int 2014;2014:265840.##Bojarska-Junak A, Hus I, Sieklucka M, Wasik-Szczepanek E, Mazurkiewicz T, Polak P, et al. Natural killer-like T CD3+/CD16+CD56+ cells in chronic lymphocytic leukemia: intracellular cytokine expression and relationship with clinical outcome. Oncol Rep 2010;24(3):803-10. ##Marin D, Li Y, Basar R, Rafei H, Daher M, Dou J, et al. Safety, efficacy and determinants of response of allogeneic CD19-specific CAR-NK cells in CD19+ B cell tumors: a phase 1/2 trial. Nat Med 2024:772-84.##Huang M, Liu Y, Yan Q, Peng M, Ge J, Mo Y, et al. NK cells as powerful therapeutic tool in cancer immunotherapy. Cellular Oncology. 2024:1-25.##Zeya HI, Keku E, Richards F, 2nd, Spurr CL. Monocyte and granulocyte defect in chronic lymphocytic leukemia. Am J Pathol 1979;95(1):43-54.##Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, Bahlo J, et al. Mutations driving CLL and their evolution in progression and relapse. Nature 2015;526(7574):525-30.##Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E, et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002;99(24):15524-9.##Dong JT, Boyd JC, Frierson HF, Jr. Loss of heterozygosity at 13q14 and 13q21 in high grade, high stage prostate cancer. Prostate 2001;49(3):166-71.##Klein U, Lia M, Crespo M, Siegel R, Shen Q, Mo T, et al. The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia. Cancer Cell 2010;17(1):28-40.##Austen B, Powell JE, Alvi A, Edwards I, Hooper L, Starczynski J, et al. Mutations in the ATM gene lead to impaired overall and treatment-free survival that is independent of IGVH mutation status in patients with B-CLL. Blood 2005;106(9):3175-82.##Zenz T, Mertens D, Kuppers R, Dohner H, Stilgenbauer S. From pathogenesis to treatment of chronic lymphocytic leukaemia. Nat Rev Cancer 2010;10(1):37-50.##Dohner H, Stilgenbauer S, James MR, Benner A, Weilguni T, Bentz M, et al. 11q deletions identify a new subset of B-cell chronic lymphocytic leukemia characterized by extensive nodal involvement and inferior prognosis. Blood 1997;89(7):2516-22.##Catovsky D, Richards S, Matutes E, Oscier D, Dyer M, Bezares RF, et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet 2007;370(9583):230-9.##Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med 2000;343(26):1910-6.##Dohner H, Fischer K, Bentz M, Hansen K, Benner A, Cabot G, et al. p53 gene deletion predicts for poor survival and non-response to therapy with purine analogs in chronic B-cell leukemias. Blood. 1995;85(6):1580-9.##Geisler CH, Philip P, Christensen BE, Hou-Jensen K, Pedersen NT, Jensen OM, et al. In B-cell chronic lymphocytic leukaemia chromosome 17 abnormalities and not trisomy 12 are the single most important cytogenetic abnormalities for the prognosis: a cytogenetic and immunophenotypic study of 480 unselected newly diagnosed patients. Leuk Res 1997;21(11-12):1011-23.##el Rouby S, Thomas A, Costin D, Rosenberg CR, Potmesil M, Silber R, et al. p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression. Blood 1993;82(11):3452-9.##Zenz T, Vollmer D, Trbusek M, Smardova J, Benner A, Soussi T, et al. TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations. Leukemia 2010;24(12):2072-9.##Trbusek M, Smardova J, Malcikova J, Sebejova L, Dobes P, Svitakova M, et al. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia. Journal of clinical oncology : official journal of the American Society of Clin Oncol 2011;29(19):2703-8.##Seiffert M, Dietrich S, Jethwa A, Glimm H, Lichter P, Zenz T. Exploiting biological diversity and genomic aberrations in chronic lymphocytic leukemia. Leuk Lymphoma 2012;53(6):1023-31.##Ljungstrom V, Cortese D, Young E, Pandzic T, Mansouri L, Plevova K, et al. Whole-exome sequencing in relapsing chronic lymphocytic leukemia: clinical impact of recurrent RPS15 mutations. Blood 2016;127(8):1007-16.##Puente XS, Pinyol M, Quesada V, Conde L, Ordonez GR, Villamor N, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature 2011;475(7354):101-5.##Puente XS, Bea S, Valdes-Mas R, Villamor N, Gutierrez-Abril J, Martin-Subero JI, et al. Non-coding recurrent mutations in chronic lymphocytic leukaemia. Nature 2015;526(7574):519-24.##LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood 2008;112(5):1570-80.##Burger JA, Chiorazzi N. B cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol 2013;34(12):592-601.##Herishanu Y, Perez-Galan P, Liu D, Biancotto A, Pittaluga S, Vire B, et al. The lymph node microenvironment promotes B-cell receptor signaling, NF-kappaB activation, and tumor proliferation in chronic lymphocytic leukemia. Blood 2011;117(2):563-74.##Chu CC, Catera R, Hatzi K, Yan XJ, Zhang L, Wang XB, et al. Chronic lymphocytic leukemia antibodies with a common stereotypic rearrangement recognize nonmuscle myosin heavy chain IIA. Blood 2008;112(13):5122-9.##Broker BM, Klajman A, Youinou P, Jouquan J, Worman CP, Murphy J, et al. Chronic lymphocytic leukemic (CLL) cells secrete multispecific autoantibodies. J Autoimmun 1988;1(5):469-81.##Damle RN, Ghiotto F, Valetto A, Albesiano E, Fais F, Yan XJ, et al. B-cell chronic lymphocytic leukemia cells express a surface membrane phenotype of activated, antigen-experienced B lymphocytes. Blood 2002;99(11):4087-93.##Lanemo Myhrinder A, Hellqvist E, Sidorova E, Soderberg A, Baxendale H, Dahle C, et al. A new perspective: molecular motifs on oxidized LDL, apoptotic cells, and bacteria are targets for chronic lymphocytic leukemia antibodies. Blood 2008;111(7):3838-48.##Hoogeboom R, van Kessel KP, Hochstenbach F, Wormhoudt TA, Reinten RJ, Wagner K, et al. A mutated B cell chronic lymphocytic leukemia subset that recognizes and responds to fungi. J Exp Med 2013;210(1):59-70.##Stadanlick JE, Kaileh M, Karnell FG, Scholz JL, Miller JP, Quinn WJ, 3rd, et al. Tonic B cell antigen receptor signals supply an NF-kappaB substrate for prosurvival BLyS signaling. Nat Immunol 2008;9(12):1379-87.##Scupoli MT, Pizzolo G. Signaling pathways activated by the B-cell receptor in chronic lymphocytic leukemia. Expert Rev Hematol 2012;5(3):341-8.##Healy JI, Goodnow CC. Positive versus negative signaling by lymphocyte antigen receptors. Annu Rev Immunol 1998;16:645-70.##Contri A, Brunati AM, Trentin L, Cabrelle A, Miorin M, Cesaro L, et al. Chronic lymphocytic leukemia B cells contain anomalous Lyn tyrosine kinase, a putative contribution to defective apoptosis. J Clin Invest 2005;115(2):369-78.##Saijo K, Schmedt C, Su IH, Karasuyama H, Lowell CA, Reth M, et al. Essential role of Src-family protein tyrosine kinases in NF-kappaB activation during B cell development. Nat Immunol 2003;4(3):274-9.##Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Sica S, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 2009;23(4):686-97.##Rinaldi A, Kwee I, Taborelli M, Largo C, Uccella S, Martin V, et al. Genomic and expression profiling identifies the B-cell associated tyrosine kinase Syk as a possible therapeutic target in mantle cell lymphoma. Br J Haematol 2006;132(3):303-16.##Chen L, Monti S, Juszczynski P, Daley J, Chen W, Witzig TE, et al. SYK-dependent tonic B-cell receptor signaling is a rational treatment target in diffuse large B-cell lymphoma. Blood 2008;111(4):2230-7.##Muzio M, Apollonio B, Scielzo C, Frenquelli M, Vandoni I, Boussiotis V, et al. Constitutive activation of distinct BCR-signaling pathways in a subset of CLL patients: a molecular signature of anergy. Blood 2008;112(1):188-95.##Hewamana S, Alghazal S, Lin TT, Clement M, Jenkins C, Guzman ML, et al. The NF-kappaB subunit Rel A is associated with in vitro survival and clinical disease progression in chronic lymphocytic leukemia and represents a promising therapeutic target. Blood 2008;111(9):4681-9.##Krysov S, Potter KN, Mockridge CI, Coelho V, Wheatley I, Packham G, et al. Surface IgM of CLL cells displays unusual glycans indicative of engagement of antigen in vivo. Blood 2010;115(21):4198-205.##Zhang XY, Zhang PY. Receptor tyrosine kinases in carcinogenesis. Oncol Lett 2020;20(5):195.##Landreth KS, Narayanan R, Dorshkind K. Insulin-like growth factor-I regulates pro-B cell differentiation. Blood 1992;80(5):1207-12.##Schillaci R, Galeano A, Becu-Villalobos D, Spinelli O, Sapia S, Bezares RF. Autocrine/paracrine involvement of insulin-like growth factor-I and its receptor in chronic lymphocytic leukaemia. Br J Haematol 2005;130(1):58-66.##Saiya-Cork K, Collins R, Parkin B, Ouillette P, Kuizon E, Kujawski L, et al. A pathobiological role of the insulin receptor in chronic lymphocytic leukemia. Clin Cancer Res 2011;17(9):2679-92.##Green JL, Kuntz SG, Sternberg PW. Ror receptor tyrosine kinases: orphans no more. Trends Cell Biol 2008;18(11):536-44.##Daneshmanesh AH, Hojjat-Farsangi M, Ghaderi A, Moshfegh A, Hansson L, Schultz J, et al. A receptor tyrosine kinase ROR1 inhibitor (KAN0439834) induced significant apoptosis of pancreatic cells which was enhanced by erlotinib and ibrutinib. PLoS One 2018;13(6):e0198038.##Hojjat-Farsangi M, Moshfegh A, Daneshmanesh AH, Khan AS, Mikaelsson E, Osterborg A, et al. The receptor tyrosine kinase ROR1--an oncofetal antigen for targeted cancer therapy. Semin Cancer Biol. 2014;29:21-31.##Hojjat-Farsangi M, Khan AS, Daneshmanesh AH, Moshfegh A, Sandin A, Mansouri L, et al. The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells. PLoS One 2013;8(10):e78339.##Hojjat-Farsangi M, Ghaemimanesh F, Daneshmanesh AH, Bayat AA, Mahmoudian J, Jeddi-Tehrani M, et al. Inhibition of the receptor tyrosine kinase ROR1 by anti-ROR1 monoclonal antibodies and siRNA induced apoptosis of melanoma cells. PLoS One 2013;8(4):e61167. Retraction.##Daneshmanesh AH, Hojjat-Farsangi M, Khan AS, Jeddi-Tehrani M, Akhondi MM, Bayat AA, et al. Monoclonal antibodies against ROR1 induce apoptosis of chronic lymphocytic leukemia (CLL) cells. Leukemia 2012;26(6):1348-55.##Daneshmanesh AH, Porwit A, Hojjat-Farsangi M, Jeddi-Tehrani M, Tamm KP, Grander D, et al. Orphan receptor tyrosine kinases ROR1 and ROR2 in hematological malignancies. Leuk Lymphoma 2013;54(4):843-50.##Shabani M, Asgarian Omran H, Farsangi MH, Vossough P, Sharifian RA, Toughe GR, et al. Comparative expression profile of orphan receptor tyrosine kinase ROR1 in Iranian patients with lymphoid and myeloid leukemias. Avicenna J Med Biotechnol 2011;3(3):119-25.##Baskar S, Kwong KY, Hofer T, Levy JM, Kennedy MG, Lee E, et al. Unique cell surface expression of receptor tyrosine kinase ROR1 in human B-cell chronic lymphocytic leukemia. Clin Cancer Res 2008;14(2):396-404.##Daneshmanesh AH, Mikaelsson E, Jeddi-Tehrani M, Bayat AA, Ghods R, Ostadkarampour M, et al. Ror1, a cell surface receptor tyrosine kinase is expressed in chronic lymphocytic leukemia and may serve as a putative target for therapy. Int J Cancer 2008;123(5):1190-5.##Hojjat-Farsangi M, Jeddi-Tehrani M, Razavi SM, Sharifian RA, Mellstedt H, Shokri F, et al. Immunoglobulin heavy chain variable region gene usage and mutational status of the leukemic B cells in Iranian patients with chronic lymphocytic leukemia. Cancer Sci 2009;100(12):2346-53.##Farsangi MH, Jeddi-Tehrani M, Sharifian RA, Razavi SM, Khoshnoodi J, Rabbani H, et al. Analysis of the immunoglobulin heavy chain variable region gene expression in Iranian patients with chronic lymphocytic leukemia. Leuk Lymphoma 2007;48(1):109-16.##Choi MY, Widhopf GF, 2nd, Ghia EM, Kidwell RL, Hasan MK, Yu J, et al. Phase I Trial: Cirmtuzumab Inhibits ROR1 Signaling and Stemness Signatures in Patients with Chronic Lymphocytic Leukemia. Cell Stem Cell 2018;22(6):951-9 e3.##Hojjat-Farsangi M, Daneshmanesh AH, Khan AS, Shetye J, Mozaffari F, Kharaziha P, et al. First-in-class oral small molecule inhibitor of the tyrosine kinase ROR1 (KAN0439834) induced significant apoptosis of chronic lymphocytic leukemia cells. Leukemia 2018;32(10):2291-5.##Veronese L, Tournilhac O, Verrelle P, Davi F, Dighiero G, Chautard E, et al. Strong correlation between VEGF and MCL-1 mRNA expression levels in B-cell chronic lymphocytic leukemia. Leuk Res 2009;33(12):1623-6.##Kay NE, Bone ND, Tschumper RC, Howell KH, Geyer SM, Dewald GW, et al. B-CLL cells are capable of synthesis and secretion of both pro- and anti-angiogenic molecules. Leukemia 2002;16(5):911-9.##Kini AR, Kay NE, Peterson LC. Increased bone marrow angiogenesis in B cell chronic lymphocytic leukemia. Leukemia 2000;14(8):1414-8.##Molica S, Vitelli G, Levato D, Ricciotti A, Digiesi G. Clinicoprognostic implications of increased serum levels of vascular endothelial growth factor and basic fibroblastic growth factor in early B-cell chronic lymphocytic leukaemia. Br J Cancer 2002;86(1):31-5.##Lai C, Lemke G. An extended family of protein-tyrosine kinase genes differentially expressed in the vertebrate nervous system. Neuron 1991;6(5):691-704.##O'Bryan JP, Frye RA, Cogswell PC, Neubauer A, Kitch B, Prokop C, et al. axl, a transforming gene isolated from primary human myeloid leukemia cells, encodes a novel receptor tyrosine kinase. Mol Cell Biol 1991;11(10):5016-31.##Hafizi S, Dahlback B. Signalling and functional diversity within the Axl subfamily of receptor tyrosine kinases. Cytokine Growth Factor Rev 2006;17(4):295-304.##Ghosh AK, Secreto C, Boysen J, Sassoon T, Shanafelt TD, Mukhopadhyay D, et al. The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy. Blood 2011;117(6):1928-37.##Cooper CS, Park M, Blair DG, Tainsky MA, Huebner K, Croce CM, et al. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 1984;311(5981):29-33.##Liu X, Yao W, Newton RC, Scherle PA. Targeting the c-MET signaling pathway for cancer therapy. Expert Opin Investig Drugs 2008;17(7):997-1011.##Eksioglu-Demiralp E, Akdeniz T, Bayik M. Aberrant expression of c-met and HGF/c-met pathway provides survival advantage in B-chronic lymphocytic leukemia. Cytometry B Clin Cytom 2011;80(1):1-7.##Shabani M, Hojjat-Farsangi M. Targeting Receptor Tyrosine Kinases Using Monoclonal Antibodies: The Most Specific Tools for Targeted-Based Cancer Therapy. Curr Drug Targets 2016;17(14):1687-703.##Hojjat-Farsangi M. Small-molecule inhibitors of the receptor tyrosine kinases: promising tools for targeted cancer therapies. Int J Mol Sci 2014;15(8):13768-801. ##Alsouqi A, Woyach JA. Covalent Bruton's Tyrosine Kinase Inhibitors in Chronic Lymphocytic Leukemia. Clin Lymphoma Myeloma Leuk 2024 May 31:S2152-2650(24)00210-6.##Freeman CL, Gribben JG. Immunotherapy in Chronic Lymphocytic Leukaemia (CLL). Curr Hematol Malig Rep 2016;11(1):29-36.##Jaglowski SM, Alinari L, Lapalombella R, Muthusamy N, Byrd JC. The clinical application of monoclonal antibodies in chronic lymphocytic leukemia. Blood 2010;116(19):3705-14.##Plosker GL, Figgitt DP. Rituximab: a review of its use in non-Hodgkin's lymphoma and chronic lymphocytic leukaemia. Drugs 2003;63(8):803-43.##Uchida J, Lee Y, Hasegawa M, Liang Y, Bradney A, Oliver JA, et al. Mouse CD20 expression and function. Int Immunol 2004;16(1):119-29.##Press OW, Howell-Clark J, Anderson S, Bernstein I. Retention of B-cell-specific monoclonal antibodies by human lymphoma cells. Blood 1994;83(5):1390-7.##Golay J, Lazzari M, Facchinetti V, Bernasconi S, Borleri G, Barbui T, et al. CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59. Blood 2001;98(12):3383-9.##Aue G, Lindorfer MA, Beum PV, Pawluczkowycz AW, Vire B, Hughes T, et al. Fractionated subcutaneous rituximab is well-tolerated and preserves CD20 expression on tumor cells in patients with chronic lymphocytic leukemia. Haematologica 2010;95(2):329-32.##Gowda A, Roda J, Hussain SR, Ramanunni A, Joshi T, Schmidt S, et al. IL-21 mediates apoptosis through up-regulation of the BH3 family member BIM and enhances both direct and antibody-dependent cellular cytotoxicity in primary chronic lymphocytic leukemia cells in vitro. Blood 2008;111(9):4723-30.##Gowda A, Ramanunni A, Cheney C, Rozewski D, Kindsvogel W, Lehman A, et al. Differential effects of IL-2 and IL-21 on expansion of the CD4+ CD25+ Foxp3+ T regulatory cells with redundant roles in natural killer cell mediated antibody dependent cellular cytotoxicity in chronic lymphocytic leukemia. MAbs 2010;2(1):35-41.##Teeling JL, French RR, Cragg MS, van den Brakel J, Pluyter M, Huang H, et al. Characterization of new human CD20 monoclonal antibodies with potent cytolytic activity against non-Hodgkin lymphomas. Blood 2004;104(6):1793-800.##Patz M, Isaeva P, Forcob N, Muller B, Frenzel LP, Wendtner CM, et al. Comparison of the in vitro effects of the anti-CD20 antibodies rituximab and GA101 on chronic lymphocytic leukaemia cells. Br J Haematol. 2011;152(3):295-306.##Bologna L, Gotti E, Manganini M, Rambaldi A, Intermesoli T, Introna M, et al. Mechanism of action of type II, glycoengineered, anti-CD20 monoclonal antibody GA101 in B-chronic lymphocytic leukemia whole blood assays in comparison with rituximab and alemtuzumab. J Immunol. 2011;186(6):3762-9.##Lei MM, Sorial MN, Lou U, Yu M, Medrano A, Ford J, et al. Real-world evidence of obinutuzumab and venetoclax in previously treated patients with chronic lymphocytic leukemia or small lymphocytic lymphoma. Leuk Lymphoma 2024;65(5):653-9.##Sachdeva M, Dhingra S. Obinutuzumab: A FDA approved monoclonal antibody in the treatment of untreated chronic lymphocytic leukemia. Int J Appl Basic Med Res 2015;5(1):54-7.##Huber H, Tausch E, Schneider C, Edenhofer S, von Tresckow J, Robrecht S, et al. Final analysis of the CLL2-GIVe trial: obinutuzumab, ibrutinib, and venetoclax for untreated CLL with del(17p)/TP53mut. Blood 2023;142(11):961-72.##Golay J, Manganini M, Rambaldi A, Introna M. Effect of alemtuzumab on neoplastic B cells. Haematologica. 2004;89(12):1476-83.##Rossmann ED, Lundin J, Lenkei R, Mellstedt H, Osterborg A. Variability in B-cell antigen expression: implications for the treatment of B-cell lymphomas and leukemias with monoclonal antibodies. Hematol J 2001;2(5):300-6.##Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Dyer MJ, et al. Levels of expression of CD52 in normal and leukemic B and T cells: correlation with in vivo therapeutic responses to Campath-1H. Leuk Res 1998;22(2):185-91.##Mone AP, Cheney C, Banks AL, Tridandapani S, Mehter N, Guster S, et al. Alemtuzumab induces caspase-independent cell death in human chronic lymphocytic leukemia cells through a lipid raft-dependent mechanism. Leukemia 2006;20(2):272-9.##Stilgenbauer S, Zenz T, Winkler D, Buhler A, Schlenk RF, Groner S, et al. Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol 2009;27(24):3994-4001.##Lozanski G, Heerema NA, Flinn IW, Smith L, Harbison J, Webb J, et al. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations and deletions. Blood. 2004;103(9):3278-81.##Tedder TF, Inaoki M, Sato S. The CD19-CD21 complex regulates signal transduction thresholds governing humoral immunity and autoimmunity. Immunity 1997;6(2):107-18.##Gladstone D, Andre M, Zaucha J, Assouline S, Bellam N, Cascavilla N, et al. Results of a phase 2 study of MEDI-551 and bendamustine vs rituximab and bendamustine in relapsed or refractory chronic lymphocytic leukemia. Am Soc Hematology 2014.##Woyach JA, Awan F, Flinn IW, Berdeja JG, Wiley E, Mansoor S, et al. A phase 1 trial of the Fc-engineered CD19 antibody XmAb5574 (MOR00208) demonstrates safety and preliminary efficacy in relapsed CLL. Blood 2014;124(24):3553-60.##Zhao X, Lapalombella R, Joshi T, Cheney C, Gowda A, Hayden-Ledbetter MS, et al. Targeting CD37-positive lymphoid malignancies with a novel engineered small modular immunopharmaceutical. Blood 2007;110(7):2569-77.##Byrd JC, Pagel JM, Awan FT, Forero A, Flinn IW, Deauna-Limayo DP, et al. A phase 1 study evaluating the safety and tolerability of otlertuzumab, an anti-CD37 mono-specific ADAPTIR therapeutic protein in chronic lymphocytic leukemia. Blood 2014;123(9):1302-8.##Hulkkonen J, Vilpo L, Hurme M, Vilpo J. Surface antigen expression in chronic lymphocytic leukemia: clustering analysis, interrelationships and effects of chromosomal abnormalities. Leukemia 2002;16(2):178-85.##Luqman M, Klabunde S, Lin K, Georgakis GV, Cherukuri A, Holash J, et al. The antileukemia activity of a human anti-CD40 antagonist antibody, HCD122, on human chronic lymphocytic leukemia cells. Blood 2008;112(3):711-20.##Byrd JC, Kipps TJ, Flinn IW, Cooper M, Odenike O, Bendiske J, et al. Phase I study of the anti-CD40 humanized monoclonal antibody lucatumumab (HCD122) in relapsed chronic lymphocytic leukemia. Leuk Lymphoma 2012;53(11):2136-42.##Lapalombella R, Gowda A, Joshi T, Mehter N, Cheney C, Lehman A, et al. The humanized CD40 antibody SGN-40 demonstrates pre-clinical activity that is enhanced by lenalidomide in chronic lymphocytic leukaemia. Br J Haematol 2009;144(6):848-55.##Furman RR, Forero-Torres A, Shustov A, Drachman JG. A phase I study of dacetuzumab (SGN-40, a humanized anti-CD40 monoclonal antibody) in patients with chronic lymphocytic leukemia. Leuk Lymphoma 2010;51(2):228-35.##Lewis JD, Reilly BD, Bright RK. Tumor-associated antigens: from discovery to immunity. Int Rev Immunol 2003;22(2):81-112.##Nagorsen D, Scheibenbogen C, Marincola FM, Letsch A, Keilholz U. Natural T cell immunity against cancer. Clin Cancer Res 2003;9(12):4296-303.##Babiak A, Steinhauser M, Gotz M, Herbst C, Dohner H, Greiner J. Frequent T cell responses against immunogenic targets in lung cancer patients for targeted immunotherapy. Oncol Rep 2014;31(1):384-90.##Liu C, Richard K, Wiggins M, Zhu X, Conrad DH, Song W. CD23 can negatively regulate B-cell receptor signaling. Sci Rep 2016;6:25629.##Fournier S, Delespesse G, Rubio M, Biron G, Sarfati M. CD23 antigen regulation and signaling in chronic lymphocytic leukemia. J Clin Invest 1992;89(4):1312-21.##Sarfati M, Chevret S, Chastang C, Biron G, Stryckmans P, Delespesse G, et al. Prognostic importance of serum soluble CD23 level in chronic lymphocytic leukemia. Blood 1996;88(11):4259-64.##Klein U, Tu Y, Stolovitzky GA, Mattioli M, Cattoretti G, Husson H, et al. Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells. J Exp Med 2001;194(11):1625-38.##Bund D, Mayr C, Kofler DM, Hallek M, Wendtner CM. CD23 is recognized as tumor-associated antigen (TAA) in B-CLL by CD8+ autologous T lymphocytes. Exp Hematol 2007;35(6):920-30.##Byrd JC, O'Brien S, Flinn IW, Kipps TJ, Weiss M, Rai K, et al. Phase 1 study of lumiliximab with detailed pharmacokinetic and pharmacodynamic measurements in patients with relapsed or refractory chronic lymphocytic leukemia. Clin Cancer Res 2007;13(15 Pt 1):4448-55. ##Byrd JC, Kipps TJ, Flinn IW, Castro J, Lin TS, Wierda W, et al. Phase 1/2 study of lumiliximab combined with fludarabine, cyclophosphamide, and rituximab in patients with relapsed or refractory chronic lymphocytic leukemia. Blood 2010;115(3):489-95.##Urso L, Calabrese F, Favaretto A, Conte P, Pasello G. Critical review about MDM2 in cancer: Possible role in malignant mesothelioma and implications for treatment. Crit Rev Oncol Hematol 2016;97:220-30.##Wade M, Li YC, Wahl GM. MDM2, MDMX and p53 in oncogenesis and cancer therapy. Nat Rev Cancer 2013;13(2):83-96.##Seliger B, Papadileris S, Vogel D, Hess G, Brendel C, Storkel S, et al. Analysis of the p53 and MDM-2 gene in acute myeloid leukemia. Eur J Haematol 1996;57(3):230-40.##Finnegan MC, Goepel JR, Royds J, Hancock BW, Goyns MH. Elevated levels of MDM-2 and p53 expression are associated with high grade non-Hodgkin's lymphomas. Cancer Lett 1994;86(2):215-21.##Watanabe T, Hotta T, Ichikawa A, Kinoshita T, Nagai H, Uchida T, et al. The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of B-cell origin. Blood 1994;84(9):3158-65.##Mayr C, Bund D, Schlee M, Bamberger M, Kofler DM, Hallek M, et al. MDM2 is recognized as a tumor-associated antigen in chronic lymphocytic leukemia by CD8+ autologous T lymphocytes. Exp Hematol 2006;34(1):44-53.##Wang H, Yu D, Agrawal S, Zhang R. Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oligonucleotides: in vitro and in vivo activities and mechanisms. Prostate 2003;54(3):194-205.##Bixby D, Kujawski L, Wang S, Malek SN. The pre-clinical development of MDM2 inhibitors in chronic lymphocytic leukemia uncovers a central role for p53 status in sensitivity to MDM2 inhibitor-mediated apoptosis. Cell Cycle 2008;7(8):971-9.##Andreeff M, Kelly KR, Yee K, Assouline S, Strair R, Popplewell L, et al. Results of the Phase I Trial of RG7112, a Small-Molecule MDM2 Antagonist in Leukemia. Clin Cancer Res 2016;22(4):868-76.##Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med 1997;3(8):917-21##Jaiswal PK, Goel A, Mittal R. Survivin: A molecular biomarker in cancer. Indian J Med Res 2015;141(4):389.##Lv Y-G, Yu F, Yao Q, Chen J-H, Wang L. The role of survivin in diagnosis, prognosis and treatment of breast cancer. J Thorac Dis 2010;2(2):100.##Granziero L, Ghia P, Circosta P, Gottardi D, Strola G, Geuna M, et al. Survivin is expressed on CD40 stimulation and interfaces proliferation and apoptosis in B-cell chronic lymphocytic leukemia. Blood 2001;97(9):2777-83.##Andersen MH, Pedersen LØ, Becker JC, thor Straten P. Identification of a cytotoxic T lymphocyte response to the apoptosis inhibitor protein survivin in cancer patients. Cancer Res 2001;61(3):869-72.##Schmitz M, Diestelkoetter P, Weigle B, Schmachtenberg F, Stevanovic S, Ockert D, et al. Generation of survivin-specific CD8+ T effector cells by dendritic cells pulsed with protein or selected peptides. Cancer Res 2000;60(17):4845-9.##Schmidt SM, Schag K, Müller MR, Weck MM, Appel S, Kanz L, et al. Survivin is a shared tumor-associated antigen expressed in a broad variety of malignancies and recognized by specific cytotoxic T cells. Blood 2003;102(2):571-6.##Reker S, Becker JC, Svane IM, Ralfkiaer E, Straten Pt, Andersen MH. HLA‐B35‐restricted immune responses against survivin in cancer patients. Int J Cancer 2004;108(6):937-41.##Miletic AV, Jellusova J, Cato MH, Lee CR, Baracho GV, Conway EM, et al. Essential role for Survivin in the proliferative expansion of progenitor and mature B cells. J Immunol 2016;196(5):2195-204.##Purroy N, Abrisqueta P, Carabia J, Carpio C, Calpe E, Palacio C, et al. Targeting the proliferative and chemoresistant compartment in chronic lymphocytic leukemia by inhibiting survivin protein. Leukemia 2014;28(10):1993-2004.##Tibes R, McDonagh K, Lekakis L, Bogenberger J, Kim S, Frazer N, et al. Phase I study of the novel Cdc2/CDK1 and AKT inhibitor terameprocol in patients with advanced leukemias. Invest New Drugs 2015;33(2):389-96.##Rostamzadeh D, Kazemi T, Amirghofran Z, Shabani M. Update on Fc receptor-like (FCRL) family: new immunoregulatory players in health and diseases. Expert Opin Ther Targets 2018;22(6):487-502.##Rostamzadeh D, Dabbaghmanesh M, Shabani M, Hosseini A, Amirghofran Z. Expression profile of human Fc receptor-like 1, 2, and 4 molecules in peripheral blood mononuclear cells of patients with Hashimoto’s thyroiditis and Graves’ disease. Horm Metab Res 2015;47(09):693-8.##Li FJ, Ding S, Pan J, Shakhmatov MA, Kashentseva E, Wu J, et al. FCRL2 expression predicts IGHV mutation status and clinical progression in chronic lymphocytic leukemia. Blood 2008;112(1):179-87.##Benedetti D, Perini C, Tissino E, Dal Bo M, Bulian P, Bomben R, et al. The B-cell receptor signaling inhibitor molecules CD305 and CD307b are markers of favorable prognosis in chronic lymphocytic leukemia with both mutated and unmutated IGHV gene status. Blood 2016 Dec 2;128(22):4358.##Oldberg A, Antonsson P, Lindblom K, Heinegård D. A collagen‐binding 59‐kd protein (fibromodulin) is structurally related to the small interstitial proteoglycans PG‐S1 and PG‐S2 (decorin). EMBO J 1989;8(9):2601-4.##Hedbom E, Heinegård D. Interaction of a 59-kDa connective tissue matrix protein with collagen I and collagen II. J Biol Chem 1989;264(12):6898-905.##Jelinek DF, Tschumper RC, Stolovitzky GA, Iturria SJ, Tu Y, Lepre J, et al. Identification of a Global Gene Expression Signature of B-Chronic Lymphocytic Leukemia1 1 Mayo Comprehensive Cancer Center, National Cancer Institute CA91542 (awarded to NE Kay), and generous philanthropic support provided by Edson Spencer. Mol Cancer Res 2003;1(5):346-61.##Vallat L, Magdelénat H, Merle-Béral Hln, Masdehors P, Potocki de Montalk G, Davi F, et al. The resistance of B-CLL cells to DNA damage–induced apoptosis defined by DNA microarrays. Blood 2003;101(11):4598-606.##Mayr C, Bund D, Schlee M, Moosmann A, Kofler DM, Hallek M, et al. Fibromodulin as a novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (CLL), which allows expansion of specific CD8+ autologous T lymphocytes. Blood 2005;105(4):1566-73.##Mikaelsson E, Jeddi-Tehrani M, Osterborg A, Shokri F, Mellstedt H, Rabbani H. Fibromodulin—a novel tumor associated antigen exclusively expressed in tumor B-cells from patients with chronic lymphocytic leukemia and mantle cell lymphoma. Leuk Lymphoma 2003;44(suppl 2):21.##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. Iran J Immunol 2019;16(2):127-41.##Yip K, Reed J. Bcl-2 family proteins and cancer. Oncogene 2008;27(50):6398-406.##Fecker LF, Geilen CC, Tchernev G, Trefzer U, Assaf C, Kurbanov BM, et al. Loss of proapoptotic Bcl-2-related multidomain proteins in primary melanomas is associated with poor prognosis. J Invest Dermatol 2006;126(6):1366-71.##Agrawal SG, Liu F-T, Wiseman C, Shirali S, Liu H, Lillington D, et al. Increased proteasomal degradation of Bax is a common feature of poor prognosis chronic lymphocytic leukemia. Blood 2008;111(5):2790-6.##Nunes CT, Miners KL, Dolton G, Pepper C, Fegan C, Mason MD, et al. A novel tumor antigen derived from enhanced degradation of bax protein in human cancers. Cancer research. 2011;71(16):5435-44.##Del Principe MI, Dal Bo M, Bittolo T, Buccisano F, Rossi FM, Zucchetto A, et al. Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia. Haematologica 2016;101(1):77.##Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013;19(2):202-8.##Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V Gene Mutation Status and CD38 Expression As Novel Prognostic Indicators in Chronic Lymphocytic Leukemia: Presented in part at the 40th Annual Meeting of The American Society of Hematology, held in Miami Beach, FL, December 4-8, 1998. Blood 1999;94(6):1840-7.##Del Poeta G, Del Principe MI, Maurillo L, Rossi FM, Buccisano F, Ammatuna E, et al. Spontaneous apoptosis and proliferation detected by BCL-2 and CD71 proteins are important progression indicators within ZAP-70 negative chronic lymphocytic leukemia. Leuk Lymphoma 2010;51(1):95-106.##Gattei V, Bulian P, Del Principe MI, Zucchetto A, Maurillo L, Buccisano F, et al. Relevance of CD49d protein expression as overall survival and progressive disease prognosticator in chronic lymphocytic leukemia. Blood 2008;111(2):865-73.##Dürig J, Dührsen U, Klein-Hitpass L, Worm J, Hansen JR, Ørum H, et al. The novel antisense Bcl-2 inhibitor SPC2996 causes rapid leukemic cell clearance and immune activation in chronic lymphocytic leukemia. Leukemia 2011;25(4):638-47.##Tilly H, Coiffier B, Michallet A, Radford J, Geisler C, Gadeberg O, et al. Phase I/II study of SPC2996, an RNA antagonist of Bcl-2, in patients with advanced chronic lymphocytic leukemia (CLL). Journal of Clinical Oncology. 2007;25(18_suppl):7036.##Furstenau M, Kater AP, Robrecht S, von Tresckow J, Zhang C, Gregor M, et al. First-line venetoclax combinations versus chemoimmunotherapy in fit patients with chronic lymphocytic leukaemia (GAIA/CLL13): 4-year follow-up from a multicentre, open-label, randomised, phase 3 trial. Lancet Oncol 2024;25(6):744-59.##de la Fuente MA, Tovar V, Villamor N, Zapater N, Pizcueta P, Campo E, et al. Molecular characterization and expression of a novel human leukocyte cell-surface marker homologous to mouse Ly-9. Blood 2001;97(11):3513-20.##Bund D, Mayr C, Kofler DM, Hallek M, Wendtner C-M. Human Ly9 (CD229) as novel tumor-associated antigen (TAA) in chronic lymphocytic leukemia (B-CLL) recognized by autologous CD8+ T cells. Exp Hematol 2006;34(7):860-9.##Wallar BJ, Alberts AS. The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol 2003;13(8):435-46.##Favaro PMB, de Souza Medina S, Traina Fo, Bassères DS, Costa FF, Saad STO. Human leukocyte formin: a novel protein expressed in lymphoid malignancies and associated with Akt. Biochem Biophys Res Commun 2003;311(2):365-71.##Krackhardt AM, Witzens M, Harig S, Hodi FS, Zauls AJ, Chessia M, et al. Identification of tumor-associated antigens in chronic lymphocytic leukemia by SEREX. Blood 2002;100(6):2123-31. ##Giannopoulos K, Li L, Bojarska-Junak A, Rolinski J, Dmoszynska A, Hus I, et al. Expression of RHAMM/CD168 and other tumor-associated antigens in patients with B-cell chronic lymphocytic leukemia. Int J Oncol 2006;29(1):95-103.##Hus I, Roliński J, Tabarkiewicz J, Wojas K, Bojarska-Junak A, Greiner J, et al. Allogeneic dendritic cells pulsed with tumor lysates or apoptotic bodies as immunotherapy for patients with early-stage B-cell chronic lymphocytic leukemia. Leukemia 2005;19(9):1621-7. ##Greiner J, Li L, Ringhoffer M, Barth TF, Giannopoulos K, Guillaume P, et al. Identification and characterization of epitopes of the receptor for hyaluronic acid–mediated motility (RHAMM/CD168) recognized by CD8+ T cells of HLA-A2–positive patients with acute myeloid leukemia. Blood 2005;106(3):938-45.##Nugent CI, Lundblad V. The telomerase reverse transcriptase: components and regulation. Genes Dev 1998;12(8):1073-85.##Jafri MA, Ansari SA, Alqahtani MH, Shay JW. Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies. Genome Med 2016;8(1):69.##Trentin L, Ballon G, Ometto L, Perin A, Basso U, Chieco‐Bianchi L, et al. Telomerase activity in chronic lymphoproliferative disorders of B‐cell lineage. Br J Haematol 1999;106(3):662-8.##Kokhaei P, Palma M, Hansson L, Österborg A, Mellstedt H, Choudhury A. Telomerase (hTERT 611–626) serves as a tumor antigen in B-cell chronic lymphocytic leukemia and generates spontaneously antileukemic, cytotoxic T cells. Exp Hematol 2007;35(2):297-304.##Rohrer J, Barsoum A, Coggin Jr J. The development of a new universal tumor rejection antigen expressed on human and rodent cancers for vaccination, prevention of cancer, and anti-tumor therapy. Mod Asp Immunobiol 2001;5:191-5.##Barsoum A, Rohrer J, Coggin J. 37kDa oncofetal antigen is an autoimmunogenic homologue of the 37kDa laminin receptor precursor. Cellular and Molecular Biology Letters 2000;2(05).##Siegel S, Wagner A, Kabelitz D, Marget M, Coggin Jr J, Barsoum A, et al. Induction of cytotoxic T-cell responses against the oncofetal antigen-immature laminin receptor for the treatment of hematologic malignancies. Blood 2003;102(13):4416-23.##Su Z, Dannull J, Heiser A, Yancey D, Pruitt S, Madden J, et al. Immunological and clinical responses in metastatic renal cancer patients vaccinated with tumor RNA-transfected dendritic cells. Cancer Res 2003;63(9):2127-33.##Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling LC, et al. SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c lysozyme-like protein of human spermatozoa. Biol Reprod 2003;68(5):1525-37.##Wang Z, Zhang Y, Mandal A, Zhang J, Giles FJ, Herr JC, et al. The spermatozoa protein, SLLP1, is a novel cancer–testis antigen in hematologic malignancies. Clin Cancer Res 2004;10(19):6544-50.##Said JW, Hoyer KK, French SW, Rosenfelt L, Garcia-Lloret M, Koh PJ, et al. TCL1 oncogene expression in B cell subsets from lymphoid hyperplasia and distinct classes of B cell lymphoma. Lab Invest 2001;81(4):555-64.##Narducci MG, Pescarmona E, Lazzeri C, Signoretti S, Lavinia AM, Remotti D, et al. Regulation of TCL1 expression in B-and T-cell lymphomas and reactive lymphoid tissues. Cancer Res 2000;60(8):2095-100.##Weng J, Rawal S, Chu F, Park HJ, Sharma R, Delgado DA, et al. TCL1: a shared tumor-associated antigen for immunotherapy against B-cell lymphomas. Blood 2012;120(8):1613-23.##Sahasrabuddhe AA, Elenitoba-Johnson KS. TCL1A expression promotes aggressive biology in CLL. Blood 2023;141(12):1371-3.##Heid HW, Moll R, Schwetlick I, Rackwitz H-R, Keenan TW. Adipophilin is a specific marker of lipid accumulation in diverse cell types and diseases. Cell Tissue Res 1998;294(2):309-21.##Buechler C, Ritter M, Duong CQ, Orso E, Kapinsky M, Schmitz G. Adipophilin is a sensitive marker for lipid loading in human blood monocytes. Biochim Biophys Acta 2001;1532(1-2):97-104.##Rae FK, Stephenson SA, Nicol DL, Clements JA. Novel association of a diverse range of genes with renal cell carcinoma as identified by differential display. Int J Cancer 2000;88(5):726-32.##Schmidt SM, Schag K, Müller MR, Weinschenk T, Appel S, Schoor O, et al. Induction of adipophilin-specific cytotoxic T lymphocytes using a novel HLA-A2-binding peptide that mediates tumor cell lysis. Cancer Res 2004;64(3):1164-70.##Bolkun L, Tynecka M, Wasiluk T, Piszcz J, Starosz A, Grubczak K, et al. A Proliferation-Inducing Ligand and B-Cell Activating Factor Are Upregulated in Patients with Essential Thrombocythemia. J Clin Med 2022;11(16):4663.##O’Donnell A, Pepper C, Mitchell S, Pepper A. NF-kB and the CLL microenvironment. Front Oncol 2023;13:1169397.##Ullah MA, Mackay F. The BAFF-APRIL System in Cancer. Cancers (Basel) 2023;15(6):1791.##Kern C, Cornuel J-F, Billard C, Tang R, Rouillard D, Stenou V, et al. Involvement of BAFF and APRIL in the resistance to apoptosis of B-CLL through an autocrine pathway. Blood 2004;103(2):679-88.##Hahne M, Kataoka T, Schröter M, Hofmann K, Irmler M, Bodmer J-L, et al. APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J Exp Med 1998;188(6):1185-90.##He B, Chadburn A, Jou E, Schattner EJ, Knowles DM, Cerutti A. Lymphoma B cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL. J Immunol 2004;172(5):3268-79.##Endo T, Nishio M, Enzler T, Cottam HB, Fukuda T, James DF, et al. BAFF and APRIL support chronic lymphocytic leukemia B-cell survival through activation of the canonical NF-κB pathway. Blood 2007;109(2):703-10.##Bojarska-Junak A, Hus I, Chocholska S, Wąsik-Szczepanek E, Sieklucka M, Dmoszyńska A, et al. BAFF and APRIL expression in B-cell chronic lymphocytic leukemia: correlation with biological and clinical features. Leuk Res 2009;33(10):1319-27.##Haiat S, Billard C, Quiney C, Ajchenbaum‐Cymbalista F, Kolb JP. Role of BAFF and APRIL in human B‐cell chronic lymphocytic leukaemia. Immunology 2006;118(3):281-92.##Tandler C, Schmidt M, Heitmann JS, Hierold J, Schmidt J, Schneider P, et al. Neutralization of B-cell activating factor (BAFF) by belimumab reinforces small molecule inhibitor treatment in chronic lymphocytic leukemia. Cancers (Basel) 2020;12(10):2725.##Luo Y, Qie Y, Gadd ME, Manna A, To T, Li S, et al. Translational development of BAFF-R-specific chimeric antigen receptor T-cell therapy targeting B-cell lymphoid malignancies. American Society of Clinical Oncology 2023.##Chen L, Mao W, Ren C, Li J, Zhang J. Comprehensive Insights that Targeting PIM for Cancer Therapy: Prospects and Obstacles. J Med Chem 2024;67(1):38-64.##Decker S, Finter J, Forde AJ, Kissel S, Schwaller J, Mack TS, et al. PIM kinases are essential for chronic lymphocytic leukemia cell survival (PIM2/3) and CXCR4-mediated microenvironmental interactions (PIM1). Mol Cancer Ther 2014;13(5):1231-45.##Kowalewski DJ, Schuster H, Berlin C, Kanz L, Salih HR, Rammensee H-G, et al. Identification of Novel Tumor-Associated Antigens for Chronic Lymphocytic Leukemia (CLL) Based On HLA Ligandome Analysis–New Targets for Peptide Based Immunotherapy. Blood 2012;120(21):4119.##Christensen DJ, Chen Y, Oddo J, Matta KM, Neil J, Davis ED, et al. SET oncoprotein overexpression in B-cell chronic lymphocytic leukemia and non-Hodgkin lymphoma: a predictor of aggressive disease and a new treatment target. Blood 2011;118(15):4150-8.##Nadeu F, Royo R, Clot G, Duran-Ferrer M, Navarro A, Martín S, et al. IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics. Blood 2021;137(21):2935-46.##Märkl F, Schultheiß C, Ali M, Chen S-S, Zintchenko M, Egli L, et al. Mutation-specific CAR T cells as precision therapy for IGLV3-21R110 expressing high-risk chronic lymphocytic leukemia. Nat Commun 2024;15(1):993.##Zhao Y, Su H, Shen X, Du J, Zhang X, Zhao Y. The immunological function of CD52 and its targeting in organ transplantation. Inflamm Res 2017;66(7):571-8.##Vojdeman FJ, Herman SEM, Kirkby N, Wiestner A, van T' Veer MB, Tjonnfjord GE, et al. Soluble CD52 is an indicator of disease activity in chronic lymphocytic leukemia. Leuk Lymphoma 2017;58(10):2356-62. ##Czuczman MS, Kahanic S, Forero A, Davis G, Munteanu M, Van Den Neste E, et al. Results of a phase II study of bendamustine and ofatumumab in untreated indolent B cell non-Hodgkin's lymphoma. Ann Hematol 2015;94(4):633-41.##Purroy N, Abrisqueta P, Carabia J, Carpio C, Calpe E, Palacio C, et al. Targeting the proliferative and chemoresistant compartment in chronic lymphocytic leukemia by inhibiting survivin protein. Leukemia 2014;28(10):1993-2004.##Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J, et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013;19(2):202-8.##Del Principe MI, Dal Bo M, Bittolo T, Buccisano F, Rossi FM, Zucchetto A, et al. Clinical significance of bax/bcl-2 ratio in chronic lymphocytic leukemia. Haematologica 2016;101(1):77-85.##

The Importance of Biosecurity in Emerging Biotechnologies and Synthetic Biology 2 2 The age of synthetic biology is ushering in new technologies for the advancement of society, human health, and agriculture. It appears that synthetic biology has integrated engineering paradigms into biological contexts. The combined use of new biotechnology and synthetic biology raises concerns about biosafety, biosecurity, and even cyberbiosecurity. For example, synthetic biology increases the possibility of designing, developing, and deploying pathogenic bioweapons in new and different ways than natural pathogens, as well as manipulating the genome. Evaluation of new technologies and platforms that enable creative or destructive manipulation of biological materials, systems, and organisms is important to identify potential security opportunities and vulnerabilities. This issue poses challenges to the medical community and civilian populations worldwide, creating a growing need to implement and enforce standardized biosafety and biosecurity regulations to protect humans, animals, plants, and the environment. It is critical to establish rules and management guidelines, provide strong leadership at the individual and institutional levels, and utilize established biosafety and biosecurity tools to mitigate the risks associated with synthetic biology. This review addresses the current state of synthetic biology, focusing on the concepts of biosafety, biosecurity, and cyber-biosecurity, as well as enhancing the standardization, regulation, and management of biosecurity in synthetic biology. In this review, the current situation in the Middle East region has been discussed and the challenges and opportunities encountered by synthetic biology researchers in this area is explored. The Middle East region is vulnerable to bioterrorism due to various factors. However, some countries in this strategically important region face challenges as they lack the necessary resources to effectively combat this significant global threat. These attacks are not limited to a specific border or area; they can affect multiple countries or have a global impact. 223 232 Mahboubeh Soleimani Sasani Chemical, Biological, Radiological & Nuclear (CBRN) Instructor, Iran's Passive Defense Organization Iran BiosecurityBiotechnologyBioterrorismSynthetic biology 60589.pdf Ahteensuu M. Synthetic Biology, Genome Editing, and the Risk of Bioterrorism. Sci Eng Ethics 2017 Dec;23(6):1541-61. ##Mingzhu DI, Bingzhi LI, Ying WA, Zexiong XI, Duo LI, Yingjin YU. Significant research progress in synthetic biology. Synthetic Biology Journal 2020 Feb 29;1(1):7.##Wang L, Jiang S, Chen C, He W, Wu X, Wang F, et al. Synthetic genomics: from DNA synthesis to genome design. Angew Chem Int Ed Engl 2018 Feb 12;57(7):1748-56.##Evans NG, Selgelid MJ. Biosecurity and Open-Source Biology: The Promise and Peril of Distributed Synthetic Biological Technologies. Sci Eng Ethics 2015 Aug;21(4):1065-83. ##Kwik Gronvall G. Biosecurity: the opportunities and threats of industrialization and personalization. Bulletin of the Atomic Scientists 2015 Nov;71(6):39-44.##National Academies of Sciences, Engineering, and Medicine; Division on Earth and Life Studies; Board on Life Sciences; Board on Chemical Sciences and Technology; Committee on Strategies for Identifying and Addressing Potential Biodefense Vulnerabilities Posed by Synthetic Biology. Biodefense in the Age of Synthetic Biology. Washington (DC): National Academies Press (US); 2018 Jun 19.##Zeng X, Jiang H, Yang G, Ou Y, Lu S, Jiang J, Lei R, Su L. Regulation and management of the biosecurity for synthetic biology. Synth Syst Biotechnol 2022 Jun;7(2):784–90. ##Li J, Zhao H, Zheng L, An W. Advances in synthetic biology and biosafety governance. Front Bioeng Biotechnol 2021 Apr 30;9:598087. ##Yeh JY, Seo HJ, Park JY, Cho YS, Cho IS, Lee JH, Hwang JM, Choi IS. Livestock agroterrorism: the deliberate introduction of a highly infectious animal pathogen. Foodborne Pathog Dis 2012 Oct 1;9(10):869-77. ##Getz LJ, Dellaire G. Angels and Devils: Dilemmas in Dual-Use Biotechnology. Trends Biotechnol 2018 Dec;36(12):1202-5. ##Cello J, Paul AV, Wimmer E. Chemical synthesis of poliovirus cDNA: generation of infectious virus in the absence of natural template. Science 2002 Aug 9;297(5583):1016-8. ##Astuto-Gribble LM, Caskey SA. Laboratory Biosafety and Biosecurity Risk Assessment Technical Guidance Document. Sandia National Lab.(SNL-NM), Albuquerque, NM (United States); 2014. ##Dutta S, Das D. Bioprocess: Control, management, and biosafety issues. InBasic Biotechniques for Bioprocess and Bioentrepreneurship: Elsevier; 2023. p. 355-364.##Dickmann P, Sheeley H, Lightfoot N. Biosafety and Biosecurity: A Relative Risk-Based Framework for Safer, More Secure, and Sustainable Laboratory Capacity Building. Front Public Health 2015 Oct 21;3:241. ##McLennan A. Biosecurity: potential for deliberate misuse of synthetic biology. Regulation of Synthetic Biology: Edward Elgar Publishing; 2018. p. 207-48.##Gaudioso J, Gribble LA, Salerno RM. Biosecurity: Progress and challenges. JALA: Journal of the Association for Laboratory Automation. 2009;14(3):141-7.##LeDuc JW, Yuan Z. Safety and security in the age of synthetic biology. Journal of Biosafety and Biosecurity. 2019 Sep 1;1(2):77-9.##Huang Y, Huang J, Xia H, Shi Y, Ma H, Yuan Z. Networking for training Level 3/4 biosafety laboratory staff. J Biosaf Biosecur 2019 Mar;1(1):46-9. ##World Health Organization. WHO consultative meeting high/maximum containment (biosafety level 4) laboratories networking: venue: International Agency on Research on Cancer (IARC), Lyon, France, 13-15 December 2017: meeting report. World Health Organization; 2018.##Trump BD, Florin MV, Perkins E, Linkov I. Biosecurity for synthetic biology and emerging biotechnologies: Critical challenges for governance. Emerging Threats of Synthetic Biology and Biotechnology: Addressing Security and Resilience Issues 2021:1-2.##Auplish A, Vu TTT, Pham Duc P, Green A, Tiwari H, Housen T, Stevenson MA, Dhand N. Capacity and needs assessment of veterinary services in Vietnam in biosecurity, biosafety and One Health. PLoS One 2024 Jan 11;19(1):e0295898. ##Xu J. Reverse microbial etiology: A research field for predicting and preventing emerging infectious diseases caused by an unknown microorganism. J Biosaf Biosecur 2019 Mar;1(1):19-21. ##Trump BD, Galaitsi SE, Appleton E, Bleijs DA, Florin MV, Gollihar JD, et al. Building biosecurity for synthetic biology. Mol Syst Biol 2020 Jul;16(7):e9723. ##Trump BD, Galaitsi S, Pollock M, Volk KM, Linkov I. Promoting Effective Biosecurity Governance: Using Tripwires to Anticipate and Ameliorate Potentially Harmful Development Trends. AB3. 2021:209. ##Noyce RS, Lederman S, Evans DH. Construction of an infectious horsepox virus vaccine from chemically synthesized DNA fragments. PloS One 2018 Jan 19;13(1):e0188453. ##Sun T, Song J, Wang M, Zhao C, Zhang W. Challenges and recent progress in the governance of biosecurity risks in the era of synthetic biology. Journal of Biosafety and Biosecurity 2022 Jun 1;4(1):59-67.##Yan W, Xin FX, Dong WL, Zhou J, Zhang WM, Jiang M. Synthetic biology and research progress. J Biol 2020;37(5):1-9.##Wang F, Zhang W. Synthetic biology: recent progress, biosafety and biosecurity concerns, and possible solutions. Journal of Biosafety and Biosecurity 2019 Mar 1;1(1):22-30. ##European research area network for the development and coordination of synthetic biology in Europe. [Internet]. 2014.##Conrad PA, Meek LA, Dumit J. Operationalizing a One Health approach to global health challenges. Comp Immunol Microbiol Infect Dis 2013 May;36(3):211-6. 2013;36(3):211-6. ##Bratlie S, Halvorsen K, Myskja BK, Mellegård H, Bjorvatn C, Frost P, Heiene G, et al. A novel governance framework for GMO: A tiered, more flexible regulation for GMOs would help to stimulate innovation and public debate. EMBO Rep 2019 May;20(5):e47812. ##Keiper F, Atanassova A. Regulation of synthetic biology: developments under the convention on biological diversity and its protocols. Front Bioeng Biotechnol 2020 Apr 9;8:310. ##Guoping ZH. Synthetic biology: unsealing the convergence era of life science research. Bulletin of Chinese Academy of Sciences (Chinese Version) 2018;33(11):1135-49.##Linkov I, Trump BD, Anklam E, Berube D, Boisseasu P, Cummings C, et al. Comparative, collaborative, and integrative risk governance for emerging technologies. Environ Syst Decis 2018 Jun;38:170-6. ##Wikmark OG, Brautaset T, Agapito-Tenfen SZ, Okoli AS, Myhr AI, Binimelis R, Ching LL. Synthetic biology-biosafety and contribution to addressing societal challenges. The Royal Academy of Engineering, London. 2016.##Shang L, Zhang W, Dando M. Biological Security After the Pandemic. Essentials of Biological Security: A Global Perspective 2024:1.##Habibi Z, Hadi NA, Kim EE, Alkhataybeh RAM, Sbeih A, Abou-Hamden A, et al. Progress in neurosurgery: Contributions of women neurosurgeons in the Middle East. J Clin Neurosci 2021 Apr;86:337-46. ##Laith AE, Alnemri M. Biosafety and biosecurity in the era of biotechnology: The Middle East region. Journal of Biosafety and Biosecurity 2022 Dec 1;4(2):130-45.##Astuto-Gribble LM, Gaudioso JM, Caskey SA, Zemlo TR. A survey of bioscience research and biosafety and biosecurity practices in Asia, Eastern Europe, Latin America, and the Middle East. Applied biosafety. 2009;14(4):181-96.##Prainsack B, Firestine O. Genetically modified survival: red and green biotechnology in Israel. Science as Culture. 2005 Dec 1;14(4):355-72.##Arsanjani MH. Negotiating the UN declaration on human cloning. American Journal of International Law 2006;100(1):164-79.##Nezhad Fard RM, Moslemy M, Golshahi H. The history of modern biotechnology in Iran: A medical review. J Biotechnol Biomater 2013;3(159):2. ##Gantait S, El-Dawayati MM, Panigrahi J, Labrooy C, Verma SK. The retrospect and prospect of the applications of biotechnology in Phoenix dactylifera L. Appl Microbiol Biotechnol 2018 Oct;102(19):8229-59. ##Jain SM. Date palm biotechnology: Current status and prospective-an overview. Emir J Food Agric 2012 Apr 4;24(5):386-99.##Shooshtari A, Hajatpour Z, Ghaffari MR, Seraji F, Loni F. Awareness of GMOs in terms of the Iran biosafety act: A case study of Tehran city. Heliyon 2024 Feb 15;10(3).##Jalali Farahani G, Bakhtiyari I. Explaining the Factors Affecting the Command and Control System of the Non-Operating Defense Organization. Strategic Defense Studies 2019 Jan 21;16(74):5-28.##Peng Y. The era of synthetic biology: biosafety, biosecurity and governance. J Int Secur Stud 2020;38:29.##Council NR, Policy, Affairs G, Security, Cooperation, Standards CoR, et al. Biotechnology research in an age of terrorism. 2004.##Hamilton RA, Mampuys R, Galaitsi SE, Collins A, Istomin I, Ahteensuu M, et al. Opportunities, Challenges, and Future Considerations for Top-Down Governance for Biosecurity and Synthetic Biology. 2021 Sep 8. In: Trump BD, Florin MV, Perkins E, Linkov I, editors. Emerging Threats of Synthetic Biology and Biotechnology: Addressing Security and Resilience Issues [Internet]. Dordrecht (DE): Springer; 2021. Chapter 3.##Arndt E, Schneider K, Bland L, Robinson A, Gibert A, Camac J, et al. A conceptual framework for measuring and improving the resilience of biosecurity systems. J Applied Ecology 2024;61(8):1749-60.##World Health Organization. WHO BioHub system: biosafety and biosecurity: criteria and operational modalities. 2022.##Peccoud J, Gallegos JE, Murch R, Buchholz WG, Raman S. Cyberbiosecurity: from naive trust to risk awareness. Trends Biotechnol 2018 Jan 1;36(1):4-7. ##Murch RS, So WK, Buchholz WG, Raman S, Peccoud J. Cyberbiosecurity: an emerging new discipline to help safeguard the bioeconomy. Front Bioeng Biotechnol 2018 Apr 5;6:39. ##World Health Organization. Biosecurity: An integrated approach to manage risk to human, animal and plant life and health. International Food Safety Authorities Network (INFOSAN). 2010.##Shinomiya N, Minehata M, Dando M. Bioweapons and dual-use research of concern. J Disaster Res 2013 Aug 1;8(4):654-66. ##Lundstrom K, Boulikas T. Viral and non-viral vectors in gene therapy: technology development and clinical trials. Technol Cancer Res Treat 2003 Oct;2(5):471-85. ##Nouri A, Chyba CF. Biotechnology and biosecurity. Global Catastrophic Risks 2008 Jul 3:450-80.##Bossi P, Garin D, Guihot A, Gay F, Crance JM, Debord T, et al. Bioterrorism: management of major biological agents. Cell Mol Life Sci 2006 Oct;63(19-20):2196-212. ##Kerwat K, Becker S, Wulf H, Densow D. [Biological weapons]. Dtsch Med Wochenschr 2010 Aug;135(33):1612-6. German. ##Mathew SA. Implications of stem cell therapy on the socio-scientific community. In: Stem Cells 2024 Jan 1 (pp. 387-394). Academic Press.##Hinchliffe S, Allen J, Lavau S, Bingham N, Carter S. Biosecurity and the topologies of infected life: from borderlines to borderlands. Transactions of the Institute of British Geographers 2013 Oct;38(4):531-43.##Reaser JK, Chitale RA, Tabor GM, Hudson PJ, Plowright RK. Looking left: ecologically based biosecurity to prevent pandemics. Health Secur 2024 Feb 1;22(1):74-81. ##Kane A, Parker MT. Screening State of Play: The Biosecurity Practices of Synthetic DNA Providers. Appl Biosaf 2024 Jun 20;29(2):85-95. ##Cowl VB, Comizzoli P, Appeltant R, Bolton RL, Browne RK, Holt WV, et al. Cloning for the Twenty-First Century and Its Place in Endangered Species Conservation. Annu Rev Anim Biosci 2024 Feb 15;12:91-112.##Trump BD, Cummings CL, Kuzma J, Linkov I, editors. Synthetic biology 2020: Frontiers in risk analysis and governance. Springer Nature 2019 Nov 28. ##Millett P, Binz T, Evans SW, Kuiken T, Oye K, Palmer MJ,et al. Developing a comprehensive, adaptive, and international biosafety and biosecurity program for advanced biotechnology: the IGEM experience. Appl Biosaf 2019 Jun 1;24(2):64-71. ##Isasi RM, Knoppers BM. Beyond the permissibility of embryonic and stem cell research: substantive requirements and procedural safeguards. Hum Reprod 2006 Oct;21(10):2474-81. ## Abelman M, Lopes M, O’Rourke PP. Human Embryonic Stem Cell Research Oversight: A Confluence of Voluntary Self-Regulation and Shifting Policy Initiatives. InResearch Regulatory Compliance: Elsevier; 2015 p. 297-320. ##Hall CM, Baird T. Ecotourism, biological invasions and biosecurity. International handbook on ecotourism: Edward Elgar Publishing; 2013. p. 66-77.##Hall CM. Biological invasion, biosecurity, tourism, and globalisation. In: Handbook of globalisation and tourism 2019 Dec 2 (pp. 114-125). Edward Elgar Publishing.##Arsanjani MH. Negotiating the UN declaration on human cloning. American Journal of International Law 2006 Jan;100(1):164-79.##Hall CM. Biosecurity, tourism and mobility: institutional arrangements for managing tourism-related biological invasions. Journal of Policy Research in Tourism, Leisure and Events 2011 Nov 1;3(3):256-80.##Sacramento O. Mass tourism, biosecurity and sustainability challenges: prospects illustrated by the current COVID-19 pandemic. Journal of Tourism Futures 2023 Mar 23.##Jappah JV, Smith DT. Global governmentality: Biosecurity in the era of infectious diseases. Glob Public Health 2015;10(10):1139-56.##Tumpey TM, Basler CF, Aguilar PV, Zeng H, Solórzano A, Swayne DE, et al. Characterization of the reconstructed 1918 Spanish influenza pandemic virus. Science 2005 Oct 7;310(5745):77-80. ##Gardner TS. Synthetic biology: from hype to impact. Trends Biotechnol 2013 Mar;31(3):123-5.##Kallergi A, Asin‐Garcia E, Martins dos Santos VA, Landeweerd L. Context matters: on the road to responsible biosafety technologies in synthetic biology. EMBO Rep 2021 Jan 7;22(1):e51227.##Trump BD, Keisler JM, Volk KM, Linkov I. Biosecurity demands resilience. ACS Publications; 2020.##

The Potential of Human Wharton’s Jelly Mesenchymal Stem Cells Secretome Based Topical Gel for Therapeutic Application 2 2 Background: Diabetic Foot Ulcer (DFU) might be worsened by neuropathy and vascular issues. This condition can cause 14.3% fatality, stressing the need for effective wound healing therapy. Wound healing is a complex biological process, and human Wharton's Jelly Mesenchymal Stem Cells (hWJMSCs) may help manage DFU treatment issues. This research focuses on utilizing a gel carrier to deliver bioactive substances from Wharton's Jelly Mesenchymal Stem Cells secretome (hWJ-MSCs-Sec) as a possible treatment for DFU. Methods: To maintain quality, hWJMSCs-Sec is thoroughly mixed with carbomer gel and freeze-dried. ELISA test is performed to determine the characterization of the gel of hWJMSCs-Sec such as Keratinocyte Growth Factor (KGF), Platelet-Derived Growth Factor (PDGF), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF), and Heparin-Binding EGF-Like Growth Factor (HB-EGF). The antioxidant activity was also measured with Hydrogen peroxide (H2O2), Nitric oxide (NO), and Ferric Reducing Antioxidant Power (FRAP) assay. Proliferation assay was utilized using WST-8 and the wound healing potential was assessed via the migration cell ability of scratched-human skin fibroblast (BJ cells). Results: The freeze-dried hWJ-MSCs-Sec showed higher levels of KGF, HGF, PDGF, EGF, HB-EGF, and the antioxidant activities compared to fresh hWJ-MSCs-Sec. Additionally, the gel of freeze-dried hWJ-MSCs-Sec exhibited higher levels compared to the gel of fresh hWJMSCs-Sec. This was evidenced by faster closure of scratched wounds on BJ cells treated with hWJMSCs-Sec and freeze-dried hWJ-MSCs-Sec gel. Conclusion: The freeze-dried hWJ-MSCs-Sec gel exhibits superior quality compared to the non-freeze-dried hWJ-MSCs-Sec gel. This demonstrates that the freeze-drying procedure can maintain the bioactive chemicals found in hWJMSCs-Sec, potentially enhancing the efficacy of this gel in promoting cell regeneration for wound healing. 233 243 Wahyu Widowati Ahmad Faried Department of Neurosurgery, Oncology & Stem Cell Working Group, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, IndonesiaDr. Hasan Sadikin Hospital Department of Neurosurgery, Oncology & Stem Cell Working Group, Faculty of Medicine, Universitas Padjadjaran, Bandung 40161, IndonesiaDr. Hasan Sadikin Hospital IndonesiaIndonesia Rimonta Febby Gunanegara Faculty of Medicine, Maranatha Christian University Faculty of Medicine, Maranatha Christian University Indonesia Fanny Rahardja Faculty of Medicine, Maranatha Christian University Faculty of Medicine, Maranatha Christian University Indonesia Fadhilah Haifa Zahiroh Biomolecular and Biomedical Research Center Bandung, Aretha Medika Utama Biomolecular and Biomedical Research Center Bandung, Aretha Medika Utama Indonesia Annisa Firdaus Sutendi Biomolecular and Biomedical Research Center Bandung, Aretha Medika Utama Biomolecular and Biomedical Research Center Bandung, Aretha Medika Utama Indonesia Rizal Azis Biomedical Engineering Department of Electrical Engineering, Faculty of Engineering, University of IndonesiaDepartment of Translational Medical Science, Division of Cancer and Stem Cell, Biodiscovery Institute 3, The University of Nottingham, University Park, United Kingdom NG72RD Biomedical Engineering Department of Electrical Engineering, Faculty of Engineering, University of IndonesiaDepartment of Translational Medical Science, Division of Cancer and Stem Cell, Biodiscovery Institute 3, The University of Nottingham, University Park, United Kingdom NG72RD IndonesiaUnited Kingdom NG72RD Renandy Kristianlie Ekajaya Biology Study Program, Faculty of Mathematics and Science Education, Universitas Pendidikan Indonesia Biology Study Program, Faculty of Mathematics and Science Education, Universitas Pendidikan Indonesia Indonesia AntioxidantsCarbomer gelFreeze dried secretome gelhWJ-MSCsWound healing 60590.pdf Miller TA, Campbell JH, Bloom N, Wurdeman SR. Racial disparities in health care with timing to amputation following diabetic foot ulcer. Diabetes Care 2022 Oct 1;45(10):2336-41. ##Kavitha KV, Tiwari S, Purandare VB, Khedkar S, Bhosale SS, Unnikrishnan AG. Choice of wound care in diabetic foot ulcer: A practical approach. World J Diabetes 2014 Aug 8;5(4):546. ##Morales-González M, Díaz LE, Dominguez-Paz C, Valero MF. Insights into the design of polyurethane dressings suitable for the stages of skin wound-healing: A systematic review. Polymers (Basel) 2022 Jul 24;14(15):2990. ##Serra MB, Barroso WA, Silva NN, Silva SD, Borges AC, Abreu IC, Borges MO. From inflammation to current and alternative therapies involved in wound healing. Int J Inflam 2017;2017(1):3406215. ##Zhao R, Liang H, Clarke E, Jackson C, Xue M. Inflammation in chronic wounds. Int J Mol Sci 2016 Dec 11;17(12):2085. ##Das K, Roychoudhury A. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. Frontiers in Environmental Science 2014 Dec 2;2:53.##Liu Z, Ren Z, Zhang J, Chuang CC, Kandaswamy E, Zhou T, Zuo L. Role of ROS and nutritional antioxidants in human diseases. Front Physiol 2018 May 17;9:360203. ##Dao DT, Anez-Bustillos L, Adam RM, Puder M, Bielenberg DR. Heparin-binding epidermal growth factor–like growth factor as a critical mediator of tissue repair and regeneration. Am J Pathol 2018 Nov 1;188(11):2446-56.##Jian K, Yang C, Li T, Wu X, Shen J, Wei J, et al. PDGF-BB-derived supramolecular hydrogel for promoting skin wound healing. J Nanobiotechnology 2022 Apr 26;20(1):201. ##Woodby B, Scott M, Bodily J. The interaction between human papillomaviruses and the stromal microenvironment. Prog Mol Biol Transl Sci 2016 Jan 1;144:169-238. ##Velardi E, Tsai JJ, van den Brink MR. T cell regeneration after immunological injury. Nat Rev Immunol 2021 May;21(5):277-91.##Park SR, Kim JW, Jun HS, Roh JY, Lee HY, Hong IS. Stem cell secretome and its effect on cellular mechanisms relevant to wound healing. Mol Ther 2018 Feb 7;26(2):606-17. ##Kamolz LP, Keck M, Kasper C. Wharton’s jelly mesenchymal stem cells promote wound healing and tissue regeneration. Stem Cell Res Ther 2014 Sep;5:1-2. ##Arifka M, Wilar G, Elamin KM, Wathoni N. Polymeric hydrogels as mesenchymal stem cell secretome delivery system in biomedical applications. Polymers (Basel) 2022 Mar 17;14(6):1218. ##Zhao G, Liu F, Lan S, Li P, Wang L, Kou J, et al. Large-scale expansion of Wharton’s jelly-derived mesenchymal stem cells on gelatin microbeads, with retention of self-renewal and multipotency characteristics and the capacity for enhancing skin wound healing. Stem Cell Res Ther 2015 Dec;6:1-6.##Firlar I, Altunbek M, McCarthy C, Ramalingam M, Camci-Unal G. Functional hydrogels for treatment of chronic wounds. Gels 2022 Feb 17;8(2):127. ##Ho AY, Wright JL, Blitzblau RC, Mutter RW, Duda DG, Norton L, et al. Optimizing radiation therapy to boost systemic immune responses in breast cancer: a critical review for breast radiation oncologists. Int J Radiat Oncol Biol Phys 2020 Sep 1;108(1):227-41. ##Molnar A, Lakat T, Hosszu A, Szebeni B, Balogh A, Orfi L, et al. Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques. Amino Acids 2021 Jun;53(6):917-28. ##Zhang Y, Ng W, Hu J, Mussa SS, Ge Y, Xu H. Formulation and in vitro stability evaluation of ethosomal carbomer hydrogel for transdermal vaccine delivery. Colloids Surf B Biointerfaces 2018 Mar 1;163:184-91. ##Widowati W, Widyastuti H, Murti H, Laksmitawati DR, Kusuma HS, Rizal R, et al. Interleukins and VEGF secretome of human Wharton’s jelly mesenchymal stem cells-conditioned medium (hWJMSCs-CM) in different passages and oxygen tensions. Biosci Res 2017 Oct 1;14(4):776-87.##Widowati W, Gunanegara RF, Rizal R, Widodo WS, Amalia A, Wibowo SH, Handono K, Marlina M, Lister IN, Chiuman L. Comparative analysis of Wharton’s Jelly mesenchymal stem cell (WJ-MSCs) isolated using explant and enzymatic methods. InJournal of Physics: Conference Series 2019 Nov 1 (Vol. 1374, No. 1, p. 012024). IOP Publishing.##Kusuma HS, Widowati W, Gunanegara RF, Juliandi B, Lister NE, Arumwardana S, et al. Effect of conditioned medium from IGF1-induced human Wharton’s jelly mesenchymal stem cells (IGF1-hWJMSCs-CM) on osteoarthritis. Avicenna J Med Biotechnol 2020 Jul;12(3):172.##Zukhiroh Z, Putra A, Chodidjah C, Sumarawati T, Subchan P, Trisnadi S, Hidayah N, et al. Effect of secretome-hypoxia mesenchymal stem cells on regulating SOD and MMP-1 mRNA expressions in skin hyperpigmentation rats. Open Access Macedonian Journal of Medical Sciences 2022 Dec 15;10(A):1-7.##Nowak D, Jakubczyk E. The freeze-drying of foods—The characteristic of the process course and the effect of its parameters on the physical properties of food materials. Foods 2020 Oct 18;9(10):1488. ##Widowati W, Widyastuti H, Murti H, Laksmitawati DR, Kusuma HS, Rizal R, et al. Interleukins and VEGF secretome of human Wharton’s jelly mesenchymal stem cells-conditioned medium (hWJMSCs-CM) in different passages and oxygen tensions. Biosci Res 2017 Oct 1;14(4):776-87.##Priyandoko D, Widowati W, Kusuma HS, Afifah E, Wijayanti CR, Wahyuni CD, et al. Antioxidant Activity of Green Tea Extract and Myricetin. In2021 IEEE International Conference on Health, Instrumentation & Measurement, and Natural Sciences (InHeNce) 2021 Jul 14 (pp. 1-5). IEEE.##Widowati W, Prahastuti S, Hidayat M, Hasianna ST, Wahyudianingsih R, Eltania TF, et al. Detam 1 black soybean against cisplatin-induced acute ren failure on rat model via antioxidant, antiinflammatory and antiapoptosis potential. J Tradit Complement Med 2022 Jul 1;12(4):426-35. ##Widowati W, Rani AP, Hamzah RA, Arumwardana S, Afifah E, Kusuma HS, et al. Antioxidant and antiaging assays of Hibiscus sabdariffa extract and its compounds. Natural Product Sciences 2017 Sep 1;23(3):192-200.##Girsang E, Lister IN, Ginting CN, Widowati W, Arumwardana S, Marthania M, et al. Chlorogenic acid in preventing and curing ultraviolet-induced damage in human skin fibroblast as an antiaging cell model. Pharmaciana 2023;13(2):159-65.##Widowati W, Priyandoko D, Lenny L, Revika R, Novianti S, Kusuma HS, et al. Camellia sinensis L. Extract Suppresses Inflammation on Acute Respiratory Distress Syndrome Cells Models via Decreasing IL-1ß, IL-6 and COX-2 Expressions. Trends in Sciences 2024;21(1):7010.##Kruse CR, Singh M, Targosinski S, Sinha I, Sørensen JA, Eriksson E, et al. The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization: In vitro and in vivo study. Wound Repair Regen 2017 Apr;25(2):260-9. ##Widowati W, Noverina R, Ayuningtyas W, Kurniawan D, Kusuma HS, Arumwardana S, et al. Proliferation, characterization and differentiation potency of adipose tissue-derived mesenchymal stem cells (AT-MSCs) cultured in fresh frozen and non-fresh frozen plasma. Int J Mol Cell Med 2019;8(4):283. ##Wallace EW, Kear-Scott JL, Pilipenko EV, Schwartz MH, Laskowski PR, Rojek AE, et al. Reversible, specific, active aggregates of endogenous proteins assemble upon heat stress. Cell 2015 Sep 10;162(6):1286-98. ##Merivaara A, Zini J, Koivunotko E, Valkonen S, Korhonen O, Fernandes FM, Yliperttula M. Preservation of biomaterials and cells by freeze-drying: Change of paradigm. J Control Release 2021 Aug 10;336:480-98. ##Kolimi P, Narala S, Nyavanandi D, Youssef AA, Dudhipala N. Innovative treatment strategies to accelerate wound healing: trajectory and recent advancements. Cells 2022 Aug 6;11(15):2439. ##Tarnawski AS, Ahluwalia A. The critical role of growth factors in gastric ulcer healing: the cellular and molecular mechanisms and potential clinical implications. Cells 2021 Aug 2;10(8):1964. ##Rezvanian M, Ng SF, Alavi T, Ahmad W. In-vivo evaluation of Alginate-Pectin hydrogel film loaded with Simvastatin for diabetic wound healing in Streptozotocin-induced diabetic rats. Int J Biol Macromol 2021 Feb 28;171:308-19. ##Ruttanapattanakul J, Wikan N, Okonogi S, Takuathung MN, Buacheen P, Pitchakarn P, et al. Boesenbergia rotunda extract accelerates human keratinocyte proliferation through activating ERK1/2 and PI3K/Akt kinases. Biomed Pharmacother 2021 Jan 1;133:111002. ##Gallo S, Sala V, Gatti S, Crepaldi T. Cellular and molecular mechanisms of HGF/Met in the cardiovascular system. Clin Sci 2015 Dec 1;129(12):1173-93. ##Shi R, Lian W, Jin Y, Cao C, Han S, Yang X, et al. Role and effect of vein-transplanted human umbilical cord mesenchymal stem cells in the repair of diabetic foot ulcers in rats. Acta Biochimica et Biophysica Sinica 2020 Jun;52(6):620-30. ##Ahangar P, Mills SJ, Cowin AJ. Mesenchymal stem cell secretome as an emerging cell-free alternative for improving wound repair. Int J Mol Sci 2020 Sep 24;21(19):7038. ##Martino MM, Briquez PS, Maruyama K, Hubbell JA. Extracellular matrix-inspired growth factor delivery systems for bone regeneration. Adv Drug Deliv Rev 2015 Nov 1;94:41-52. ##Stunova A, Vistejnova L. Dermal fibroblasts—A heterogeneous population with regulatory function in wound healing. Cytokine Growth Factor Rev 2018 Feb 1;39:137-50. ##Kuo YR, Wang CT, Cheng JT, Kao GS, Chiang YC, Wang CJ. Adipose-derived stem cells accelerate diabetic wound healing through the induction of autocrine and paracrine effects. Cell Transplantation 2016 Jan;25(1):71-81.##White MJ, Briquez PS, White DA, Hubbell JA. VEGF-A, PDGF-BB and HB-EGF engineered for promiscuous super affinity to the extracellular matrix improve wound healing in a model of type 1 diabetes. NPJ Regen Med 2021 Nov 18;6(1):76. ##Zhu G, Wang Q, Lu S, Niu Y. Hydrogen peroxide: a potential wound therapeutic target. Medical Principles and Practice 2017 Apr 5;26(4):301-8.##Malone‐Povolny MJ, Maloney SE, Schoenfisch MH. Nitric oxide therapy for diabetic wound healing. Advanced Healthcare Materials 2019 Jun;8(12):1801210.##Fu X, Liu G, Halim A, Ju Y, Luo Q, Song G. Mesenchymal stem cell migration and tissue repair. Cells 2019 Jul 28;8(8):784. ##Kardas G, Daszyńska-Kardas A, Marynowski M, Brząkalska O, Kuna P, Panek M. Role of platelet-derived growth factor (PDGF) in asthma as an immunoregulatory factor mediating airway remodeling and possible pharmacological target. Frontiers in Pharmacology 2020 Feb 14;11:47.##Augustine R, Hasan A, Dalvi YB, Rehman SR, Varghese R, Unni RN, et al. Growth factor loaded in situ photocrosslinkable poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/gelatin methacryloyl hybrid patch for diabetic wound healing. Materials Science and Engineering: C 2021 Jan 1;118:111519.##Oyinloye TM, Yoon WB. Effect of freeze-drying on quality and grinding process of food produce: A review. Processes 2020 Mar 20;8(3):354.##

5-Fluorouracil Effectively Depletes Tumor Induced Myeloid Derived Suppressor Cells in 4T1 Mammary Carcinoma Model 2 2 Background: Myeloid Derived Suppressor Cells (MDSCs) are capable of inhibiting both innate and adaptive immune responses and accumulate in the microenvironment of breast tumors. Hence, MDSC depletion by chemotherapeutic agents can improve clinical efficacy of cancer immunotherapy. The effects of 5-FU and doxorubicin agents on MDSC reduction in 4T1 breast cancer murine model were evaluated. Methods: 5×105 of 4T1 tumor cells were injected into mammary fat pad of BALB/c female mice. Tumor bearing mice were randomly divided into 4 groups: PBS receiving control group, doxorubicin receiving groups at doses of 2.5 and 5 mg/kg, and 5-FU receiving group at dose of 50 mg/kg. Doxorubicin and 5-FU agents were intraperitoneally administrated at three doses with 5-day intervals and five doses for three times a week, respectively. Then, on day 20 post tumor cells injection, spleens and tumors were isolated to determine frequency of CD11b+ Gr1+ MDSCs by flow cytometry analysis. Results: 5-FU was able to reduce significantly both splenic and interatumoral MDSCs comparing to control group (p=0.0276 and p=0.0067, respectively). Also, Doxorubicin treatment at dose of 50 mg/kg was associated to a significant reduction of splenic MDSCs in comparison to untreated group (p=0.0382). However, only 5-FU injection led to inhibit notably tumor growth in comparison to control group (p=0.0139). Conclusion: Findings show that 5-FU has inhibitory effects on MDSCs and tumor growth in 4T1 tumor model. So, more investigations are needed to study combination of 5-FU with immune based approaches to enhance the efficacy of cancer therapies. 244 250 Khadijeh Ramezani-Aliakbari Department of Immunology, School of Medicine, Shahid Beheshti University of Medical SciencesDepartment of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University Department of Immunology, School of Medicine, Shahid Beheshti University of Medical SciencesDepartment of Pathobiology, Faculty of Veterinary Science, Bu-Ali Sina University IranIran Seyed Amir Jalali Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Maedeh Alinejad Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Mahmood Jeddi-Tehrani Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mahdi Shabani Breast cancerFluorouracilImmunotherapyMammary neoplasmTumor microenvironmentTreatment outcome 60591.pdf Damuzzo V, Pinton L, Desantis G, Solito S, Marigo I, Bronte V. Complexity and challenges in defining myeloid-derived suppressor cells. Cytom B ClinCytom 2015; 88(2):77–91.##Kumar V, Patel S, Tcyganov E & Gabrilovich DI. The nature of myeloid- derived suppressor cells in the tumor microenvironment. Trends Immunol 2016;37(3):208–20.##Youn JI, Nagaraj S, Collazo M, Gabrilovich DI. Subsets of myeloid derived suppressor cells in tumor-bearing mice. J Immunol 2008;181(8):5791–802.##Peranzoni E, Zilio S, Marigo I, Dolcetti L, Zanovello P, Mandruzzato S, et al. Myeloid-derived suppressor cell heterogeneity and subset definition. Curr Opin Immunol 2010;22:238–44. ##Ostrand-Rosenberg S. Myeloid-derived suppressor cells: more mechanisms for inhibiting antitumor immunity. Cancer Immunol Immunother 2010;59(10):1593–600.##Wang Z, Zhang Y, Liu Y, Wang L, Zhao L, Yang T, et al. Association of myeloid-derived suppressor cells and efficacy of cytokine-induced killer cell immunotherapy in metastatic renal cell carcinoma patients. J Immunother 2014;37(1):43–50.##Schneider T, Sevko A, Heussel CP, Umansky L BP, Dienemann H, Safi S, et al. Serum inflammatory factors and circulating immunosuppressive cells are predictive markers for efficacy of radiofrequency ablation in nonsmall-cell lung cancer. Clin Exp Immunol 2015;180(3):467–74. ##Tang H, Li H, Sun Z. Targeting myeloid-derived suppressor cells for cancer therapy The role of MDSCs in the TME Classification of MDSCs. Cancer Biol Med 2021; 18(4):992–1009. ##Qin H, Lerman B, Sakamaki I, Wei G, Cha SC, Rao SS, et al. Generation of a new therapeutic peptide that depletes myeloid-derived suppressor cells in tumor-bearing mice. Nat Med 2014;20(6):676–81.##Draghiciu O, Lubbers J, Nijman HW, Daemen T. Myeloid derived suppressor cells-An overview of combat strategies to increase immunotherapy efficacy. Oncoimmunology 2015;4(1):e954829.##Ko HJ, Kim YJ, Kim YS, Chang WS, Ko SY, Chang SY, et al. A combination of chemoimmunotherapies can efficiently break self-tolerance and induce antitumor immunity in a tolerogenic murine tumor model. Cancer Res 2007;67(15):7477–86. ##Ding ZC, Lu X, Yu M, Lemos H, Huang L, Chandler P, et al. Immunosuppressive myeloid cells induced by chemotherapy attenuate antitumor CD4C T-cell responses through the PD-1-PD-L1 axis. Cancer Res 2014;74(13):3441–53. ##Alizadeh D, Trad M, Hanke NT, Larmonier CB, Janikashvili N, Bonnotte B, et al. Doxorubicin eliminates myeloid-derived suppressor cells and enhances the efficacy of adaptive T-cell transfer in breast cancer. Cancer Res 2014;74(1):104–18.##Vincent J, Mignot G, Chalmin F, Ladoire S, Bruchard M, Chevriaux A, et al. 5-Fluorouracil selectively kills tumor-associated myeloid-derived suppressor cells resulting in enhanced T cell-dependent antitumor immunity. Cancer Res 2010;70(8):3052–61.##Bosiljcic M. Myeloid-derived suppressor cell accumulation in secondary target organs promotes metastatic growth in breast cancer. University of British Columbia; 2014. ##Dijkgraaf EM, Santegoets SJ, Reyners AK, Goedemans R, Nijman HW, van Poelgeest MI, et al. A phase 1/2 study combining gemcitabine, Pegintron and p53 SLP vaccine in patients with platinum-resistant ovarian cancer. Oncotarget 2015;6(31):32228–43.##Liu Y, Wei G, Cheng WA, Dong Z, Sun H, Lee VY, et al. Targeting myeloid-derived suppressor cells for cancer immunotherapy. Cancer Immunol Immunother 2018; 67(8):1181–95.##Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and cancer. J Immunol 2009;182(8):4499–506. ##Limagne E, Euvrard R, Thibaudin M, Rébé C, Derangère V, Chevriaux A, et al. Accumulation of MDSC and Th17 cells in patients with metastatic colorectal cancer predicts the efficacy of a FOLFOX-bevacizumab drug treatment regimen. Cancer Res 2016;76(18):5241–52.##Almand B, Clark JI, Nikitina E, van Beynen J, English NR, Knight SC, et al. Increased production of immature myeloid cells in cancer patients: a mechanism of immunosuppression in cancer. J Immunol 2001;166(1):678–89. ##De Cicco P, Ercolano G, Ianaro A. The New Era of Cancer Immunotherapy: Targeting Myeloid-Derived Suppressor Cells to Overcome Immune Evasion. Front Immunol 2020;11:1680.##Srivastava MK, Zhu L, Harris-White M, Kar U, Huang M, Johnson MF, et al. Myeloid suppressor cell depletion augments antitumor activity in lung cancer. PLoS One 2012;7(7):e40677.##Hurez V, Daniel BJ, Sun L, Liu AJ, Ludwig SM, Kious MJ, et al. Mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice. Cancer Res 2012;72(8):2089–99.##Ramezani-Aliakbari K, Khaki-Bakhtiarvand V, Mahmoudian J, Asgarian-Omran H, Shokri F, Hojjat-Farsangi M, et al. Evaluation of the anti-tumor effects of an anti-Human Epidermal growth factor receptor 2 (HER2) monoclonal antibody in combination with CD11b+/Gr-1+ myeloid cells depletion using a recombinant peptibody in 4 T1-HER2 tumor model. Int Immunopharmacol 2023;121:110463.##Zitvogel L, Apetoh L, Ghiringhelli F, Kroemer G. Immunological aspects of cancer chemotherapy. Nat Rev Immunol 2008;8(1):59–73.##Ghiringhelli F, Puig PE, Roux S, Parcellier A, Schmitt E, Solary E, et al. Tumor cells convert immaturemyeloid dendritic cells into TGF-β-secreting cells inducing CD4+CD25+ regulatory T cell proliferation. J Exp Med 2005;202(7):919–29.##Shurin GV, Tourkova IL, Kaneno R, Shurin MR. Chemotherapeutic agents in noncytotoxic concentrations increase antigen presentation by dendritic cells via an IL-12-dependent mechanism. J Immunol 2009;183(1):137–44.##Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux S, Martin F, et al. Metronomicm cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother 2007;56(5):641–8.##Bailly C, Thuru X, Quesnel B. Combined cytotoxic chemotherapy and immunotherapy of cancer: modern times. NAR Cancer. 2020 Feb 17;2(1):zcaa002.##Longley DB, Harkin DP, Johnston PG. 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer 2003;3(5):330–8.##Gmeiner WH. Fluoropyrimidine modulation of the antitumor immune response-prospects for improved colorectal cancer treatment. Cancers (Basel) 2020;12(6):1641–59.##Apetoh L, Vegran F, Ladoire S, Ghiringhelli F. Restoration of antitumor immunity through selective inhibition of myeloid derived suppressor cells by anticancer therapies. Curr Mol Med 2011;11(5):365–72.##Namdar A, Mirzaei HR, Jadidi-Niaragh F, Ashourpour M, Ajami M, Hadjati J, et al. Multiple low doses of 5-fluorouracil diminishes immunosuppression by myeloid derived suppressor cells in murine melanoma model. Iran J Immunol 2015;12(3):176–87.##Khosravianfar N, Hadjati J, Namdar A, Boghozian R, Hafezi M. Myeloid-derived Suppressor Cells Elimination by 5-Fluorouracil Increased Dendritic Cell-based Vaccine Function and Improved Immunity in Tumor Mice. Iran J Immunol 2018;17(1):47–55. ##Balog J, Hackler L, Kovács AK, Neuperger P, Alföldi R, Nagy LI, et al. Single cell mass cytometry revealed the immunomodulatory effect of cisplatin via downregulation of splenic cd44+, il-17a+ mdscs and promotion of circulating ifn-γ+ myeloid cells in the 4t1 metastatic breast cancer model. Int J Mol Sci 2019;21(1):170.##Le HK, Graham L, Cha E, Morales JK, Manjili MH, Bear HD. Gemcitabine directly inhibits myeloid derived suppressor cells in BALB/c mice bearing 4T1 mammary carcinoma and augments expansion of T cells from tumor-bearing mice. Int Immunopharmacol 2009; 9(7–8):900–9.##Geng F, Bao X, Dong L, Guo Q, Guo J, Xie Y, et al. Doxorubicin pretreatment enhances FAP α / survivin co-targeting DNA vaccine anti-tumor activity primarily through decreasing peripheral MDSCs in the 4T1 murine breast cancer model. Oncoimmunology 2020;9(1):1747350.##Fang H, Fen W, Zong N, Liu C, Wu L, Qian Z, et al. Histone deacetylase inhibitors deplete myeloid ‑ derived suppressor cells induced by 4T1 mammary tumors in vivo and in vitro. Cancer Immunol Immunother 2017;66(3):355–66.##Fang H, Ang B, Xu X, Huang X, Wu Y, SunY, et al. TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells. Cell Mol Immunol 2014; 11:150–9.##Legrand AJ, Konstantinou M, Goode EF, Meier P. The diversification of cell death and immunity: Memento Mori. Mol Cell 2019;76(2):232–42. ##Otsubo D, Yamashita K, Fujita M, Nishi M, Kimura Y, Hasegawa H, et al. Early-phase treatment by low-dose 5-fluorouracil or primary tumor resection inhibits MDSC-mediated lung metastasis formation. Anticancer Res 2015;35(8):4425–31.##Tao K, Fang M, Alroy J, Sahagian GG. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer 2008;8:1–20.##DuPre’ SA, Hunter Jr KW. Murine mammary carcinoma 4T1 induces a leukemoid reaction with splenomegaly: Association with tumor-derived growth factors. Exp Mol Pathol 2007;82(1):12–24.##

Immunogenic Consideration of a Designed Polypeptide Against Brucellosis Compared to RB51: An In Vivo Study 2 2 Background: Brucellosis in livestock and its transmission to humans through the consumption of contaminated dairy products is an important issue. The introduction of new approaches using immunogenic proteins against and diagnosing brucellosis is a serious issue in human health. Methods: Brucella abortus contains five proteins including: MOXR family ATPase-α2, T9SS C-terminal target domain-containing protein, Cobyric acid synthase, Hypothetical protein, and VirB11 type IV Secretion protein, which were considered and the designed recombinant polypeptide was produced and evaluated. The pure recombinant protein ABOR with 549aa in combination with chitin as an adjuvant was injected subcutaneously into guinea pigs to evaluate their immunity responses. Results: The results indicated that the ABOR recombinant protein induced Th1 immunity with high levels of specific IgG (IgG2a) as well as Interferon-γ (IFN-γ), Interleukin-2 (IL-2), IL-12, and Tumor Necrosis Factor-alpha (TNF-α), compared to the control group. Th1/Th2 ratio analysis demonstrated the efficacy of ABOR protein combined with chitin in stimulating cellular immunity in the animals. Conclusion: Designed recombinant polypeptide combined with chitin showed ability for induction of cellular and humoral immunity an guinea pigs compared to RB51 vaccine. 251 259 Mina Saadat Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences Iran Mojgan Bandehpour Department of Embryology and Andrology, Avicenna Research Institute, ACECR Iran Bahram Kazemi Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences Iran Nariman Mosaffa Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences Iran Brucella abortusGuinea pigsImmunityRecombinant proteins 60592.pdf Suárez-Esquivel M, Chaves-Olarte E, Moreno E, Guzmán-Verri C. Brucella Genomics: Macro and Micro Evolution. Int J Mol Sci 2020;21(20):7749. ##Baldwin CL, Goenka R. Host immune responses to the intracellular bacteria Brucella: does the bacteria instruct the host to facilitate chronic infection? Crit Rev Immunol 2006;26(5):407-42.##Skendros P, Pappas G, Boura P. Cell-mediated immunity in human brucellosis. Microbes Infect 2011;13(2):134-42.##Saadat M, Gandomkar M, Bandehpour M, Bandehpour M, Kazemi B. Immunomics Approach to Develop an Immunogenic Polypeptide from Brucella abortus: Design and Recombinant Expression: Develop an immunogenic polypeptide from Brucella abortus. Trends in Peptide and Protein Sciences.;8(1):1-7.##Delpino MV, Estein SM, Fossati CA, Baldi PC, Cassataro J. Vaccination with Brucella recombinant DnaK and SurA proteins induces protection against Brucella abortus infection in BALB/c mice. Vaccine 2007;25(37-38):6721-9. ##Byndloss MX, Tsolis RM. Brucella spp. virulence factors and immunity. Ann Rev Anim Biosci 2016;4:111-27. ##Zhan Y, Liu Z, Cheers C. Tumor necrosis factor alpha and interleukin-12 contribute to resistance to the intracellular bacterium Brucella abortus by different mechanisms. Infec Immun 1996;64(7):2782-6. ##Azizi M, Yousefi R, Yeganeh F, Hoseini MH. Study of the immune adjuvant effect of chitin microparticles on plasma levels of IgG1 and IgG2a during immunization against Leishmania infection in BALB/c mice. J Res Med Sci 2019;43(2):64-70.##Goodwin ZI, Pascual DW. Brucellosis vaccines for livestock, Vet Immunol Immunopathol 2016;181:51-8. ##Lalsiamthara J, Lee JH. Development and trial of vaccines against Brucella. J Vet Sci 2017;18(S1):281-90. ##Hill AV. Vaccines against malaria. Philos Trans R Soc Lond B Biol Sci 2011;366(1579):2806-14.##Watson PS, Novy PL, Friedland LR. Potential benefits of using a multicomponent vaccine for prevention of serogroup B meningococcal disease. Inter J Infect Dis 2019;85:22-7. ##Sarmadi M, Gheibi A, Khanahmad H, Khorramizadeh MR, Hejazi SH, Zahedi N, et al. Design and Characterization of a Recombinant Brucella abortus RB51 Vaccine That Elicits Enhanced T Cell-Mediated Immune Response. Vaccines (Basel) 2022;10(3):388. ##Kaushik P, Singh DK, Kumar SV, Tiwari AK, Shukla G, Dayal S, et al. Protection of mice against Brucella abortus 544 challenge by vaccination with recombinant OMP28 adjuvanted with CpG oligonucleotides. Vet Res Commun 2010;34(2):119-32.##Golshani M, Buozari S. A review of brucellosis in Iran: epidemiology, risk factors, diagnosis, control, and prevention. Iran Biomed J 2017;21(6):349-59. ##Lalsiamthara J, Lee JH. Development and trial of vaccines against Brucella. J Vet Sci 2017; 18(S1):281-90. ##Bugybayeva D, Kydyrbayev Z, Zinina N, Assanzhanova N, Yespembetov B, Kozhamkulov Y, et al. A new candidate vaccine for human brucellosis based on influenza viral vectors: a preliminary investigation for the development of an immunization schedule in a guinea pig model. Infect Dis Poverty 2021;10(1):13.##Yin D, Li L, Song D, Liu Y, Ju W, Song X, et al. A novel recombinant multi-epitope protein against Brucella melitensis infection. Immunol Lett 2016;175:1-7. ##Del Giudice G, Rappuoli R, Didierlaurent AM. Correlates of adjuvanticity: a review on adjuvants in licensed vaccines. Semin Immunol 2018;39:14-21. ##Da Silva CA, Hartl D, Liu W, Lee CG, Elias JA. TLR-2 and IL-17A in chitin-induced macrophage activation and acute inflammation. J Immunol 2008;181(6):4279-86. ##Reese TA, Liang HE, Tager AM, Luster AD, Van Rooijen N, Voehringer D, et al. Chitin induces accumulation in tissue of innate immune cells associated with allergy. Nature 2007; 447(7140):92-6.##Da Silva CA, Pochard P, Lee CG, Elias JA. Chitin particles are multifaceted immune adjuvants. Am J Respir Crit Care Med 2010;182(12):1482-91. ##Demirdag K, Ozden M, Kalkan A, Godekmerdan A, Kilic SS. Serum cytokine levels in patients with acute brucellosis and their relation to the traditional inflammatory markers. FEMS Immunol Med Microbiol 2003;39(2):149-53. ##Kidd P. Th1/Th2 Balance: The Hypothesis, its Limitations, and Implications for Health and Disease. Altern Med Rev 2003;8(9):223-46. ##Avila-Calderón ED, Lopez-Merino A, Sriranganathan N, Boyle SM, A Contreras-Rodríguez. A history of the development of Brucella vaccines. BioMed Res Int 2013;2013:743509. ##He Y, Vemulapalli R, Zeytun A, Schurig GG. Induction of specific cytotoxic lymphocytes in mice vaccinated with Brucella abortus RB51. Infect Immun 2001;69(9):5502-8. ##Fu S, Xu J, Li X, Xie Y, Qiu Y, Du X, et al. Immunization of mice with recombinant protein CobB or AsnC confers protection against Brucella abortus infection. PloS One 2012;7(2):e29552. ##Eze MO, Yuan L, Crawford RM, Paranavitana CM, Hadfield TL, Bhattacharjee AK, et al. Effects of opsonization and gamma interferon on growth of Brucella melitensis 16M in mouse peritoneal macrophages in vitro. Infect Immun 2000;68(1):257-63.##Sieira R, Comerci DJ, Sánchez DO, Ugalde RA. A homologue of an operon required for DNA transfer in Agrobacterium is required in Brucella abortus for virulence and intracellular multiplication. J Bacteriol 2000;182(17):4849-55. ##Dorneles EM, Teixeira-Carvalho A, Araújo MS, Sriranganathan N, Lage AP. Immune response triggered by Brucella abortus following infection or vaccination. Vaccine 2015;33(31):3659-66. ##

The Effect of Simulated Physiological Oocyte Maturation (SPOM) and L-Carnitine on Bovine Oocyte Developmental Competence 2 2 Background: Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation in the presence of cAMP modulators. These modulators increase the intracellular concentrations of cAMP, which inhibits the immediate resumption of meiosis and gives the oocyte more time to gain optimal developmental competence. In addition, L-carnitine helps to increase the energy supply of cells through the β-oxidation of fatty acids. This study aimed to investigate the effect of SPOM and L-carnitine supplementation during In Vitro Maturation (IVM) and In Vitro Culture (IVC) on the developmental competence of bovine oocytes. Methods: Ovarian Cumulus Complexes (COCs) were cultured in the presence or absence of forskolin+IBMX during the first 2 hr of IVM (pre-IVM) with or without L-carnitine (LC) during IVM or IVC in six experimental groups as follows: I) pre-IVM (pre-IVM group), II) pre-IVM with L-carnitine supplementation during IVM (pre-IVM/LC group), III) L-carnitine supplementation during IVM (IVM/LC group), IV) L-carnitine supplementation during in vitro culture (IVC/LC group), V) pre-IVM+ IVC/LC group, and VI) no treatment during IVM and IVC (Control group). The cleavage and blastocyst rates, the blastocysts’ total cells number, and the expression of Nanog, Bax, Oct4, Cdx2, and Ifnt genes in resulting blastocysts were assessed. To assess differences among experimental groups, a one-way analysis of variance was initially employed, followed by post hoc Fisher LSD. The difference between groups was considered statistically significant when p<0.05. Results: The cleavage and blastocyst rates in the Pre-IVM and Pre-IVM/LC groups was higher than control group and other groups (p≤0.05) except for IVC/LC and IVM/LC groups, respectively. The number of blastocyst’s Inner Cell Mass (ICM) in pre-IVM and Pre-IVM/LC groups as well as the ratio of ICM/TE were higher than control group (p<0.05). The expression of OCT4, CDX2, and IFNT increased in both the pre-IVM and pre-IVM/LC groups compared to the control group (p<0.05). Conclusion: In conclusion, the application of SPOM-adapted IVM and L-carnitine during IVM of bovine oocyte improves the quantity and quality of the resulting embryos. 260 267 Ali Malekpour Department of Embryology and Andrology, Avicenna Research Institute, ACECRSina Fanavaran Mandegar Company, Alborz Science and Technology Park Sina Fanavaran Mandegar Company, Alborz Science and Technology Park IranIran Abolfazl Shirazi Sina Fanavaran Mandegar Company, Alborz Science and Technology Park Iran Sara Borjian Boroujeni Department of Embryology and Andrology, Avicenna Research Institute, ACECRSina Fanavaran Mandegar Company, Alborz Science and Technology Park IranIran Ali Sarvari Department of Embryology and Andrology, Avicenna Research Institute, ACECRSina Fanavaran Mandegar Company, Alborz Science and Technology Park IranIran Mohammad Mehdi Naderi Department of Embryology and Andrology, Avicenna Research Institute, ACECRSina Fanavaran Mandegar Company, Alborz Science and Technology Park IranIran Mostafa Pournourali Department of Embryology and Andrology, Avicenna Research Institute, ACECRSina Fanavaran Mandegar Company, Alborz Science and Technology Park Sina Fanavaran Mandegar Company, Alborz Science and Technology Park IranIran Bahareh Behzadi Department of Embryology and Andrology, Avicenna Research Institute, ACECR Iran Mohammad Mehdi Mehrazar Department of Embryology and Andrology, Avicenna Research Institute, ACECR Iran 3-Isobutyl-l-methylxanthinebcl-2-associated X proteinBlastocystCarnitineCattleMeiosisOocytesPre-IVM 60593.pdf Pournourali M, Tarang A, Mashayekhi F. Chromosomal analysis of two buffalo breeds of Mazani and Azeri from Iran. Iranian Journal of Veterinary Science and Technology 2015;7(1):22-31.##Shirazi A, Naderi MM, Hassanpour H, Heidari M, Borjian S, Sarvari A, Akhondi MM. The effect of ovine oocyte vitrification on expression of subset of genes involved in epigenetic modifications during oocyte maturation and early embryo development. Theriogenology 2016;86(9):2136-46. ##Amini MS, Naderi MM, Shirazi A, Aminafshar M, Boroujeni SB, Pournourali M, et al. Bioactive Materials Derived from Menstrual Blood Stem Cells Enhance the Quality of In Vitro Bovine Embryos. Avicenna J Med Biotechnol 2022;14(4):287-93.##Naranjo-Gómez JS, Uribe-García HF, Herrera-Sánchez MP, Lozano-Villegas KJ, Rodríguez-Hernández R, Rondón-Barragán IS. Heat stress on cattle embryo: gene regulation and adaptation. Heliyon 2021;7(3):e06570. ##Nejat-Dehkordi S, Ahmadi E, Shirazi A, Nazari H, Shams-Esfandabadi N. Embryo co-culture with bovine amniotic membrane stem cells can enhance the cryo-survival of IVF-derived bovine blastocysts comparable with co-culture with bovine oviduct epithelial cells. Zygote 2021;29(2):102-7. ##Nazari H, Esfandabadi N, Shirazi A, Ahadi AM, Ahmadi E, Safdarinejad A. Effect of Oocyte Maturation Period on Developmental Rate and Sex ratio Distribution of in vitro Produced Bovine Embryos. Iranian Journal of Applied Animal Science 2022; 12(2):249-53.##Saleh AC, Sabry R, Mastromonaco GF, Favetta LA. BPA and BPS affect the expression of anti-Mullerian hormone (AMH) and its receptor during bovine oocyte maturation and early embryo development. Reprod Biol Endocrinol 2021;19(1):119. ##Turhan A, Pereira MT, Schuler G, Bleul U, Kowalewski MP. Hypoxia-inducible factor (HIF1alpha) inhibition modulates cumulus cell function and affects bovine oocyte maturation in vitro. Biol Reprod 2021;104(2):479-91. ##Abdulkarim A, Balboula A, Badr M, Bedir W, Elmetwally M, Zaabel S. Comparing in vitro maturation rates in buffalo and cattle oocytes and evaluating the effect of cAMP modulators on maturation and subsequent developmental competence. Mansoura Veterinary Medical Journal 2021; 22(3):136-40.##Roelen BA. Bovine oocyte maturation: acquisition of developmental competence. Reprod Fertil Dev 2020;32(2):98-103. ##Hao T, Xu X, Hao H, Du W, Pang Y, Zhao S, et al. Melatonin improves the maturation and developmental ability of bovine oocytes by up-regulating GJA4 to enhance gap junction intercellular communication. Reprod Fertil Dev 2021;33(14):760-71.##Razza EM, Pedersen HS, Stroebech L, Fontes PK, Kadarmideen HN, Callesen H, et al. Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos. J Assist Reprod Genet 2019;36(3):413-24. ##Yu BY, Subudeng G, Du CG, Liu ZH, Zhao YF, Namei E, Bai Y, Yang BX, Li HJ. Plasminogen activator, tissue type regulates germinal vesicle breakdown and cumulus expansion of bovine cumulus–oocyte complex in vitro. Biol Reprod 2019;100(6):1473-81. ##Carolan, C, Lonergan P, Monget P, Monniaux D, Mermillod P. Effect of follicle size and quality on the ability of follicular fluid to support cytoplasmic maturation of bovine oocytes. Mol Reprod Dev 1996;43(4):477-83. ##Albuz FK, Sasseville M, Lane M, Armstrong DT, Thompson JG, Gilchrist RB. Simulated physiological oocyte maturation (SPOM): a novel in vitro maturation system that substantially improves embryo yield and pregnancy outcomes. Hum Reprod 2010;25(12): 2999–3011.##Raza SH, Abd El‐Aziz AH, Abdelnour SA, Easa AA, Alagawany M, Farag MR, et al. The role of forskolin as a lipolytic stimulator during in vitro oocyte maturation and the in vitro embryo production of livestock. Reprod Domest Anim 2021;56(12):1486-96. ##Thongkittidilok C, Doriguzzi N, Nagashima J, Brown M, Chansaenroj A, Songsasen N. Cilostamide and forskolin maintain gap junction function of incubated dog follicles. Theriogenology 2020; 142:222-8. ##Oliveira CS, da Silva Feuchard VL, de Souza Marques SC, Saraiva NZ. Modulation of lipid metabolism through multiple pathways during oocyte maturation and embryo culture in bovine. Zygote 2022;30(2):258-66. ##Zhenwei J, Xianhua Z. Pre-IVM treatment with C-type natriuretic peptide in the presence of cysteamine enhances bovine oocytes antioxidant defense ability and developmental competence in vitro. Iran J Vet Res 2019;20(3):173-179. ##Jasim SA, Ali SA, Fadhil OQ, Rakhmatova MK, Kzar HH, Margiana R, et al. Investigating the Effects of Hydro-alcoholic Urtica Dioica Extract and Retinoic Acid on Follicular Development: An Animal Study. Med J Islam Repub Iran 2023;37:1. ##Jiang WJ, Yao XR, Zhao YH, Gao QS, Jin QG, Li YH, XU YN. L-carnitine prevents bovine oocyte aging and promotes subsequent embryonic development. J Reprod Dev 2019;65(6):499-506. ##Jiang W, Li Y, Zhao Y, Gao Q, Jin Q, Yan C, et al. L-carnitine supplementation during in vitro culture regulates oxidative stress in embryos from bovine aged oocytes. Theriogenology 2020;143:64-73. ##Catandi GD, Cheng MH, Chicco AJ, Chen T, Carnevale EM. L-carnitine enhances developmental potential of bovine oocytes matured under high lipid concentrations in vitro. Anim Reprod Sci 2023;252:107249. ##Carrillo-González DF, Hernández-Herrera DY, Maldonado-Estrada JG. The role of L-carnitine in bovine embryo metabolism. A review of the effect of supplementation with a metabolic modulator on in vitro embryo production. Anim Biotechnol 2023;34(2):413-23.##Chankitisakul V, Somfai T, Inaba Y, Techakumphu M, Nagai T. Supplementation of maturation medium with L-carnitine improves cryo-tolerance of bovine in vitro matured oocytes. Theriogenology 2013;79(4):590-8. ##Demyda-Peyrás S, Dorado J, Hidalgo M, Anter J, De Luca L, Genero E, et al. Effects of oocyte quality, incubation time and maturation environment on the number of chromosomal abnormalities in IVF-derived early bovine embryos. Reprod Fertil Dev 2013;25(7):1077-84.##Leal GR, Graciosa MAG, Monteiro CAS, Pasolini R, Dos Reis Camargo AJ, Oliveira CS, et al. The SPOM-adapted IVM system improves in vitro production of bovine embryos. Theriogenology 2020 Dec;158:277-82. ##Li HJ, Sutton-McDowall ML, Wang X, Sugimura S, Thompson JG, Gilchrist RB. Extending prematuration with cAMP modulators enhances the cumulus contribution to oocyte antioxidant defense and oocyte quality via gap junctions. Hum Reprod 2016;31(4):810-21. ##Razza EM, Pedersen HS, Stroebech L, Fontes PK, Kadarmideen HN, Callesen H, et al. Simulated physiological oocyte maturation has side effects on bovine oocytes and embryos. J Assist Reprod Genet 2019;36(3):413-24. ##Zhao B, Li H, Zhang H, Ren S, Li Y, Wang X, et al. The effect of L-carnitine supplementation during in vitro maturation on oocyte maturation and somatic cloned embryo development. Reprod Biol 2024;24(2):100853. ##Phongnimitr T, Liang Y, Srirattana K, Panyawai K, Sripunya N, Treetampinich C, et al. Effect of L-carnitine on maturation, cryo-tolerance and embryo developmental competence of bovine oocytes. Anim Sci J 2013;84(11):719-25. ##Carrillo-González DF, Rodríguez-Osorio N, Long CR, Vásquez-Araque NA, Maldonado-Estrada JG. L-carnitine supplementation during in vitro maturation and in vitro culture does not affect the survival rates after vitrification and warming but alters Inf-T and ptgs2 gene expression. Int J Mol Sci 2020 Aug 5;21(16):5601. ##Ghanem N. L-carnitine improved bovine blastocyst rate and quality when supplemented at different preimplantation stages. Egyptian Journal of Animal Production 2015;52(2):89-99.##Shafiei G, Almasi M, Nikzad H, Miyan J, Mahabadi JA, Moshkdanian G. L-carnitine reduces the adverse effects of ROS and up-regulates the expression of implantation related genes in in vitro developed mouse embryos. Theriogenology 2020 Mar 15:145:59-66.##Franciosi F, Coticchio G, Lodde V, Tessaro I, Modina SC, Fadini R, et al. Natriuretic peptide precursor C delays meiotic resumption and sustains gap junction-mediated communication in bovine cumulus-enclosed oocytes. Biol Reprod 2014 Sep;91(3):61. ##Sutton-McDowall ML, Feil D, Robker RL, Thompson JG, Dunning KR. Utilization of endogenous fatty acid stores for energy production in bovine preimplantation embryos. Theriogenology 2012;77(8):1632-41. ##Wu GQ, Jia BY, Li JJ, Fu XW, Zhou GB, Hou YP, et al. L-carnitine enhances oocyte maturation and development of parthenogenetic embryos in pigs. Theriogenology 2011;76(5):785-93. ##Simmet K, Zakhartchenko V, Philippou-Massier J, Blum H, Klymiuk N, Wolf E. OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proc Natl Acad Sci USA 2018 Mar 13;115(11):2770-5. ##Kelleher AM, O'Neil E, Benne J, Cecil R, Spencer TE. NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo. Mol Reprod Dev 2020;87(1):152-60. ##Pournourali M, Tarang A, Farzadi Haghighi S, Yousefi M, Bahadori MH. Polymorphism variant of MnSOD A16V and risk of female infertility in northern Iran. Taiwan J Obstet Gynecol 2016;55(6):801-803. ##Salimi M, Shirazi A, Norouzian M, Mehrazar MM, Naderi MM, Shokrgozar MA, et al. Histone modifications of H3K4me3, H3K9me3 and lineage gene expressions in chimeric mouse embryo. Cell J 2020;22(1):96-105. ##

Generation of Optimized Consensus Sequences for Hepatitis C virus (HCV) Envelope 2 Gly-coprotein (E2) by a Modified Algorithm: Implication for a Pan-genomic HCV Vaccine 2 2 Background: Despite the success of "direct-acting antivirals" in treating Hepatitis C Virus (HCV) infection, invention of a preventive HCV vaccine is crucial for global elimination of the virus. Recent data indicated the importance of the induction of Pan-genomic neutralizing Antibodies (PnAbs) against heterogenic HCV Envelope 2(E2), the cellular receptor binding antigen, by any HCV vaccine candidate. To overcome HCVE2 heterogeneity, "generation of consensus HCVE2 sequences" is proposed. However, Consensus Sequence (CS) generating algorithms such as "Threshold" and "Majority" have certain limitations including "Threshold-rigidity" which leads to induction of undefined residues and insensitivity of the "Majority" towards the "evolutionary cost of residual substitutions". Methods: Herein, first a modification to the "Majority" algorithm was introduced by incorporating BLOSUM matrices. Secondly, the HCVE2 sequences generated by the "Fitness" algorithm (using 1698 sequences from genotypes 1, 2, and 3) was compared with those generated by the "Majority" and "Threshold" algorithms using several in silico tools. Results: Results indicated that only "Fitness" provided completely defined, gapless HCVE2s for all genotypes/subtypes, while considered the evolutionary cost of amino acid replacements (main "Majority/Threshold" limitations) by substitution of several residues within the generated consensuses. Moreover, "Fitness-generated HCVE2 CSs" were superior for antigenic/immunogenic characteristics as an antigen, while their positions within the phylogenetic trees were still preserved. Conclusion: "Fitness" algorithm is capable of generating superior/optimum HCVE2 CSs for inclusion in a pan-genomic HCV vaccine and can be similarly used in CS generation for other highly variable antigens from other heterogenic pathogens. 268 278 Reyhaneh Mohabati Department of Molecular Virology, Pasteur Institute of Iran Department of Molecular Virology, Pasteur Institute of Iran Iran Reza Rezaei School of Biology, College of Science, University of Tehran School of Biology, College of Science, University of Tehran Iran Nasir Mohajel Department of Molecular Virology, Pasteur Institute of Iran Iran Mohammad Mehdi Ranjbar Department of FMD Vaccine Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO) Department of FMD Vaccine Production, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO) Iran Katayoun Samimi-Rad 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 Kayhan Azadmanesh Farzin Roohvand Amino acidsAntibodiesAntiviral AgentsConsensus sequenceGenomicsGenotypeHepacivirusHepatitic CInventionsPhylogenyVaccinesVirus diseases 60594.pdf Blach S, Terrault NA, Tacke F, Gamkrelidze I, Craxi A, Tanaka J, et al. Global change in hepatitis C virus prevalence and cascade of care between 2015 and 2020: a modelling study. Lancet Gastroenterol Hepatol 2022 May;7(5):396-415.##Spearman CW, Dusheiko GM, Hellard M, Sonderup M. Hepatitis C. Lancet 2019;394(10207):1451–66.##Pedrana A, Munari S, Stoové M, Doyle J, Hellard M. The phases of hepatitis C elimination : achieving WHO elimination targets. Lancet Gastroenterol Hepatol 2021;6(January):6–8.##Naggie S, Wyles D. Direct-acting antiviral therapy for hepatitis C virus infection: Fulfilling the potential on the road to elimination. J Infect Dis 2020;222(Suppl 9):S741–4.##El-Sayed MH, Feld JJ. Vaccination at the forefront of the fight against hepatitis B and C. Nat Rev Gastroenterol Hepatol 2022;19(2):87–8.##Zibbell JE, Iqbal K, Patel RC, Suryaprasad A, Sanders KJ, Moore-Moravian L, et al. Increases in hepatitis C virus infection related to injection drug use among persons aged ≤30 years - Kentucky, Tennessee, Virginia, and West Virginia, 2006-2012. MMWR Morb Mortal Wkly Rep 2015;64(17):453–8.##Lewis KC, Barker LK, Jiles RB, Gupta N. Estimated Prevalence and Awareness of Hepatitis C Virus Infection Among US Adults: National Health and Nutrition Examination Survey, January 2017–March 2020. Clin Infect Dis [Internet] 2023;77(10):1413–5.##Heaton NS, Sachs D, Chen CJC-J, Hai R, Palese P. Genome-wide mutagenesis of influenza virus reveals unique plasticity of the hemagglutinin and NS1 proteins. Proc Natl Acad Sci USA 2013;110(50):20248–53.##Zhao Q, He K, Zhang X, Xu M, Zhang X, Li H. Production and immunogenicity of different prophylactic vaccines for hepatitis C virus (Review). Exp Ther Med 2022;24(1):474.##Gomez-Escobar E, Roingeard P, Beaumont E. Current Hepatitis C Vaccine Candidates Based on the Induction of Neutralizing Antibodies. Viruses 2023;15(5):1151.##Cuypers L, Li G, Libin P, Piampongsant S, Vandamme AM, Theys K. Genetic diversity and selective pressure in hepatitis C virus genotypes 1–6: Significance for direct-acting antiviral treatment and drug resistance. Viruses 2015;7(9):5018–39.##Page K, Melia MT, Veenhuis RT, Winter M, Rousseau KE, Massaccesi G, et al. Randomized Trial of a Vaccine Regimen to Prevent Chronic HCV Infection. N Engl J Med 2021;384(6):541–9.##Mankowski MC, Kinchen VJ, Wasilewski LN, Flyak AI, Ray SC, Crowe JE, et al. Synergistic anti-HCV broadly neutralizing human monoclonal antibodies with independent mechanisms. Proc Natl Acad Sci USA 2018;115(1):E82–91.##Waterman MS, Jones R. Consensus methods for DNA and protein sequence alignment. Methods Enzymol 1990;183(C):221–37.##Hertz GZ, Hartzell GW, Stormo GD. Identification of consensus patterns in unaligned DNA sequences known to be functionally related. Comput Appl Biosci 1990;6(2):81–92.##Bailey TL, Williams N, Misleh C, Li WW. MEME: Discovering and analyzing DNA and protein sequence motifs. Nucleic Acids Res 2006;34(Web Server issue):W369–73.##Iserte JA, Stephan BI, Goñi SE, Borio CS, Ghiringhelli PD, Lozano ME. Family-Specific Degenerate Primer Design: A Tool to Design Consensus Degenerated Oligonucleotides. Biotechnol Res Int 2013;2013:1–9.##Weaver EA, Lu Z, Camacho ZT, Moukdar F, Liao H-XH, Ma B-JB, et al. Cross-Subtype T-Cell Immune Responses Induced by a Human Immunodeficiency Virus Type 1 Group M Consensus Env Immunogen. J Virol 2006;80(14):6745–56.##Liao H, Sutherland LL, Xia S, Brock ME, Scearce RM, Vanleeuwen S, et al. A Group M Consensus Envelope Glycoprotein Induces Antibodies That Neutralize Subsets of Subtype B and C HIV-1 Primary Viruses. Virology 2006;353(2):268–82.##Laddy DJ, Yan J, Corbitt N, Kobasa D, Kobinger GP, Weiner DB. Immunogenicity of Novel Consensus-based DNA Vaccines Against Avian Influenza. Vaccine 2007;25(16):2984–9.##Chen MW, Cheng TJR, Huang Y, Jan JT, Ma SH, Yu AL, et al. A consensus-hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses. Proc Natl Acad Sci USA 2008;105(36):13538–43.##Latimer B, Toporovski R, Yan J, Pankhong P, Morrow MP, Khan AS, et al. Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine. Hum Vaccines Immunother 2014;10(8):2357–65.##Okonechnikov K, Golosova O, Fursov M, Varlamov A, Vaskin Y, Efremov I, et al. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012;28(8):1166–7.##Kuiken C, Yusim K, Boykin L, Richardson R. The Los Alamos hepatitis C sequence database. Bioinformatics 2005;21(3):379–84.##Henikoff S, Henikoff JG. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 1992;89(22):10915–9.##Eddy SR. Where did the BLOSUM62 alignment score matrix come from? Nat Biotechnol 2004;22(8):1035–6.##Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A, et al. Biopython : freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 2009;25(11):1422–3.##Pickett BE, Sadat EL, Zhang Y, Noronha JM, Squires RB, Hunt V, et al. ViPR: An open bioinformatics database and analysis resource for virology research. Nucleic Acids Res 2012;40(Database issue):D593–8.##Katoh K, Standley DM. MAFFT Multiple Sequence Alignment Software Version 7 : Improvements in Performance and Usability Article Fast Track. Mol Biol Evol 2013;30(4):772–80.##Hall T, Biosciences I, Carlsbad C. BioEdit: An important software for molecular biology. GERF Bull Biosci 2011;2(June):60–1.##Magnan CN, Zeller M, Kayala MA, Vigil A, Randall A, Felgner PL, et al. High-throughput prediction of protein antigenicity using protein microarray data. Bioinformatics 2010;26(23):2936–43.##Potter JA, Owsianka AM, Jeffery N, Matthews DJ, Keck Z-Y, Lau P, et al. Toward a Hepatitis C Virus Vaccine: the Structural Basis of Hepatitis C Virus Neutralization by AP33, a Broadly Neutralizing Antibody. J Virol 2012;86(23):12923–32.##Desombere I, Fafi-Kremer S, Van Houtte F, Pessaux P, Farhoudi A, Heydmann L, et al. Monoclonal anti-envelope antibody AP33 protects humanized mice against a patient-derived hepatitis C virus challenge. Hepatology 2016;63(4):1120–34.##Krey T, Meola A, Keck Z yong, Damier-Piolle L, Foung SKHH, Rey FA. Structural Basis of HCV Neutralization by Human Monoclonal Antibodies Resistant to Viral Neutralization Escape. PLoS Pathog 2013;9(5):e1003364.##Gupta R, Brunak S. Prediction of glycosylation across the human proteome and the correlation to protein function. Pac Symp Biocomput 2002;322:310–22.##Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol 2021;38(7):3022–7.##Lefkowitz EJ, Dempsey DM, Hendrickson RC, Orton RJ, Siddell SG, Smith DB. Virus taxonomy: The database of the International Committee on Taxonomy of Viruses (ICTV). Nucleic Acids Res 2018;46(D1):D708–17.##Carter DM, Darby CA, Lefoley BC, Crevar CJ, Alefantis T, Oomen R, et al. Design and Characterization of a Computationally Optimized Broadly Reactive Hemagglutinin Vaccine for H1N1 Influenza Viruses. J Virol 2016;90(9):4720–34.##Giles BM, Crevar CJ, Carter DM, Bissel SJ, Schultz-Cherry S, Wiley CA, et al. A computationally optimized hemagglutinin virus-like particle vaccine elicits broadly reactive antibodies that protect nonhuman primates from H5N1 infection. J Infect Dis 2012;205(10):1562–70.##Wong TM, Allen JD, Bebin-Blackwell AG, Carter DM, Alefantis T, DiNapoli J, et al. Computationally Optimized Broadly Reactive Hemagglutinin Elicits Hemagglutination Inhibition Antibodies against a Panel of H3N2 Influenza Virus Cocirculating Variants. J Virol 2017;91(24):e01581-17.##Tarr AW, Backx M, Hamed MR, Urbanowicz RA, Mcclure P, Brown RJP, et al. Immunization with a synthetic consensus hepatitis C virus E2 glycoprotein ectodomain elicits virus-neutralizing antibodies. Antiviral Res 2018;160(2018):25–37.##Linchangco Jr GV, Foley B, Leitner T. Updated HIV-1 consensus sequences change but stay within similar distance from worldwide samples. Front Microbiol 2022;12(January):1–7.##Weaver EA, Camacho ZT, Gao F. Similar T-Cell Immune Responses Induced by Group M Consensus Env Immunogens with Wild-Type or Minimum Consensus Variable Regions. AIDS Res Hum Retroviruses 2010;26(5):577–84.##Laddy DJ, Yan J, Corbitt N, Kobasa D, Kobinger GP, Weiner DB. Immunogenicity of novel consensus-based DNA vaccines against avian influenza. Vaccine 2007;25(16):2984–9.##Azhari Kemal R, Ivan J, Lili Sandjaja EB, Putra Santosa A. Computational Design of Ancestral and Consensus Asian Dengue Envelope Protein for Vaccine Candidate. KnE Life Sci 2020;2020:53–64.##Bhattacharya S, Banerjee A, Ray S. Development of new vaccine target against SARS-CoV2 using envelope (E) protein: An evolutionary, molecular modeling and docking based study. Int J Biol Macromol 2020;172(January):74–81.##Kato N, Sekiya H, Ootsuyama Y, Nakazawa T. Humoral Immune Response to Hypervariable Region 1 of the Putative Envelope Glycoprotein ( gp7O ) of Hepatitis C Virus. J Virol 1993;67(7):3923–30.##Farci T, Shimoda A, Wong D, Cabezon T, De Gioannis D, Strazzera A, et al. Prevention of hepatitis C virus infection in chimpanzees by hyperimmune serum against the hypervariable region 1 of the envelope 2 protein. Proc Natl Acad Sci USA 1996 Dec;93(26):15394–9.##Orlova O V, Drutsa VL, Spirin P V, Prasolov VS, Rubtsov PM, Kochetkov SN. The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells. Acta Naturae 2015;7(24):87–97.##Pantophlet R, Wilson IA, Burton DR. Hyperglycosylated Mutants of Human Immunodeficiency Virus (HIV) Type 1 Monomeric gp120 as Novel Antigens for HIV Vaccine Design. J Virol 2003;77(10):5889–901. ##Kumari S, Kessel A, Singhal D, Kaur G, Bern D, Lemay-St-Denis C, Singh J, Jain S. Computational identification of a multi-peptide vaccine candidate in E2 glycoprotein against diverse hepatitis c virus genotypes. J Biomol Struct Dyn 2023;41(20):11044–61.##Ahmad S, Demneh FM, Rehman B, Almanaa TN, Akhtar N, Pazoki-Toroudi H, et al. In silico design of a novel multi-epitope vaccine against HCV infection through immunoinformatics approaches. Int J Biol Macromol 2024 May 1;267:131517.##Shukla P, Pandey P, Prasad B, Robinson T, Purohit R, D’Cruz MMT LG, et al. Immuno-informatics analysis predicts B and T cell consensus epitopes for designing peptide vaccine against SARS-CoV-2 with 99.82% global population coverage. Brief Bioinform 2022 Jan 17;23(1):bbab496.##Comparative Genomics: Volumes 1 and 2, Bergman NH, editor. Totowa (NJ): Humana Press; 2007. Chapter 10 PSI-BLAST Tutorial, Medha Bhagwat and L. Aravind.##Mohammadi A, Zahiri J, Mohammadi S, Khodarahmi M, Arab SS. PSSMCOOL: a comprehensive R package for generating evolutionary-based descriptors of protein sequences from PSSM profiles. Biol Methods Protoc 2022 Mar 30;7(1):bpac008.##

Optimization of the Production of Soluble Recombinant TEV Protease in Two E. coli Strains 2 2 Background: The low solubility of Tobacco Etch Virus (TEV) protease, a functional enzyme that cleaves protein tags without significant modification in its sequence, is one of the most important limitations of this enzyme. In this study, the aim was to increase the solubility of TEV by changing the expression conditions and designing lysis buffer with various solubilizing agents to improve its solubility. Methods: Escherichia coli (E. coli) BL21 (DE3) and E. coli origami harboring wild type TEV-pKR793 and mutant N23F TEV-pKR793 plasmids were used for the expression. Response surface methodology was used to determine the best culture conditions (IPTG concentration, incubation time and incubation temperature) of soluble expression. Furthermore, eight different solubilizing agents were added separately to the lysis buffer to check their effect on the protein solubility. Results: The production of soluble N23F in E. coli BL21 (DE3) was two-folds more than the wild type and the inclusion body formation in the mentioned form was diminished as about 25% in comparison to the wild type. Finally, betaine had the most effects for enhancing the soluble expression of N23F in both host cells. For the wild type, sodium selenite, xylitol, and glycine showed the most effects on soluble production. Conclusion: The solubility of the mutant form of TEV protease increased in E. coli BL21 (DE3) compared to its wild form. Also, using additives such as betaine to the lysis buffer, increased the solubility of N23F in E. coli BL21 (DE3) and origami strains. 279 283 Matineh Shahriari Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Fatemeh Shafiee Bioinformatics Research Center, School of pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Bioinformatics Research Center, School of pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Fatemeh Moazen Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Hamid Mir Mohammad Sadeghi Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences Iran Escherichia coli (E. coli)SolubilitySolubilizing agentsTEV protease 60595.pdf Mohammadian N, Mohammadian H, Moazen F, Pakdel MH, Jahanian-Najafabadi A, Mir Mohammad Sadeghi H. Optimization of solvent media to solubilize TEV protease using response surface method. Res Pharm Sci 2020;15(4):331-9. ##Blommel PG, Fox BG. A combined approach to improving large-scale production of tobacco etch virus protease. Protein Expr Purif 2007;55(1):53-68.##Renicke C, Spadaccini R, Taxis C. A tobacco etch virus protease with increased substrate tolerance at the P1' position. PLoS One 2013;8(6):e67915. ##Henrichs T, Mikhaleva N, Conz C, Deuerling E, Boyd D, Zelazny A, et al. Target-directed proteolysis at the ribosome. Proc Natl Acad Sci USA 2005;102(12):4246-51. ##Pauli A, Althoff F, Oliveira RA, Heidmann S, Schuldiner O, Lehner CF, et al. Cell-type-specific TEV protease cleavage reveals cohesin functions in Drosophila neurons. Dev Cell 2008;14(2):239-51. ##Rawlings ND, Barrett AJ. Evolutionary families of peptidases. Biochem J 1993;290 ( Pt 1)(Pt 1):205-18.##Copeland MF, Politz MC, Johnson CB, Markley AL, Pfleger BF. A transcription activator-like effector (TALE) induction system mediated by proteolysis. Nat Chem Biol 2016;12(4):254-60.##Sun P, Austin BP, Tozser J, Waugh DS. Structural determinants of tobacco vein mottling virus protease substrate specificity. Protein Sci 2010;19(11):2240-51. ##Faber KN, Kram AM, Ehrmann M, Veenhuis M. A novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease. J Biol Chem 2001;276(39):36501-7. ##Cabrita LD, Gilis D, Robertson AL, Dehouck Y, Rooman M, Bottomley SP. Enhancing the stability and solubility of TEV protease using in silico design. Protein Sci 2007;16(11):2360-7. ##Tropea JE, Cherry S, Waugh DS. Expression and purification of soluble His(6)-tagged TEV protease. Methods Mol Biol 2009;498:297-307.##de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B. Chaperone-based procedure to increase yields of soluble recombinant proteins produced in E. coli. BMC Biotechnol 2007;7:32. ##Esposito D, Chatterjee DK. Enhancement of soluble protein expression through the use of fusion tags. Curr Opin Biotechnol 2006;17(4):353-8.##Papaneophytou CP, Kontopidis G. Statistical approaches to maximize recombinant protein expression in Escherichia coli: a general review. Protein Expr Purif 2014;94:22-32. ##Sorensen HP, Mortensen KK. Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli. Microb Cell Fact 2005;4(1):1. ##Cesaratto F, Burrone OR, Petris G. Tobacco Etch Virus protease: A shortcut across biotechnologies. J Biotechnol 2016;231:239-49. ##Mohammadian H, Mahnam K, Sadeghi HM, Ganjalikhany MR, Akbari V. Rational design of a new mutant of tobacco etch virus protease in order to increase the in vitro solubility. Res Pharm Sci 2020;15(2):164-73. ##Golovanov AP, Hautbergue GM, Wilson SA, Lian LY. A simple method for improving protein solubility and long-term stability. J Am Chem Soc 2004;126(29):8933-9. ##Tsumoto K, Umetsu M, Kumagai I, Ejima D, Philo JS, Arakawa T. Role of arginine in protein refolding, solubilization, and purification. Biotechnol Prog 2004;20(5):1301-8.##Long X, Gou Y, Luo M, Zhang S, Zhang H, Bai L, et al. Soluble expression, purification, and characterization of active recombinant human tissue plasminogen activator by auto-induction in E. coli. BMC Biotechnol 2015;15:13. ##