Integrating Psychiatry and Medical Biotechnology as a Way to Achieve Scientific, Precision, and Personalized Psychiatry 2 2 Besides concerns about the increasing prevalence of psychiatric disorders and the significant burdens and costs, there are concerns about its validity. The dilemma of validity went so far that studies described the diagnoses in psychiatry as scientifically worthless. We suggest integrating psychiatry and medical biotechnology and using biotechnological products in psychiatric aspects help psychiatry become more precise, strengthen its position among other sciences, and increase its scientific credibility by giving examples. For this matter, we need different inputs to choose between the vast outputs. The most common inputs are clinical symptoms, cognitive function, individual and environmental risk factors, molecular markers, genetic markers, neuroimaging signs, and big data. Some molecular markers have been shown to have a relationship with psychiatric disorders such as Interleukin-6 (IL-6) and Tumor Necrosis Factor-α (TNF-α). Genetic studies might evolve the most accurate part of precision psychiatry. Currently, and through the developments in technology, genome-wide association studies have become available. In neuroimaging signs, psychiatric disorders are associated with generalized rather than focal brain network dysfunction, and functional magnetic resonance imaging could be performed to study them. It would exhibit different aberrancies in various psychiatric disorders. In big data, the constitution of predictive models and movement toward precision psychiatry can be led by using artificial intelligence and machine learning. 172 175 Ahmad Shamabadi School of Medicine, Tehran University of Medical SciencesPsychiatric Research Center, Roozbeh Psychiatric Hospital, Tehran University of Medical Sciences School of Medicine, Tehran University of Medical SciencesPsychiatric Research Center, Roozbeh Psychiatric Hospital, Tehran University of Medical Sciences IranIran Alireza Hasanzadeh Psychiatric Research Center, Roozbeh Psychiatric Hospital, Tehran University of Medical Sciences Psychiatric Research Center, Roozbeh Psychiatric Hospital, Tehran University of Medical Sciences Iran Shahin Akhondzadeh Behavioral sciencesCytokinesIntegrative medicineKnowledgeTechnology 40485.pdf Akhondzadeh S. Hippocampal synaptic plasticity and cognition. J Clin Pharm Ther 1999; 24(4):241-8.##Akhondzadeh S. The 5-HT hypothesis of schizophrenia. 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Influence of comorbid psychiatric disorders on the risk of development of alcohol dependence by genetic variations of ALDH2 and ADH1B. Alcohol Clin Exp Res 2020;44(11):2275-82.##Chung KH, Li CY, Kuo SY, Sithole T, Liu WW, Chung MH. Risk of psychiatric disorders in patients with chronic insomnia and sedative-hypnotic prescription: a nationwide population-based follow-up study. J Clin Sleep Med 2015;11(5):543-51.##Myllyaho T, Siira V, Wahlberg KE, Hakko H, Tikkanen V, Läksy K, et al. Dysfunctional family functioning in high socioeconomic status families as a risk factor for the development of psychiatric disorders in adoptees: the Finnish Adoptive Family Study of Schizophrenia. Soc Psychiatry Psychiatr Epidemiol 2021.##Paananen R, Tuulio-Henriksson A, Merikukka M, Gissler M. Intergenerational transmission of psychiatric disorders: the 1987 Finnish Birth Cohort study. Eur Child Adolesc Psychiatry 2021;30(3):381-9.##Dewa CS, Lin E, Kooehoorn M, Goldner E. 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Schizophr Bull 2018;44(1):75-83.##Bekhbat M, Chu K, Le NA, Woolwine BJ, Haroon E, Miller AH, et al. Glucose and lipid-related biomarkers and the antidepressant response to infliximab in patients with treatment-resistant depression. Psychoneuroendocrinology 2018;98:222-9.##Miller BJ, Buckley P, Seabolt W, Mellor A, Kirkpatrick B. Meta-analysis of cytokine alterations in schizophrenia: clinical status and antipsychotic effects. Biol Psychiatry 2011;70(7):663-71.##Reale M, Costantini E, Greig NH. Cytokine imbalance in schizophrenia. From research to clinic: potential implications for treatment. Front Psychiatry 2021;12:536257.##Psychiatric GWAS Consortium Bipolar Disorder Working Group. Large-scale genome-wide association analysis of bipolar disorder identifies a new susceptibility locus near ODZ4. Nat Genet 2011;43(10):977-83.##Schizophrenia Working Group of the Psychiatric Genomics Consortium. Biological insights from 108 schizophrenia-associated genetic loci. 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DNA methylation associated with persistent ADHD suggests TARBP1 as novel candidate. Neuropharmacology 2021;184:108370.##Bassett AS, Chow EW, AbdelMalik P, Gheorghiu M, Husted J, Weksberg R. The schizophrenia phenotype in 22q11 deletion syndrome. Am J Psychiatry 2003;160(9):1580-6.##Barch DM. Resting-state functional connectivity in the human connectome project: current status and relevance to understanding psychopathology. Harv Rev Psychiatry 2017;25(5):209-17.##Gratton C, Kraus BT, Greene DJ, Gordon EM, Laumann TO, Nelson SM, et al. Defining individual-specific functional neuroanatomy for precision psychiatry. Biol Psychiatry 2020;88(1):28-39.##Keshavan MS, Collin G, Guimond S, Kelly S, Prasad KM, Lizano P. Neuroimaging in schizophrenia. Neuroimaging Clin N Am 2020;30(1):73-83##Haijma SV, Van Haren N, Cahn W, Koolschijn PC, Hulshoff Pol HE, Kahn RS. Brain volumes in schizophrenia: a meta-analysis in over 18 000 subjects. 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Apoptosis Induction with Combined Use of Cisplatin and Fisetin in Cisplatin-resistance A2780 Ovarian Cancer Cells 2 2 Background: Ovarian cancer is the leading cause of death caused by genital cancers. One of the most common treatments for this type of cancer is chemotherapy by cisplatin, which induces apoptosis in cancer cells. Apoptosis is a type of physiological cell death. Cisplatin chemotherapy usually has several side effects and cellular resistance to cisplatin is a common incidence. In order to overcome these problems, the use of combination therapies using natural substances has been considered. Fisetin is a flavonoid with anti-cancer activity which induces apoptosis. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated. Methods: In the present experimental study, the effect of combined use of Fisetin and cisplatin on ovarian cancer cell lines (A2780) was investigated by using MTT assay. Cell death was also determined by DAPI, acridine orange/propidium iodide, and Annexin/PI assay. Apoptotic gene expression of Bax, BCL-2, caspase 3, and caspase 9 was also assessed by real time PCR. Results: The results of MTT assay indicated that the combined treatment of Fisetin and cisplatin effectively inhibits proliferation of A2780 cells. The results of DAPI staining showed that fragmentation of chromatin in cells occurred in the combined treatment. Acridine orange-propidium iodide staining and Annexin/PI staining showed an increase in the rate of apoptotic cells in cells under combined treatment. The results of the study regarding changes in gene expression also indicated that Bax pro-apoptotic gene expression and BCL-2 anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of caspases 3 and 9 significantly increased as well. Conclusion: According to the findings of this study, the combined use of cisplatin and Fisetin increases the induction of apoptosis in cisplatin-resistant ovarian cancer cells (A2780); therefore, the combined use of cisplatin and Fisetin can be considered a promising strategy in the treatment of ovarian cancer. 176 182 Samira Jafarzadeh Department of Biology, Mashhad branch, Islamic Azad University Department of Biology, Mashhad branch, Islamic Azad University Iran Javad Baharara Research Center for Animal Development Applied of Biology, Mashhad branch, Islamic Azad University Research Center for Animal Development Applied of Biology, Mashhad branch, Islamic Azad University Iran Maryam Tehranipour Department of Biology, Mashhad branch, Islamic Azad University Department of Biology, Mashhad branch, Islamic Azad University Iran ApoptosisCisplatinFisetinOvarian neoplasms 40474.pdf Sudhakar A. History of cancer, ancient and modern treatment methods. J Cancer Sci Ther 2009;1(2):1-4.##Schirrmacher V. From chemotherapy to biological therapy: A review of novel concepts to decrease the side effects of systemic cancer treatment. Int J Oncol 2019;54(2):407-19.##Allen TM. Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer 2002;2(10):750-63.##Marin JJG, Al-Abdulla R, Lozano E, Briz O, Bujanda L, M Banales J, et al. Mechanisms of resistance to chemotherapy in gastric cancer. Anti-Cancer Agents Med Chem 2016;16(3):318-34.##Zhao L, Liu S, Liang D, Jiang T, Yan X, Zhao S, et al. Resensitization of cisplatin resistance ovarian cancer cells to cisplatin through pretreatment with low dose fraction radiation. Cancer Med 2019;8(5):2442-8.##Hu C-MJ, Zhang L. Nanoparticle-based combination therapy toward overcoming drug resistance in cancer. Biochem Pharmacol 2012;83(8):1104-11.##Ahmed K, Zaidi SF, Cui ZG, Zhou D, Saeed SA, Inadera H. Potential proapoptotic phytochemical agents for the treatment and prevention of colorectal cancer. Oncol Lett 2019;18(1):487-98.##Lall RK, Adhami VM, Mukhtar H. Dietary flavonoid fisetin for cancer prevention and treatment. Mol Nutr Food Res 2016;60(6):1396-405.##Kashyap D, Sharma A, Sak K, Tuli HS, Buttar HS, Bishayee A. Fisetin: A bioactive phytochemical with potential for cancer prevention and pharmacotherapy. Life Sci 2018;194:75-87.##Lu X, Jung J in, Cho HJ, Lim DY, Lee HS, Chun HS, et al. Fisetin inhibits the activities of cyclin-dependent kinases leading to cell cycle arrest in HT-29 human colon cancer cells. J Nutr 2005;135(12):2884-90.##Haddad AQ, Venkateswaran V, Viswanathan L, Teahan SJ, Fleshner NE, Klotz LH. Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines. Prostate Cancer Prostatic Dis 2006;9(1):68-76.##Kashyap D, Garg VK, Tuli HS, Yerer MB, Sak K, Sharma AK, et al. Fisetin and quercetin: promising flavonoids with chemopreventive potential. Biomolecules 2019;9(5):174.##Khorasani N, Baharara J, Iranbakhsh AR, Ramezani T. Apoptotic effects of silver nanoparticles coated with Zataria multiflora leaves extract on HepG2 cell line. Feyz J 2016;19(6):457-67.##Klimaszewska-Winiewska A, Halas-Winiewska M, Grzanka A, Grzanka D. Evaluation of anti-metastatic potential of the combination of fisetin with paclitaxel on A549 non-small cell lung cancer cells. Int J Mol Sci 2018;19(3):661.##Adan A, Baran Y. The pleiotropic effects of fisetin and hesperetin on human acute promyelocytic leukemia cells are mediated through apoptosis, cell cycle arrest, and alterations in signaling networks. Tumor Biol 2015;36(11):8973-84.##Chen Q, Qin R, Fang Y, Li H. Berberine sensitizes human ovarian cancer cells to cisplatin through miR-93/PTEN/Akt signaling pathway. Cell Physiol Biochem 2015;36(3):956-65.##Gong LH, Chen XX, Wang H, Jiang QW, Pan SS, Qiu JG, et al. Piperlongumine induces apoptosis and synergizes with cisplatin or paclitaxel in human ovarian cancer cells. Oxid Med Cell Longev 2014;2014.##Kim KH, Lee B, Kim YR, Kim MA, Ryu N, Kim UK, et al. Evaluating protective and therapeutic effects of alpha-lipoic acid on cisplatin-induced ototoxicity. Cell Death Dis 2018;9(8):1-13.##Sahu BD, Kalvala AK, Koneru M, Kumar JM, Kuncha M, Rachamalla SS, et al. Ameliorative effect of fisetin on cisplatin-induced nephrotoxicity in rats via modulation of NF- B activation and antioxidant defence. PLoS One 2014;9(9):e105070.##Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics and apoptosis. Genes Dev 2008;22(12):1577-90.##Letai AG. Diagnosing and exploiting cancer’s addiction to blocks in apoptosis. Nat Rev Cancer 2008;8(2):121-32.##Wang SH, Shih YL, Lee CC, Chen WL, Lin CJ, Lin YS, et al. The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis. Chem Biol Interact 2009;181(1):45-51.##Pérez EH, Luna JM, Rojas LM, Kouri JB. Chondroptosis: an immunohistochemical study of apoptosis and Golgi complex in chondrocytes from human osteoarthritic cartilage. Apoptosis 2005;10(5):1105-10.##Tommasi S, Pilato B, Pinto R, Monaco A, Bruno M, Campana M, et al. Molecular and in silico analysis of BRCA1 and BRCA2 variants. Mutat Res 2008;644(1-2):64-70.##Tavsan Z, Kayali HA. Flavonoids showed anticancer effects on the ovarian cancer cells: Involvement of reactive oxygen species, apoptosis, cell cycle and invasion. Biomed Pharmacother 2019;116:109004.##Li J, Wang Y, Lei JC, Hao Y, Yang Y, Yang CX, et al. Sensitisation of ovarian cancer cells to cisplatin by flavonoids from Scutellaria barbata. Nat Prod Res 2014;28(10):683-9.##

A Cross-Sectional Study for Evaluation of KRAS and BRAF Mutations by Reverse Dot Blot, PCR-RFLP, and Allele-Specific PCR Methods Among Patients with Colorectal Cancer 2 2 Background: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods. Methods: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively. Results: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.  Conclusion: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method. 183 191 Fatemeh Sheikhsofla Department of Cellular and Molecular Biology, University of Mazandaran Department of Cellular and Molecular Biology, University of Mazandaran Iran Behzad Poopak Sajjad Firuzyar Razi Vaccine and Serum Research Institute of Karaj Iran Fatemeh Roudbari Department of Virology, University of Mazandaran Department of Virology, University of Mazandaran Iran Mojtaba Ghadiany Department of Hematology and Oncology, Shahid Beheshti University of Medical Sciences Department of Hematology and Oncology, Shahid Beheshti University of Medical Sciences Iran Allele-Specific PCRBRAFColorectal neoplasmsKRASPCR-RFLPReverse dot blot 40475.pdf Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69(1):7-34.##Van Abbema D, Vissers P, de Vos-Geelen J, Lemmens V, Janssen-Heijnen M, Tjan-Heijnen V. Trends in overall survival and treatment patterns in two large population-based cohorts of patients with breast and colorectal cancer. Cancers (Basel) 2019;11(9):1239.##Lubomierski N, Plotz G, Wormek M, Engels K, Kriener S, Trojan J, et al. BRAF mutations in colorectal carcinoma suggest two entities of microsatellite-unstable tumors. Cancer 2005;104(5):952-61.##Zacharakis M, Xynos ID, Lazaris A, Smaro T, Kosmas C, Dokou A, et al. Predictors of survival in stage IV metastatic colorectal cancer. Anticancer Res 2010;30(2):653-60.##Benavides M, Díaz-Rubio E, Carrato A, Abad A, Guillén C, Garcia-Alfonso P, et al. Tumour location and efficacy of first-line EGFR inhibitors in KRAS/RAS wild-type metastatic colorectal cancer: retrospective analyses of two phase II randomised Spanish TTD trials. ESMO Open 2019;4(6):e000599.##Solassol J, Vendrell J, Märkl B, Haas C, Bellosillo B, Montagut C, et al. Multi-center evaluation of the fully automated PCR-based Idylla™ KRAS mutation assay for rapid KRAS mutation status determination on formalin-fixed paraffin-embedded tissue of human colorectal cancer. PLoS One 2016;11(9):e0163444.##Ogino S, Meyerhardt JA, Irahara N, Niedzwiecki D, Hollis D, Saltz LB, et al. KRAS mutation in stage III colon cancer and clinical outcome following intergroup trial CALGB 89803. Clin Cancer Res 2009;15(23):7322-9.##Midthun L, Shaheen S, Deisch J, Senthil M, Tsai J, Hsueh CT. Concomitant KRAS and BRAF mutations in colorectal cancer. J Gastrointest Oncol 2019;10(3):577-81.##Garcia-Carbonero N, Martinez-Useros J, Li W, Orta A, Perez N, Carames C, et al. KRAS and BRAF mutations as prognostic and predictive biomarkers for standard chemotherapy response in metastatic colorectal cancer: a single institutional study. Cells 2020;9(1):219.##Vacante M, Borzì AM, Basile F, Biondi A. Biomarkers in colorectal cancer: Current clinical utility and future perspectives. World J Clin Cases 2018;6(15):869-81.##Matsunaga M, Kaneta T, Miwa K, Ichikawa W, Fujita KI, Nagashima F, et al. A comparison of four methods for detecting KRAS mutations in formalin-fixed specimens from metastatic colorectal cancer patients. Oncol Lett 2016;12(1):150-6.##Nagakubo Y, Hirotsu Y, Amemiya K, Oyama T, Mochizuki H, Omata M. Accurate detection of KRAS, NRAS and BRAF mutations in metastatic colorectal cancers by bridged nucleic acid-clamp real-time PCR. BMC Med Genomics 2019;12(1):162.##Hashim HO, Al-Shuhaib MB. Exploring the potential and limitations of PCR-RFLP and PCR-SSCP for SNP detection: a review. J Applied Biotechnology Reports 2019;6(4):137-44.##Bluth MJ, Bluth MH. Molecular pathology techniques: Advances in 2018. Clin Lab Med 2018;38(2):215-36.##Gold B. Origin and utility of the reverse dot–blot. Expert Rev Mol Diagn2003;3(2):143-52.##Hayama T, Hashiguchi Y, Okamoto K, Okada Y, Ono K, Shimada R, et al. G12V and G12C mutations in the gene KRAS are associated with a poorer prognosis in primary colorectal cancer. Int J Colorectal Dis 2019;34(8):1491-6.##Yang MM, Wang J, Dong L, Teng Y, Liu P, Fan JJ, et al. Lack of association of C3 gene with uveitis: additional insights into the genetic profile of uveitis regarding complement pathway genes. Sci Rep 2017;7(1):879.##Li W, Liu Y, Cai S, Yang C, Lin Z, Zhou L, et al. Not all mutations of KRAS predict poor prognosis in patients with colorectal cancer. Int J Clin Exp Pathol 2019;12(3):957-67.##Velho S, Moutinho C, Cirnes L, Albuquerque C, Hamelin R, Schmitt F, et al. BRAF, KRAS and PIK3CA mutations in colorectal serrated polyps and cancer: primary or secondary genetic events in colorectal carcinogenesis?. BMC Cancer 2008;8(1):255.##Li Z, Chen Y, Wang D, Wang G, He L, Suo J. Detection of KRAS mutations and their associations with clinicopathological features and survival in Chinese colorectal cancer patients. J Int Med Res 2012;40(4):1589-98.##Brink M, de Goeij AF, Weijenberg MP, Roemen GM, Lentjes MH, Pachen MM, et al. K-ras oncogene mutations in sporadic colorectal cancer in The Netherlands Cohort Study. Carcinogenesis 2003;24(4):703-10.##Van Cutsem E, Kohne CH, Láng I, Folprecht G, Nowacki MP, Cascinu S, et al. Cetuximab plus irinotecan, fluorouracil, and leucovorin as first-line treatment for metastatic colorectal cancer: updated analysis of overall survival according to tumor KRAS and BRAF mutation status. J Clin Oncol 2011;29(15):2011-9.##Garcia-Carbonero N, Martinez-Useros J, Li W, Orta A, Perez N, Carames C, et al. KRAS and BRAF mutations as prognostic and predictive biomarkers for standard chemotherapy response in metastatic colorectal cancer: A single institutional study. Cells 2020;9(1):219.##Janku F, Lee JJ, Tsimberidou AM, Hong DS, Naing A, Falchook GS, et al. PIK3CA mutations frequently coexist with RAS and BRAF mutations in patients with advanced cancers. PloS One 2011;6(7):e22769.##Chiu JW, Krzyzanowska MK, Serra S, Knox JJ, Dhani NC, Mackay H, et al. Molecular profiling of patients with advanced colorectal cancer: princess margaret cancer centre experience. Clin Colorectal Cancer 2018;17(1):73-9.##Loupakis F, Ruzzo AN, Cremolini C, Vincenzi B, Salvatore L, Santini D, et al. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer 2009;101(4):715-21.##Hutchins G, Southward K, Handley K, Magill L, Beaumont C, Stahlschmidt J, et al. Value of mismatch repair, KRAS, and BRAF mutations in predicting recurrence and benefits from chemotherapy in colorectal cancer. J Clin Oncol 2011;29(10):1261-70.##Ince WL, Jubb AM, Holden SN, Holmgren EB, Tobin P, Sridhar M, et al. Association of k-ras, b-raf, and p53 status with the treatment effect of bevacizumab. J Natl Cancer Inst 2005;97(13):981-9.##Cabart M, Frenel JS, Campion L, Ramee JF, Dupuis O, Senellart H, et al. [KRAS mutation does not influence oxaliplatin or irinotecan efficacy, in association with bevacizumab, in first line treatment of metastatic colorectal cancer]. Bull Cancer 2016;103(6):541-51. French.##Ergun Y, Acikgoz Y, Bal O, Ucar G, Dirikoc M, Caliskan Yildirim E, et al. KRAS codon 12 and 13 mutations may guide the selection of irinotecan or oxaliplatin in first-line treatment of metastatic colorectal cancer. Expert Rev Mol Diagn 2019;19(12):1131-40.##Hatzaki A, Razi E, Anagnostopoulou K, Iliadis K, Kodaxis A, Papaioannou D, et al. A modified mutagenic PCR-RFLP method for K-ras codon 12 and 13 mutations detection in NSCLC patients. Mol Cell Probes 2001;15(5):243-7.##De Roock W, Jonker DJ, Di Nicolantonio F, Sartore-Bianchi A, Tu D, Siena S, et al. Association of KRAS p. G13D mutation with outcome in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab. JAMA 2010;304(16):1812-20.##Tejpar S, Celik I, Schlichting M, Sartorius U, Bokemeyer C, Van Cutsem E. Association of KRAS G13D tumor mutations with outcome in patients with metastatic colorectal cancer treated with first-line chemotherapy with or without cetuximab. J Clin Oncol 2012;30(29):3570-7.##Al‐Mulla F, Milner‐White EJ, Going JJ, Birnie GD. Structural differences between valine‐12 and aspartate‐12 Ras proteins may modify carcinoma aggression. J Pathol 1999;187(4):433-8.##Guerrero S, Casanova I, Farré L, Mazo A, Capellà G, Mangues R. K-ras codon 12 mutation induces higher level of resistance to apoptosis and predisposition to anchorage-independent growth than codon 13 mutation or proto-oncogene overexpression. 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The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 2005 ;5(9):689-98.##Arrington AK, Heinrich EL, Lee W, Duldulao M, Patel S, Sanchez J, et al. Prognostic and predictive roles of KRAS mutation in colorectal cancer. Int J Mol Sci 2012;13(10):12153-68.##Osumi H, Shinozaki E, Osako M, Kawazoe Y, Oba M, Misaka T, et al. Cetuximab treatment for metastatic colorectal cancer with KRAS p. G13D mutations improves progression-free survival. Mol Clin Oncol 2015;3(5):1053-7.##Chen CC, Er TK, Liu YY, Hwang JK, Barrio MJ, Rodrigo M, et al. Computational analysis of KRAS mutations: implications for different effects on the KRAS p. G12D and p. G13D mutations. PloS One 2013;8(2):e55793.##

Production of PEGylated G-CSF from non-classical inclusion bodies expressed in Escherichia coli 2 2 Background: The recombinant human granulocyte colony stimulating factor conjugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimulating Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the conjugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli). 192 200 Nguyen Thi My Trinh Department of Molecular and Environmental Biotechnology, University of ScienceVietnam National University Department of Molecular and Environmental Biotechnology, University of ScienceVietnam National University VietnamVietnam Tran Linh Thuoc Department of Molecular and Environmental Biotechnology, University of ScienceeVietnam National University Department of Molecular and Environmental Biotechnology, University of ScienceeVietnam National University VietnamVietnam Dang Thi Phuong Thao Department of Molecular and Environmental Biotechnology, University of Science Department of Molecular and Environmental Biotechnology, University of Science Vietnam Granulocyte colony stimulating factor (G-CSF)Inclusion bodiesPolyethylene glycol 40477.pdf Basu S, Dunn A, Ward A. G-CSF: function and modes of action. Int J Mol Med 2002;10(1):3-10.##Mehta HM, Malandra M, Corey SJ. G-csf and gm-csf in neutropenia. J Immunol 2015;195(4):1341-9.##Gillis S, Urdal D, Clevenger W, Klinke R, Sassenfeld H, Price V, et al. Production of recombinant human colony stimulating factors in yeast. Behring Inst Mitt 1988(83):1-7.##Monaco L, Tagliabue R, Giovanazzi S, Bragonzi A, Soria MR. Expression of recombinant human granulocyte colony-stimulating factor in CHO dhfr− cells: new insights into the in vitro amplification expression system. Gene 1996;180(1-2):145-50.##Babaeipour V, Khanchezar S, Mofid MR, Abbas MPH. Efficient process development of recombinant human granulocyte colony-stimulating factor (rh-GCSF) production in Escherichia coli. Iran Biomed J 2015;19(2):102-10.##Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 2014;5:172.##Slouka C, Kopp J, Spadiut O, Herwig C. Perspectives of inclusion bodies for bio-based products: curse or blessing? Appl Microbiol Biotechnol 2019;103(3):1143-53.##Jevsevar S, Gaberc-Porekar V, Fonda I, Podobnik B, Grdadolnik J, Menart V. Production of nonclassical inclusion bodies from which correctly folded protein can be extracted. Biotechnol Prog 2005;21(2):632-9.##Peternel Š, Grdadolnik J, Gaberc-Porekar V, Komel R. Engineering inclusion bodies for non denaturing extraction of functional proteins. Microb Cell Fact 2008;7(1):34.##Ashrafi F, Salmasi M. Comparison of the effects of pegylated granulocyte-colony stimulating factor and granulocyte-colony stimulating factor on cytopenia induced by dose-dense chemotherapy in breast cancer patients. J Res Med Sci 2018;23:73.##Molineux G. The design and development of pegfilgrastim (PEG-rmetHuG-CSF, Neulasta®). Curr Pharm Des 2004;10(11):1235-44.##Pfeiffer N. Pegylated G-CSF may need only one dose per treatment cycle. Oncology Times 2001;23(8):43-4.##Rajan RS, Li T, Aras M, Sloey C, Sutherland W, Arai H, et al. Modulation of protein aggregation by polyethylene glycol conjugation: GCSF as a case study. Protein Sci 2006;15(5):1063-75.##Kinstler OB, Brems DN, Lauren SL, Paige AG, Hamburger JB, Treuheit MJ. Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm Res 1996;13(7):996-1002.##Tonon G, Orsini G, Schrepfer R, Taylor G, Sergi M; Bio Ker SRL assignee. G-CSF site-specific mono-conjugates. Canada patents CA2660615A1. 2008 Feb 14.##Maullu C, Raimondo D, Caboi F, Giorgetti A, Sergi M, Valentini M, et al. Site‐directed enzymatic PEGylation of the human granulocyte colony-stimulating factor. FEBS J 2009;276(22):6741-50.##Scaramuzza S, Tonon G, Olianas A, Messana I, Schrepfer R, Orsini G, et al. A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: production and physicochemical characterization. J Control Release 2012;164(3):355-63.##Grigoletto A, Mero A, Yoshioka H, Schiavon O, Pasut G. Covalent immobilisation of transglutaminase: stability and applications in protein PEGylation. J Drug Target 2017;25(9-10):856-64.##Brunelle JL, Green R. Coomassie blue staining. Methods Enzymol 2014;541:161-7.##Kavran JM, Leahy DJ. Silver staining of SDS-polyacrylamide Gel. Methods Enzymol 2014;541:169-76.##Tiwari K, Shebannavar S, Kattavarapu K, Pokalwar S, Mishra MK, Chauhan UK. Refolding of recombinant human granulocyte colony stimulating factor: Effect of cysteine/cystine redox system. Indian J Biochem Biophys 2012;49(4):285-8.##Kim CK, Lee CH, Lee SB, Oh JW. Simplified large-scale refolding, purification, and characterization of recombinant human granulocyte-colony stimulating factor in Escherichia coli. PLoS One 2013;8(11):e80109.##Kosobokova E, Skrypnik K, Pinyugina M, Shcherbakov A, Kosorukov V. Optimization of the refolding of recombinant human granulocyte-macrophage colony-stimulating factor immobilized on affinity sorbent. Applied Biochemistry and Microbiology 2014;50(8):773-9.##Mero A, Spolaore B, Veronese FM, Fontana A. Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG. Bioconjug Chem 2009;20(2):384-9.##

Fabrication of Calcium Sulfate Coated Selenium Nanoparticles and Corresponding In-Vitro Cytotoxicity Effects Against 4T1 Breast Cancer Cell Line 2 2 Background: The inhibitory effect of selenium nanoparticles (SeNPs) on cancer cells has been reported in many studies. In this study,  the purpose was to compare the in vitro effects of SeNPs and calcium sulfate coated selenium nanoparticles (CaSO4@ SeNPs) on breast cancer cells. Methods: CaSO4@SeNPs and SeNPs were chemically synthesized and characterized with Field Emission Scanning Electron Microscope (FESEM) and energy-dispersive X-ray spectroscopy (EDX). By applying MTT assay, the cytotoxicity effect of both nanomaterials on the 4T1 cancer cells was investigated. Results: While LD50 of SeNPs on 4T1 cancer cells was 80 µg, the LD50 of CaSO4@SeNPs was reported to be only 15 µg. The difference between the inhibition rates obtained for SeNPs and CaSO4@SeNPs was statistically significant (p=0.05). In addition, at higher concentrations (50 µg) of CaSO4@SeNPs, the cytotoxicity was 100% more than SeNPs alone.  Conclusion: According to the result of the present work, it can be concluded that decoration of SeNPs with calcium sulfate leads to an increase in potency by decreasing the effective dose. This effect can be attributed to activation of intrinsic apoptosis signaling and/or pH regulatory properties of CaSO4@SeNPs. However, further studies are still needed to determine the exact corresponding mechanisms of this synergistic effect. 201 206 Elnaz Faghfuri Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Ramak Ajideh Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Faranak Shahverdi Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Mina Hosseini Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Faranak Mavandadnejad Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences Iran Mohammad Hossein Yazdi Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical SciencesDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences Recombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical SciencesDepartment of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences IranIran Ahmad Reza Shahverdi Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical SciencesRecombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences Biotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical SciencesRecombinant Vaccine Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences IranIran ApoptosisBreast neoplasmsCalcium sulfateNanoparticlesSelenium 40478.pdf Gupta A, Shridhar K, Dhillon P. A review of breast cancer awareness among women in India: Cancer literate or awareness deficit? Eur J Cancer 2015;51(14):2058-66.##Uysal E. Top 100 cited classic articles in breast cancer research. Eur J Breast Health 2017;13(3):129-37.##Montagna E, Cancello G, Dellapasqua S, Munzone E, Colleoni M. Metronomic therapy and breast cancer: a systematic review. Cancer Treat Rev 2014;40(8):942-50.##Sharma R. Global, regional, national burden of breast cancer in 185 countries: evidence from GLOBOCAN 2018. Breast Cancer Res Treat 2021.##Sonkusre P, Nanduri R, Gupta P, Cameotra SS. Improved extraction of intracellular biogenic selenium nanoparticles and their specificity for cancer chemoprevention. J Nanomed Nanotechnol 2014;5(2):1.##Maiyo F, Singh M. Selenium nanoparticles: Potential in cancer gene and drug delivery. Nanomedicine (Lond) 2017;12(9):1075-89.##Mao J, Pop VJ, Bath SC, Vader HL, Redman CW, Rayman MP. Effect of low-dose selenium on thyroid autoimmunity and thyroid function in UK pregnant women with mild-to-moderate iodine deficiency. Eur J Nutr 2016;55(1):55-61.##Wang H, Zhang J, Yu H. Elemental selenium at nano size possesses lower toxicity without compromising the fundamental effect on selenoenzymes: comparison with selenomethionine in mice. Free Radic Biol Med 2007;42(10):1524-33.##Tabinda S. Selenium role in reproduction, pregnant/postpartum women and neonates: A current study. Current Nutrition & Food Science 2021;17(1):28-37.##Rock C, Moos PJ. Selenoprotein P regulation by the glucocorticoid receptor. Biometals 2009;22(6):995.##Fang W, Han A, Bi X, Xiong B, Yang W. Tumor inhibition by sodium selenite is associated with activation of c‐Jun NH2‐terminal kinase 1 and suppression of β‐catenin signaling. Int J Cancer 2010;127(1):32-42.##Reid ME, Duffield-Lillico AJ, Garland L, Turnbull BW, Clark LC, Marshall JR. Selenium supplementation and lung cancer incidence: an update of the nutritional prevention of cancer trial. Cancer Epidemiol Biomarkers Prev 2002;11(11):1285-91.##Sygit K, Cipora E, Smorawiński J. The role of selenium in inherited breast cancer in women. Journal of Health Inequalities 2020;6(2):160-5.##Zhang JS, Gao XY, Zhang LD, Bao YP. Biological effects of a nano red elemental selenium. Biofactors 2001;15(1):27-38.##Zhang J, Wang X, Xu T. Elemental selenium at nano size (Nano-Se) as a potential chemopreventive agent with reduced risk of selenium toxicity: comparison with se-methylselenocysteine in mice. Toxicol Sci. 2007;101(1):22-31.##Lu J, Berndt C, Holmgren A. Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase. Biochim Biophys Acta 2009;1790(11):1513-9.##Nayak V, Singh K, Singh A, Singh R. Potentialities of selenium nanoparticles in biomedical sciences. New Journal of Chemistry 2021;45(6):2849-78.##Yazdi MH, Masoudifar M, Varastehmoradi B, Mohammadi E, Kheradmand E, Homayouni S, et al. Effect of oral supplementation of biogenic selenium nanoparticles on white blood cell profile of BALB/c mice and mice exposed to X-ray radiation. Avicenna J Med Biotechnol 2013;5(3):158-67.##Wu JH, Deng YL, Liu Q, Yu JC, Liu YL, He ZQ, et al. Induction of apoptosis and autophagy by calcifying nanoparticles in human bladder cancer cells. Tumor Biol 2017;39(6):1010428317707688.##Pourbaghi-Masouleh M, Hosseini V. Amorphous calcium phosphate nanoparticles could function as a novel cancer therapeutic agent by employing a suitable targeted drug delivery platform. Nanoscale Res Lett 2013;8(1):449.##Khakinezhad Tehrani F, Ranji N, Kouhkan F, Hosseinzadeh S. Apoptosis induction and proliferation inhibition by silibinin encapsulated in nanoparticles in MIA PaCa-2 cancer cells and deregulation of some miRNAs. Iran J Basic Med Sci 2020;23(4):469-82.##Shahverdi AR, Shahverdi F, Faghfuri E, Reza khoshayand M, Mavandadnejad F, Yazdi MH, et al. Characterization of folic acid surface-coated selenium nanoparticles and corresponding in vitro and in vivo effects against breast cancer. Arch Med Res 2018;49(1):10-7.##Xuan G, Zhang M, Chen Y, Huang S, Lee I. Design and characterization of a cancer-targeted drug co-delivery system composed of liposomes and selenium Nanoparticles. J Nanosci Nanotechnol 2020;20(9):5295-304.##Maleki Dizaj S, Barzegar-Jalali M, Zarrintan MH, Adibkia K, Lotfipour F. Calcium carbonate nanoparticles as cancer drug delivery system. Expert Opin Drug Deliv 2015;12(10):1649-60.##Khalifehzadeh R, Arami H. Biodegradable calcium phosphate nanoparticles for cancer therapy. Adv Colloid Interface Sci 2020;279:102157.##Abdel-Gawad EI, Hassan AI, Awwad SA. Efficiency of calcium phosphate composite nanoparticles in targeting Ehrlich carcinoma cells transplanted in mice. J Adv Res 2016;7(1):143-54.##Pi J, Jin H, Liu R, Song B, Wu Q, Liu L, et al. Pathway of cytotoxicity induced by folic acid modified selenium nanoparticles in MCF-7 cells. Appl Microbiol Biotechnol 2013;97(3):1051-62.##Soumya RS, Vineetha VP, Reshma PL, Raghu KG. Preparation and characterization of selenium incorporated guar gum nanoparticle and its interaction with H9c2 cells. PloS One 2013;8(9):e74411.##Dennert G, Zwahlen M, Brinkman M, Vinceti M, Zeegers MP, Horneber M. Selenium for preventing cancer. Cochrane Database Syst Rev 2011;(5):CD005195.##Tapiero H, Townsend D, Tew K. The antioxidant role of selenium and seleno-compounds. Biomed Pharmacother 2003;57(3-4):134-44.##Huang Y, He L, Liu W, Fan C, Zheng W, Wong YS, et al. Selective cellular uptake and induction of apoptosis of cancer-targeted selenium nanoparticles. Biomaterials 2013;34(29):7106-16.##Criddle DN, Gerasimenko JV, Baumgartner HK, Jaffar M, Voronina S, Sutton R, et al. Calcium signalling and pancreatic cell death: apoptosis or necrosis? Cell Death Differ 2007;14(7):1285-94.##Wyllie AH, Morris RG, Smith AL, Dunlop D. Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. J Pathol 1984;142(1):67-77.##Zhang Y, Li X, Huang Z, Zheng W, Fan C, Chen T. Enhancement of cell permeabilization apoptosis-inducing activity of selenium nanoparticles by ATP surface decoration. Nanomedicine 2013;9(1):74-84.##Ren Y, Zhao T, Mao G, Zhang M, Li F, Zou Y, et al. Antitumor activity of hyaluronic acid–selenium nanoparticles in Heps tumor mice models. Int J Biol Macromol 2013;57:57-62.##Uğuz AC, Nazıroğlu M, Espino J, Bejarano I, González D, Rodríguez AB, et al. Selenium modulates oxidative stress-induced cell apoptosis in human myeloid HL-60 cells through regulation of calcium release and caspase-3 and-9 activities. J Membr Biol 2009;232(1-3):15-23.##Masouleh MP, Hosseini V, Pourhaghgouy M, Bakht MK. 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Molecular Mechanisms of Anti-inflammatory Activities of the Extracts of Ocimum gratissimum and Thymus vulgaris 2 2 Background: A large body of literature suggests that the extracts of Ocimum gratissimum (O. gratissimum) and Thymus vulgaris (T. vulgaris) play protective roles against various inflammatory disorders. However, the possible mechanism of action with reference to the interactions of their respective phytochemical compositions with pro-inflammatory mediators as the indication of their therapeutic effects is less clear. Therefore, the immunomodulatory properties of O. gratissimum and T. vulgaris were investigated in this study. Methods: The in vitro lipoxygenase inhibitory potentials of methanolic extracts of the selected plants were assessed through colorimetric analysis. The pharmacokinetics of some identified compounds in the botanicals were investigated via the Swiss ADME server while the molecular interactions of the compounds with lipoxygenase, IL-1, IL-6, TNF-α, IL-8, and CCL-2 were performed through molecular docking. Results: The assessment of the lipoxygenase inhibition revealed the extracts could possess anti-inflammatory agents. The pharmacokinetic results of some selected compounds identified in the botanicals showed moderate toxic effects compared to indomethacin. The molecular docking study substantiated the report of the in vitro analysis as indicated in the binding score of all the selected compounds compared to indomethacin. Conclusion: The phytochemical components of the extracts of O. gratissimum and T. vulgaris could be effective as anti-inflammatory agents that could be explored in preventing disorders associated with excessive activities of pro-inflammatory mediators. 207 216 Ige Francis Olaoye Department of Biochemistry, McPherson University, Seriki Sotayo Department of Biochemistry, McPherson University, Seriki Sotayo Nigeria Babatunde Joseph Oso Adepeju Aberuagba Department of Biochemistry, University of Ilorin, Ilorin Department of Biochemistry, University of Ilorin, Ilorin Nigeria Anti-inflammatory agentsLipoxygenaseOcimumPhytochemicalsPlant extractsThymus plant 40479.pdf Cheung CY, Poon LLM, Ng IHY, Luk W, Sia SF, et al. Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis. J Virol 2005;79(12):7819-26.##Lau SKP, Lau CCY, Chan KH, Li CPY, Chen H, Jin DY, et al. Delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel Middle East respiratory syndrome coronavirus: implications for pathogenesis and treatment. J Gen Virol 2013;94(12):2679-90.##Chen L, Liu HG, Liu W, Liu J, Liu K, Shang J.[Analysis of clinical features of 29 patients with 2019 novel coronavirus pneumonia]. Zhonghua Jie He He Hu Xi Za Zhi 2020;6;43(0):E005. Chinese.##Radi ZA, Meyerholz DK, Ackermann MR. Pulmonary cyclooxygenase-1 (COX-1) and COX-2 cellular expression and distribution after respiratory syncytial virus and parainfluenza virus infection. Viral Immunol 2010;23(1):43-8.##Amri O, Zekhnini A, Bouhaimi A, Tahrouch S, Hatimi A. Anti-inflammatory activity of methanolic extract from Pistacia atlantica Desf. leaves. Pharmacog J 2018;10(1):71-6.##Oso BJ, Oyewo EB, Oladiji AT. Ethanolic, N-hexane and aqueous partitioned extracts of Xylopia aethiopica fruit modulated inflammatory responses in turpentine oil induced acute inflammation in male wistar rats. Int J Res Health Sci 2017; 5(2):1-10.##Basch E, Ulbricht C, Hammerness P, Blevins A, Sollars D. Thyme (Thymus vulgaris L.) thymol. J Herb Pharmacother 2004;4(1):49-67.##Shinde UA, Phadke AS, Nair AM, Mungantiwar AA, Dikshit VJ, Saraf MN. Membrane stabilizing activity - a possible mechanism of action for the anti-inflammatory activity of Cedrus deodara wood oil Author links open overlay panel. Fitoterapia 1999;70(3):251-7.##Sorkun MC, Khetan A, Süleyman Er. AqSolDB, a curated reference set of aqueous solubility and 2D descriptors for a diverse set of compounds. Sci Data 2019;6(1):143.##Oso BJ, Karigidi KO. Inhibitory action of dried leaf of Cassia alata (Linn.) Roxb against lipoxygenase activity and nitric oxide generation. Scien Agropec 2019;10(2):185-90.##Daina A, Michielin O, Zoete V. 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Prevention of NSAID-associated Gastrointestinal Lesions: A comparison study pantoprazole versus Omeprazole. Am J Gastroenterol 2006;101(8):1747-55.##Wei A, Shibamoto T. Antioxidant/Lipoxygenase inhibitory activities and chemical compositions of selected essential oils. J Agric Food Chem 2010;58(12):7218-25.##Oso BJ, Olaoye IF. Comparative in vitro studies of antiglycemic potentials and molecular docking of Ageratum conyzoides L. and Phyllanthus amarus L. methanolic extracts. SN App Sci 2020;2:629.##Brylinski M. Aromatic interactions at the ligand-protein interface: Implications for the development of docking scoring functions. Chem Biol Drug Des 2018;91(2):380-90.##Arthur DE, Uzairu A. Molecular docking studies on the interaction of NCI anticancer analogues with human Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit. J King Saud University Science 2019;31(4):1151-66.##Yadav DK, Kumar S, Saloni, Misra S, Yadav L, Teli M, et al. Molecular insights into the interaction of RONS and Thieno[3,2-c]pyran analogs with SIRT6/COX-2: A molecular dynamics study. Sci Rep 2018;8(1):4777.##Fonteh P, Elkhadir A, Omondi B, Guzei I, Darkwa J, Meyer D. Impedance technology reveals correlations between cytotoxicity and lipophilicity of mono and bimetallic phosphine complexes. Biometals 2015;28(4):653-67.##Daly AK, Rettie AE, Fowler DM, Miners JO. Pharmacogenomics of CYP2C9: Functional and clinical considerations. J Pers Med 2018;8(1):1.##Zhao L, Cuff CA, Moss E, Wille U, Cyrus T, Klein EA, et al. Selective interleukin-12 synthesis defect in 12/15-lipoxygenase-deficient macrophages associated with reduced atherosclerosis in a mouse model of familial hypercholesterolemia. J Biol Chem 2002;277(38):35350-6.##Lin HC, Lin TH, Wu MY, Chiu YC, Tang CH, Hour MJ, et al. 5-Lipoxygenase inhibitors attenuate TNF-α-Induced inflammation in human synovial fibroblasts. 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Construction and Evaluation of Short Hairpin RNAs for Knockdown of Metadherin mRNA 2 2 Background: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes’ function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. Methods: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. Results: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. Conclusion: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application. 217 222 Farahnaz Zare Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences IranIran Sedigheh Sharifzadeh Abbas Behzad-Behbahani Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences IranIran Gholamreza Rafiei Dehbidi Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Iran Zahra Yousefi School of Allied Medical Sciences, Shahroud University of Medical Sciences School of Allied Medical Sciences, Shahroud University of Medical Sciences Iran Reza Ranjbaran Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Diagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Iran Noorossadat Seyyedi Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences Division of Medical Biotechnology, Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Shiraz University of Medical SciencesDiagnostic Laboratory Sciences and Technology Research Center, Faculty of Paramedical Sciences, Shiraz University of Medical Sciences IranIran ApoptosisGene expressionGene silencingJurkat cellsRNA 40480.pdf Carthew RW, Sontheimer EJ. Origins and Mechanisms of miRNAs and siRNAs. Cell 2009;136(4):642-55.##Størvold GL, Gjernes E, Askautrud HA, Børresen-Dale AL, Perou CM, Frengen E. A retroviral vector for siRNA expression in mammalian cells. Mol Biotechnol 2007;35(3):275-82.##Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391(6669):806-11.##Maczuga P, Koornneef A, Borel F, Petry H, van Deventer S, Ritsema T, et al. Optimization and comparison of knockdown efficacy between polymerase II expressed shRNA and artificial miRNA targeting luciferase and Apolipoprotein B100. BMC Biotechnol 2012;12:42.##Chen X, Mangala LS, Rodriguez-Aguayo C, Kong X, Lopez-Berestein G, Sood AK. RNA interference-based therapy and its delivery systems. Cancer Metastasis Rev 2018;37(1):107-24.##Kim B, Park JH, Sailor MJ. Rekindling RNAi therapy: materials design requirements for in vivo siRNA delivery. 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Evaluation of miR-222 Expression in HBV Infected Patients in Comparison with HDV and HBV Co-infected Patients 2 2 Background: Liver disease is more severe in HDV+HBV co-infected patients than HBV infected patients which seems to be related to differences in the expression of genes and other factors such as MicroRNAs (miRNAs). The aim of this study was to investigate miR-222 expression in HBV infected patients in comparison with HDV+HBV co-infected patients. Methods: First, total RNA was extracted from the serum samples and then, complementary DNA (cDNA) was produced using cDNA synthesis kit. Finally, miR-222 gene expression was measured using U6 as the internal control by quantitative PCR (qPCR). Results: The level of miR-222 expression in HDV+HBV co-infected samples was significantly up regulated. The fold change of the miR-222 expression between two groups was 3.3 (95% CI; 0.011- 17.63) with p<0.001. Conclusion: The expression of miR-222 was higher in HBV+HDV co-infected patients than HBV infected patients. Further studies should be conducted to confirm whether miR-222 can be a biomarker for prognosis of severe liver diseases. 223 225 Zahra Sokhanvar Department of Microbiology, Faculty of Biological Sciences, Alzahra University Department of Microbiology, Faculty of Biological Sciences, Alzahra University Iran Ameneh Elikaei Department of Microbiology, Faculty of Biological Sciences, Alzahra University Department of Microbiology, Faculty of Biological Sciences, Alzahra University Iran Zohreh Sharifi Co-infection with hepatitis B and DHepatitis B virusHepatitis D virusMiR-222 40481.pdf European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017;67(2):370-98.##Lee WM. Hepatitis B virus infection. N Engl J Med 1997;337(24):1733-45.##WHO. 2015 [Available from: https://www.who.int.##Kim BH, Kim WR. Epidemiology of hepatitis B virus infection in the United States. Clin Liver Dis (Hoboken) 2018;12(1):1-4.##Rizzetto M. Hepatitis D: thirty years after. J Hepatol 2009;50(5):1043-50.##Hadler SC, De Monzon M, Ponzetto A, Anzola E, Rivero D, Mondolfi A, et al. Delta virus infection and severe hepatitis: an epidemic in the Yucpa Indians of Venezuela. Ann Intern Med 1984;100(3):339-44.##Govindarajan S, Chin KP, Redeker AG, Peters RL. Fulminant B viral hepatitis: role of delta agent. Gastroenterology 1984;86(6):1417-20.##Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T, et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006;25(17):2537-45.##Ross SA, Davis CD. The emerging role of microRNAs and nutrition in modulating health and disease. Annu Rev Nutr 2014;34:305-36.##Zhang B, Pan X, Cobb GP, Anderson TA. microRNAs as oncogenes and tumor suppressors. Dev Biol 2007;302(1):1-12.##Bandopadhyay M, Bharadwaj M. Exosomal miRNAs in hepatitis B virus related liver disease: a new hope for biomarker. Gut Pathog 2020;12:23.##Tao J, Jiang L, Chen X. Roles of microRNA in liver cancer. Liver Research 2018;2(2):61-72.##Wang XW, Forrester K, Yeh H, Feitelson MA, Gu JR, Harris CC. Hepatitis B virus X protein inhibits p53 sequence-specific DNA binding, transcriptional activity, and association with transcription factor ERCC3. Proc Natl Acad Sci USA 1994;91(Suppl 6):2230-4.##Galardi S, Mercatelli N, Giorda E, Massalini S, Frajese GV, Ciafrè SA, et al. miR-221 and miR-222 expression affects the proliferation potential of human prostate carcinoma cell lines by targeting p27 Kip1. J Biol Chem 2007;282(32):23716-24.##Pollicino T, Raffa G, Santantonio T, Gaeta GB, Iannello G, Alibrandi A, et al. Replicative and transcriptional activities of hepatitis B virus in patients coinfected with hepatitis B and hepatitis delta viruses. J Virol 2011;85(1):432-9.##Mentha N, Clément S, Negro F, Alfaiate D. A review on hepatitis D: From virology to new therapies. J Adv Res 2019;17:3-15.##

Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs 2 2 Background: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed. One such method is Restriction Enzyme (RE) mapping. Restriction enzyme mapping can be used to screen for numerous plasmid recombinants simultaneously. Methods: In this study, the sgRNA templates were initially cloned into the above PX459 plasmids. Subsequently, the accuracy of the constructs was determined by RE mapping. Results: This method was established to screen for sgRNA-bearing PX459 plasmids. However, numerous anomalies were detected after ligation of sgRNA templates into RE digested PX459 plasmids. Conclusion: Our data suggest that RE mapping is only appropriate as an initial screen and that the identity of all plasmids with the correctly identified RE maps should be confirmed by Sanger sequencing. 226 229 Mehdi Hassani Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Sara Hesami Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Nahal Maroofi Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Mehdi Banan CRISPR-Cas systemsGene editingHealth servicesPlasmidsRestriction mapping 40483.pdf Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013;31(3):230-2.##Sander JD, Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol 2014;32(4):347-55.##Xu X, Wan T, Xin H, Li D, Pan H, Wu J, et al. Delivery of CRISPR/Cas9 for therapeutic genome editing. J Gene Med 2019;21(7):e3107.##Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819-23.##Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 2013;8(11):2281-308.##Güssow D, Clackson T. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucleic Acids Res 1989;17(10):4000.##Agrawal V, Roy N. Contaminating insert degradation by preincubation colony PCR: A method for avoiding false positives in transformant screening. Anal Biochem 2008;375(1):159-61.##Bimboim H, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 1979;7(6):1513-23.##Chang AY, Chau V, Landas JA, Pang Y. Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI Methods 2017;1:22-5.##Green MR, Sambrook J. Preparation of plasmid DNA by alkaline lysis with sodium dodecyl sulfate: minipreps. Cold Spring Harb Protoc 2016;2016(10).##

Identification of a Novel Homozygous Mutation in BBS10 Gene in an Iranian Family with Bardet-Biedl Syndrome 2 2 Background: Bardet–Biedl Syndrome (BBS) is a rare pleiotropic autosomal recessive disease related to ciliopathies with approximately 25 causative genes. BBS is a multisystemic disorder with wide spectrum of manifestations including truncal obesity, retinal dystrophy, male hypogenitalism, postaxial polydactyly, learning difﬁculties, and renal abnormalities. Methods: A consanguineous Iranian family with a 28-year-old daughter affected with BBS, resulting from a first cousin marriage, was examined. After clinical examination, Whole Exome Sequencing (WES) was applied. Following the analysis of exome data, Sanger sequencing was used to confirm as well as to co-segregate the candidate variant with the phenotype. Results: A novel homozygous variant [c. 2035G>A (p.E679K)] in exon 2 of the BBS10 gene was found which was categorized as likely pathogenic based on American College of Medical Genetics and Genomics (ACMG) guidelines and criteria. In this study, the variant was fully co-segregated with the phenotype in the family. Conclusion: Despite overlapping with other ciliopathies in terms of the phenotype, the BBS has high genetic heterogeneity and clinical variability even among affected members of a family. The symptoms observed in patients are largely related to the genes involved and the type of mutations in the BBS. In this study, in addition to phenotype description of the proband harboring a novel disease-causing variant in BBS10 gene, the spectrum of BBS symptoms was expanded. The findings of this study can be useful in genetic counseling, especially for risk estimation and prenatal diagnosis. 230 233 Mohammad Dehani Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Davood Zare-Abdollahi Ata Bushehri Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Azadeh Dehghani Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University Medical Biotechnology Research Center, Ashkezar Branch, Islamic Azad University Iran Jalil Effati Meybod Genetic Research Center, State Welfare Organization Iran Seyed Ali Mohammad Miratashi Department of Ophthalmology, Shahid Sadoughi University of Medical Sciences Department of Ophthalmology, Shahid Sadoughi University of Medical Sciences Iran Hamid Reza Khorram Khorshid Fetal Health Research Center, Hope Generation Foundation Iran Bardet–Biedl syndromeMutationWhole exome sequencing 40482.pdf M'hamdi O, Redin C, Stoetzel C, Ouertani I, Chaabouni M, Maazoul F, et al. Clinical and genetic characterization of Bardet–Biedl syndrome in Tunisia: defining a strategy for molecular diagnosis. Clin Genet 2014;85(2):172-7.##Khan S, Ullah I, Touseef M, Basit S, Khan MN, Ahmad W. Novel homozygous mutations in the genes ARL6 and BBS10 underlying Bardet–Biedl syndrome. Gene 2013;515(1):84-8.##Kurata K, Hosono K, Hikoya A, Kato A, Saitsu H, Minoshima S, et al. Clinical characteristics of a Japanese patient with Bardet-Biedl syndrome caused by BBS10 mutations. Jpn J Ophthalmol 2018;62(4):458-66.##Forsythe E, Sparks K, Best S, Borrows S, Hoskins B, Sabir A, et al. Risk factors for severe renal disease in bardet–biedl syndrome. J Am Soc Nephrol 2017;28(3):963-70.##base Rd. https://sph.uth.edu/retnet/notes.htm. 2019.##Niederlova V, Modrak M, Tsyklauri O, Huranova M, Stepanek O. Meta‐analysis of genotype‐phenotype associations in Bardet‐Biedl syndrome uncovers differences among causative genes. Hum Mutat 2019;40(11):2068-87.##Fattahi Z, Rostami P, Najmabadi A, Mohseni M, Kahrizi K, Akbari MR, et al. Mutation profile of BBS genes in Iranian patients with Bardet–Biedl syndrome: genetic characterization and report of nine novel mutations in five BBS genes. J Hum Genet. 2014;59(7):368.##Katsanis N. The oligogenic properties of Bardet–Biedl syndrome. Hum Mol Genet 2004;13(suppl_1):R65-R71.##Nishimura DY, Swiderski RE, Searby CC, Berg EM, Ferguson AL, Hennekam R, et al. Comparative genomics and gene expression analysis identifies BBS9, a new Bardet-Biedl syndrome gene. Am J Hum Genet 2005;77(6):1021-33.##Stoetzel C, Muller J, Laurier V, Davis EE, Zaghloul NA, Vicaire S, et al. Identification of a novel BBS gene (BBS12) highlights the major role of a vertebrate-specific branch of chaperonin-related proteins in Bardet-Biedl syndrome. Am J Hum Genet 2007;80(1):1-11.##Chandrasekar SP, Namboothiri S, Sen P, Sarangapani S. 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The Potential Anti-inflammatory Effects of Zerumbone in COVID-19 Patients 2 2 No Abstract 234 236 Razieh Dehghan Research Center for Molecular Medicine, Faculty of Medicine, Hamadan University of Medical Sciences Research Center for Molecular Medicine, Faculty of Medicine, Hamadan University of Medical Sciences Iran Mohammad Hasan Soheilifar Department of Medical Laser, Medical Laser Research Center, Yara Institute, ACECR Iran Farid Azizi Jalilian Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences Department of Virology, Faculty of Medicine, Hamadan University of Medical Sciences Iran Rezvan Najafi Research Center for Molecular Medicine, Faculty of Medicine, Hamadan University of Medical Sciences Research Center for Molecular Medicine, Faculty of Medicine, Hamadan University of Medical Sciences Iran Razieh Amini Editorial 40484.pdf García LF. Immune response, inflammation, and the clinical spectrum of COVID-19. FrontI mmunol 2020;11:1441.##Chien TY, Kuan-Hua Huang S, Lee CJ, Tsai PW, Wang CC. Antinociceptive and anti-inflammatory effects of zerumbone against mono-iodoacetate-induced arthritis. Int J Mol Sci 2016;17(2):249.##Huang Q, Wu X. Targeting inflammation and cytokine storm in COVID-19. Pharmacol Res 2020;159:105051.##Chu H, Chan JFW, Wang Y, Yuen TTT, Chai Y, Hou Y, et al. Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19. Clin Infect Dis 2020;71(6):1400-9.##Blanco-Melo D, Nilsson-Payant BE, Liu WC, Uhl S, Hoagland D, Møller R, et al. Imbalanced host response to SARS-CoV-2 drives development of COVID-19. Cell 2020;181(5):1036-45.##Merad M, Martin JC. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nature Reviews Immunology 2020;20:355-62.##Haque MA, Jantan I, Harikrishnan H. Zerumbone suppresses the activation of inflammatory mediators in LPS-stimulated U937 macrophages through MyD88-dependent NF-κB/MAPK/PI3K-Akt signaling pathways. Int Immunopharmacol 2018;55:312-22.##Gopalsamy B, Omar Farouk AA, Shah Tengku Mohamad TA, Sulaiman MR, Perimal EK. Antiallodynic and antihyperalgesic activities of zerumbone via the suppression of IL-1β, IL-6, and TNF-α in a mouse model of neuropathic pain. J Pain Res 2017;10:2605-19.##Tzeng TF, Liou SS, Chang CJ, Liu IM. Zerumbone, a tropical ginger sesquiterpene, ameliorates streptozotocin-induced diabetic nephropathy in rats by reducing the hyperglycemia-induced inflammatory response. Nutr Metab 2013;10(1):64.##Murakami A, Hayashi R, Tanaka T, Kwon KH, Ohigashi H, Safitri R. Suppression of dextran sodium sulfate-induced colitis in mice by zerumbone, a subtropical ginger sesquiterpene, and nimesulide: separately and in combination. Biochem Pharmacol 2003;66(7):1253-61.##Szabolcs A, Tiszlavicz L, Kaszaki J, Pósa A, Berkó A, Varga IS, et al. Zerumbone exerts a beneficial effect on inflammatory parameters of cholecystokinin octapeptide-induced experimental pancreatitis but fails to improve histology. Pancreas 2007;35(3):249-55.##Abdelwahab SI, Abdul AB, Mohd Zain ZN, Hadi AHA. Zerumbone inhibits interleukin-6 and induces apoptosis and cell cycle arrest in ovarian and cervical cancer cells. Int Immunopharmacol 2012; 12(4):594-602.##Jahani M, Dokaneheifard S, Mansouri K. Hypoxia: A key feature of COVID-19 launching activation of HIF-1 and cytokine storm. J Inflamm (Lond) 2020;17(1):33.##Sung B, Jhurani S, Ahn KS, Mastuo Y, Yi T, Guha S, et al. Zerumbone down-regulates chemokine receptor CXCR4 expression leading to inhibition of CXCL12-induced invasion of breast and pancreatic tumor cells. Cancer Res 2008;68(21):8938-44.##Hwang S, Jo M, Hong JE, Park CO, Lee CG, Yun M, et al. Zerumbone suppresses enterotoxigenic bacteroides fragilis infection-induced colonic inflammation through inhibition of NF-κΒ. Int J Mol Sci 2019;20(18):4560.##Nayak S, Singh S. Zerumbone, a natural plant dietary compound induces expression of interleukin-12P70 cytokine in human peripheral blood mononuclear cells. Asian J Pharm Clin Res 2016;9(3):312-5.##