Microbiota and Autism spectrum disorder 2 2 Autism spectrum disorder (ASD) represents a neurodevelopmental condition characterized by two main deficits: impaired social communication and interaction; restricted and repetitive patterns of interests, behaviors or activities 1 with prevalence ranges from 2 to 20 per 1,000, worldwide 1,2. Presently, core symptoms of autism have no approved treatment. Autistic disorder management emphasis is on behavioral and educational modalities that target the core symptoms 3. Psychopharmacologic interventions are introduced to improve daily function and treat associated behavioral problems including hyperactivity, irritability, and aggression; leading to support the implementation of behavioral approaches through reducing the interfering symptoms 3. In recent years, a growing number of studies have found evidence implicating dysregulation of immune responses and neuroinflammatory mechanisms in patients with ASD 3. Dysregulation of T helper cells 4, increased plasma levels of proinflammatory cytokines such as interleukin 1, 6 and 8 1-3, increased proliferation and activation of B cells and natural killer cells 4, decreased serum levels of immunoglobulin G and M 5 in presence of immunoglobulin G autoantibodies against neuron-axon filament and glial fibrillary acidic proteins 6, and increased microglial and astrocytic density and activation 5 have been documented. Risperidone, as a serotonin 5-HT (2A) receptor antagonist that can attenuate dopamine release as well 3, is the only drug approved by the Food and Drug Administration (FDA) for the treatment of irritability in children with ASD. In recent years, research correlates risperidone use with significant weight gain 3 and with high rate of relapse after discontinuation of the medication in children with ASD 4. Despite this, due to lack of insight into the exact pathogenesis of ASD, no new medication could be approved as an adjuvant or standalone treatment for patients with ASD.  The fact that diet has a huge influence on our health should be common knowledge by now. But what research has been showing us in recent years is just how fundamental the influence of diet on our health can be. Surprising links between diet and a number of previously unsuspected diseases are being continuously established. But food does not affect us only after we are born, it actually starts to shape our health during pre-natal development. Many of these associations between diet and disease, including neurological diseases, are now known to be modulated by the gut microbiota. Research on the gut-brain axis is a blooming field where new and important findings keep streaming. Individuals with ASD often also have gastrointestinal problems and dysbiosis of the gut microbiota, being unclear whether an altered gut flora is a cause of a comorbidity of ASD. It has been suggested that changes in the gut microbiota may indeed play a role in the development of the behavioral symptoms associated with ASD, but the possible mechanisms of this link remain unknown 6. As for autism, this link may come down to a particular molecule called interleukin-17a (or IL-17a), which is produced by the immune system. The molecule has already been associated with conditions like rheumatoid arthritis, multiple sclerosis, and psoriasis, and has been shown to serve an important role in preventing infections, notably those of the fungal kind. Importantly, it can also influence the way the brain develops in the womb. The effects of the microbiome on the development of MIA-induced autism could be prevented either by modifying the pregnant mother’s microbiome, or by directly blocking IL-17a signaling. Although ASD primarily impacts the brain, over recent years, links with other systems have become clear, in particular, gastrointestinal (issues seem to occur more often in individuals with ASD than in the rest of the population 6. 129 129 Shahin Akhondzadeh Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences Psychiatric Research Center, Roozbeh Hospital, Tehran University of Medical Sciences Iran Editorial 10348.pdf Zhang XC, Shu LQ, Zhao XS, Li XK. Autism spectrum disorders: autistic phenotypes and complicated mechanisms. World J Pediatr 2019;15(1):17-25. ##Khalaj M, Saghazadeh A, Shirazi E, Shalbafan MR, Alavi K, Shooshtari MH, et al. Palmitoylethanolamide as adjunctive therapy for autism: Efficacy and safety results from a randomized controlled trial. 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Review of Different Sequence Motif Finding Algorithms 2 2 The DNA motif discovery is a primary step in many systems for studying gene function.  Motif discovery plays a vital role in identification of Transcription Factor Binding Sites (TFBSs) that help in learning the mechanisms for regulation of gene expression. Over the past decades, different algorithms were used to design fast and accurate motif discovery tools. These algorithms are generally classified into consensus or probabilistic approaches that many of them are time-consuming and easily trapped in a local optimum. Nature-inspired algorithms and many of combinatorial algorithms are recently proposed to overcome these problems. This paper presents a general classification of motif discovery algorithms with new sub-categories that facilitate building a successful motif discovery algorithm. It also presents a summary of comparison between them. 130 148 Fatma Hashim Department of Biomedical Engineering, Helwan University Department of Biomedical Engineering, Helwan University Egypt Mai Mabrouk Department of Biomedical Engineering, Misr University for Science and Technology (MUST) Department of Biomedical Engineering, Misr University for Science and Technology (MUST) Egypt Walid Al-Atabany Department of Biomedical Engineering, Helwan University Department of Biomedical Engineering, Helwan University Egypt AlgorithmsBioinformaticsConsensusGene expression regulationNucleotide motifProtein binding 10368.pdf Xiong J. Essential bioinformatics. United Kingdom: Cambridge University Press; 2006. 339 p.##Zhang X, Zhou X, Wang X. Basics for Bioinformatics. In: Jiang R, Zhang X, Zhang MQ, (eds). Basics of Bioinformatics. Beijing, Heidelberg: Tsinghua University Press and Springer-Verlag; 2013. p. 1-25.##Al Bataineh MF, Al-qudah Z, Al-Zaben A. 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CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the Mouse NIH/3T3 Cells 2 2 Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting Rag genes to produce mutant mouse NIH/3T3 cell line. The Rag1 and Rag2 genes are essential for generation of mature B and T lymphocytes. Disruption of Rag genes causes disease like Severe Combined Immunodeficiency syndrome (SCID). Here, the efficiency and specificity of CRISPR system were tested with highly active sgRNAs to generate novel mutations in the NIH/3T3 mouse cell line. Methods: Four single guide RNAs were designed to target sequences in the coding region of the Rag1 and Rag2 genes. Four sgRNA-CAS9 plasmids were tested to target Rag1 and Rag2.  Results: Based on T7 endonuclease assay and sequencing analysis, the expression of sgRNAs targeting two sites in Rag1 resulted in deletion of the intervening DNA fragment. The expression of sgRNAs with Cas9 targeting two sites in Rag2 gene resulted in indel mutations at both sites. In this report, fragment deletion in Rag1 gene was detected in about 50% of transfected cells. Conclusion: Therefore, CRISPR/Cas9 system can be highly efficient and specific when gRNAs are designed rationally and provides a powerful approach for genetic engineering of cells and model animals. 149 155 Maryam Mehravar Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Abolfazl Shirazi Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRResearch Institute of Animal Embryo Technology, Shahrekord University Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECRResearch Institute of Animal Embryo Technology, Shahrekord University IranIran Mohammad Mehdi Mehrazar Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR Iran Mahboobeh Nazari Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR Iran Mehdi Banan Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Genetics Research Center, University of Social Welfare and Rehabilitation Sciences Iran Cell lineDeletionGene editingMice 10369.pdf Carroll D, Morton JJ, Beumer KJ, Segal DJ. Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 2006;1(3):1329-1341. ##Sanjana NE, Cong L, Zhou Y, Cunniff MM, Feng G, Zhang F. A transcription activator-like effector toolbox for genome engineering. Nat Protoc 2012;7(1):171-192. ##Pennisi E. The CRISPR craze. Science 2013;341(6148):833-836. ##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):819-823. ##Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339(6121):823-826. ##Baker M. Gene editing at CRISPR speed. Nat Biotechnol 2014;32(4):309-313. ##Jore MM, Lundgren M, Van Duijn E, Bultema JB, Westra ER, Waghmare SP, et al. Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 2011;18(5):529-536. ##Canver MC, Bauer DE, Dass A, Yien YY, Chung J, Masuda T, et al. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells. J Biol Chem 2014;289(31):21312-21324. ##DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 2013;41(7):4336-4343. ##Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci USA 2012;109(39):E2579-E2586. ##Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells.Elife 2013;2:e00471.##Delerue F, Ittner LM. Genome editing in mice using CRISPR/Cas9: achievements and prospects. Clon Transgen 2015;4:135.##Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. 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Nat Biotechnol 2013;31(9):822-826. ##Belizário JE. Immunodeficient mouse models: an over-view. The Open Immunology J 2009;2(1):79-85.##Chapdelaine P, Pichavant C, Rousseau J, Paques F, Tremblay J. Meganucleases can restore the reading frame of a mutated dystrophin. Gene Ther 2010;17(7):846-858. ##Ménoret S, Fontanière S, Jantz D, Tesson L, Thinard R, Rémy S, et al. Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases. FASEB J 2013;27(2):703-711. ##Niu Y, Shen B, Cui Y, Chen Y, Wang J, Wang L, et al. Generation of gene-modified cynomolgus monkey via Cas9/RNA-mediated gene targeting in one-cell embryos. Cell 2014;156(4):836-843. ##Ji Y, Resch W, Corbett E, Yamane A, Casellas R, Schatz DG. The in vivo pattern of binding of RAG1 and RAG2 to antigen receptor loci. Cell 2010;141(3):419-431. ##Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, et al. Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation. 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Comparison of T7E1 and surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3 (Bethesda) 2015;5(3):407-415. ##Xiao A, Wang Z, Hu Y, Wu Y, Luo Z, Yang Z, et al. Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish. Nucleic Acids Res 2013;41(14):e141-e141. ##Zhou J, Wang J, Shen B, Chen L, Su Y, Yang J, et al. Dual sgRNAs facilitate CRISPR/Cas9‐mediated mouse genome targeting. FEBS J 2014;281(7):1717-1725. ##Zhang L, Jia R, Palange NJ, Satheka AC, Togo J, An Y, et al. Large genomic fragment deletions and insertions in mouse using CRISPR/Cas9. PloS One 2015;10(3):e0120396. ##Bauer DE, Canver MC, Orkin SH. Generation of genomic deletions in mammalian cell lines via CRISPR/Cas9. Journal of visualized experiments: J Vis Exp 2015;(95):52118.##

Expression Patterns for TETs, LGR5 and BMI1 in Cancer Stem-like Cells Isolated from Human Colon Cancer 2 2 Background: Colon tumor is generated and maintained by a small subset of chemo-resistant cancer cells known as Cancer Stem-like Cells (CSCs) that are able to self-renew and differentiate into various cell types within the cancer milieu. CSCs are identified through expression of CD133 that is the most important surface marker of these cells. Epithelial Cell Adhesion Molecule (EpCAM) is another colon CSCs marker. Other markers that are probably involved in colon tumorigenesis are Leucine-rich repeat-containing G-protein-coupled Receptor 5 (LGR5), B cell-specific Moloney murine leukemia virus insertion site 1 (BMI1) and Ten-Eleven Translocations (TETs).  Methods: Here, mRNA expression rates of LGR5, BMI1 and TETs were surveyed by real-time PCR. After collection and digestion, colon samples were used to isolate CD133 and EpCAM positive CSCs through evaluation of AC133 EpCAM by Magnetic Activated Cell Sorting (MACS) and flow cytometry. Real-time PCR was carried out for assessing expressions of LGR5, BMI1 and TETs.  Results: High expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM positive CSCs (p≤0.05 vs. non-CSCs) were found. TET3, however, showed no significant changes for mRNA expression in the CSCs.  Conclusion: In conclusion, high mRNA expressions for LGR5, BMI1, TET1 and TET2 in the CD133 and EpCAM positive CSCs may be a useful criterion for better identification of the cells involved in colon cancer in order to specify therapeutic targets against this type of cancer. 156 161 Nader Atlasy Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Iran Fardin Amidi Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Iran Keywan Mortezaee Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences Department of Anatomy, Faculty of Medicine, Kurdistan University of Medical Sciences Iran Mohammad Sadegh Fazeli Department of Surgery, Imam Khomeini Hospital, Tehran University of Medical Sciences Department of Surgery, Imam Khomeini Hospital, Tehran University of Medical Sciences Iran Seyed Javad Mowla Department of Genetics, Faculty of Life Sciences, Tarbiat Modares University Department of Genetics, Faculty of Life Sciences, Tarbiat Modares University Iran Fatemeh Malek Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences Iran ColonFlow cytometryMolony murine leukemia virusNeoplastic stem cells 10361.pdf Wahab SR, Islam F, Gopalan V, Lam AK-y. The identifications and clinical implications of cancer stem cells in colorectal cancer. Clin Colorectal Cancer 2017;16(2):93-102.##Sahlberg SH, Spiegelberg D, Glimelius B, Stenerlöw B, Nestor M. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells. PloS One 2014;9(4):e94621. ##O’Brien CA, Pollett A, Gallinger S, Dick JE. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445(7123):106-110.##Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2006;445 (7123):111-115.##de Sousa e Melo F, Kurtova AV, Harnoss JM, Kljavin N, Hoeck JD, Hung J, et al. A distinct role for Lgr5+ stem cells in primary and metastatic colon cancer. Nature 2017;543(7647):676-680.##Fathi A, Mosaad H, Hussein S, Roshdy M, Ismail EI. Prognostic significance of CD133 and ezrin expression in colorectal carcinoma. IUBMB Life 2017;69(5):328-340.##Sureban SM, Qu D, Houchen CW. Regulation of miRNAs by agents targeting the tumor stem cell markers DCLK1, MSI1, LGR5, and BMI1. Curr Pharmacol Rep 2015;1(4):217–222.##Wu XS, Xi HQ, Chen L. Lgr5 is a potential marker of colorectal carcinoma stem cells that correlates with patient survival. World J Surg Oncol 2012;10(1):244.##Scourzic L, Mouly E, Bernard OA. TET proteins and the control of cytosine demethylation in cancer. Genome Med 2015;7(1):9. ##Kinney SRM, Pradhan S. Ten eleven translocation enzymes and 5-hydroxymethylation in mammalian development and cancer. In: Epigenetic Alterations in Oncogenesis: Springer; 2013. p. 57-79.##Kemper K, Sprick MR, de Bree M, Scopelliti A, Vermeulen L, Hoek M, et al. The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation. Cancer Res 2010;70(2):719-729.##Saigusa S, Tanaka K, Toiyama Y, Yokoe T, Okugawa Y, Ioue Y, et al. Correlation of CD133, OCT4, and SOX2 in rectal cancer and their association with distant recurrence after chemoradiotherapy. Ann Surg Oncol 2009;16(12):3488-3498.##Kemper K, Versloot M, Cameron K, Colak S, de Sousa e Melo F, de Jong JH, et al. Mutations in the Ras-Raf Axis underlie the prognostic value of CD133 in colorectal cancer. Clin Cancer Res 2012;18(11):3132-3141.##Gazzaniga P, Gradilone A, Petracca A, Nicolazzo C, Raimondi C, Iacovelli R, et al. Molecular markers in circulating tumour cells from metastatic colorectal cancer patients. J Cell Mol Med 2010;14(8):2073-2077.##Wu F, Zhu J, Mao Y, Li X, Hu B, Zhang D. Associations between the epithelial-mesenchymal transition phenotypes of circulating tumor cells and the clinicopathological features of patients with colorectal cancer. Dis Markers 2017;2017:9474532.##Zhou F, Qi Y, Xu H, Wang Q, Gao X, Guo H. Expression of EpCAM and Wnt/beta-catenin in human colon cancer. Genet Mol Res 2015;14(2):4485-4494.##Hanusova V, Skalova L, Kralova V, Matouskovs P. The effect of flubendazole on adhesion and migration in SW-480 and SW620 colon cancer cells. Anticancer Agents Med Chem 2018;18(6):837-846.##Lugli A, Iezzi G, Hostettler I, Muraro MG, Mele V, Tornillo L, et al. Prognostic impact of the expression of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and ALDH1 in colorectal cancer. Br J Cancer 2010;103(3):382-390.##Liu Z, Dai W, Jiang L, Cheng Y. Over-expression of LGR5 correlates with poor survival of colon cancer in mice as well as in patients. Neoplasma 2014;61(2):177-185.##Oost KC, van Voorthuijsen L, Fumagalli A, Lindeboom RGH, Sprangers J, Omerzu M, et al. Specific labeling of stem cell activity in human colorectal organoids using an ASCL2-responsive minigene. Cell Rep 2018;22(6):1600-1614.##Takahashi H, Ishii H, Nishida N, Takemasa I, Mizushima T, Ikeda M, et al. Significance of Lgr5+ ve cancer stem cells in the colon and rectum. Annals Surg Oncol 2011;18(4):1166-1174.##Liu Z, Dai W, Jiang L, Cheng Y. Over-expression of LGR5 correlates with poor survival of colon cancer in mice as well as in patients. Neoplasma 2013;61(2):177-185.##Fesler A, Liu H, Ju J. Modified miR-15a has therapeutic potential for improving treatment of advanced stage colorectal cancer through inhibition of BCL2, BMI1, YAP1 and DCLK1. Oncotarget 2018;9(2):2367.##Espersen MLM, Olsen J, Linnemann D, Høgdall E, Troelsen JT. Clinical implications of intestinal stem cell markers in colorectal cancer. Clin Colorectal Cancer 2015;14(2):63-71.##Kreso A, van Galen P, Pedley NM, Lima-Fernandes E, Frelin C, Davis T, et al. Self-renewal as a therapeutic target in human colorectal cancer. Nat Med 2014;20(1):29-36.##López-Arribillaga E, Rodilla V, Pellegrinet L, Guiu J, Iglesias M, Roman AC, et al. Bmi1 regulates murine intestinal stem cell proliferation and self-renewal downstream of Notch. Development 2015;142(1):41-50.##Asfaha S, Hayakawa Y, Muley A, Stokes S, Graham TA, Ericksen R, et al. Krt19+/Lgr5− cells are radioresistant cancer initiating stem cells in the colon and intestine. Cell Stem Cell 2015;16(6):627-638.##Neri F, Dettori D, Incarnato D, Krepelova A, Rapelli S, Maldotti M, et al. TET1 is a tumour suppressor that inhibits colon cancer growth by derepressing inhibitors of the WNT pathway. Oncogene 2015;34(32):4168-4176.##Chen ZX, Riggs AD. DNA methylation and demethylation in mammals. J Biol Chem 2011;286(21):18347-18353.##Hu X, Zhang L, Mao SQ, Li Z, Chen J, Zhang RR, et al. Tet and TDG mediate DNA demethylation essential for mesenchymal-to-epithelial transition in somatic cell reprogramming. Cell Stem Cell 2014;14(4):512-522.##Zheng X, Pang B, Gu G, Gao T, Zhang R, Pang Q, et al. Melatonin inhibits glioblastoma stem-like cells through suppression of EZH2-notch1 signaling axis. Int J Biol Sci 2017;13(2):245-253.##Akbarzadeh M, Movassaghpour AA, Ghanbari H, Kheirandish M, Fathi Maroufi N, Rahbarghazi R, et al. The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells. Scientific Reports 2017;7(1):17062.##

Optimization of Fermentation Conditions for Reteplase Expression by Escherichia coli Using Response Surface Methodology 2 2 Background: Expression of heterologous proteins at large scale is often a challenging job due to plasmid instability, accumulation of acetate and oxidative damage in bioreactors. Therefore, it is necessary to optimize parameters influencing cell growth and expression of recombinant protein.   Methods: In the present study, the optimal culture conditions for expression of reteplase by Escherichia coli (E. coli) BL21 (DE3) in a bench-top bioreactor was determined. Response Surface Methodology (RSM) based on Box-Behnken design was used to evaluate the effect of three variables (i.e., temperature, shaking speed and pH) and their interactions with cellular growth and protein production. The obtained data were analyzed by Design Expert software. Results: Based on results of 15 experiments, a response surface quadratic model was developed which was used to explain the relation between production of reteplase and three investigated variables. The high value of "R-Squared" (0.9894) and F-value of 51.99 confirmed the accuracy of this model. According to the developed model, the optimum fermentation conditions for reteplase expression were temperature of 32°C, shaking speed of 210 rpm, and pH of 8.4. This predicted condition was applied for the production of reteplase in the bioreactor leading to a protein yield of 188 mg/l. Conclusion: Our results indicate the significant role of culture conditions (e.g., pH, temperature and oxygen supply) in protein expression at large scale and confirm the need for optimization. The proposed strategy here can also be applied to experimental set-up of optimization for fermentation of other proteins. 162 168 Hamze Zare Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran Hamid Mir Mohammad Sadeghi Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran Vajihe Akbari Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences Iran BioreactorsEscherichia coliFermentationReteplase 10371.pdf Campbell J, Hilleman DE. 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Development of Sensitive and Rapid RNA Transcription-based Isothermal Amplification Method for Detection of Mycobacterium tuberculosis 2 2 Background: The accurate and early diagnosis of tuberculosis is important for its effective management. During the last decade, several molecular methods for detection of Tuberculosis (TB) have been developed. Since RNA especially mRNA has a generally much shorter half-life than DNA, its detection may be useful for the assessment of viability of bacteria. This research is a Nucleic Acid Sequence Based Amplification-Enzyme Linked Immunosorbent Assay (NASBA-ELISA) which was designed and developed for rapid detection of viable Mycobacterium tuberculosis (M. tuberculosis).  Methods: Oligonucleotide primers targeting tuf gene encoding viability marker EF-Tu mRNAs were selected and used for the amplification of mycobacterial RNA by the isothermal NASBA Digoxigenin (DIG) labeling process and incorporated with DIG-UTP, reverse transcriptase and T7 RNA polymerase.  Results: Using the NASBA-ELISA system, as little as 17.5 pg of RNA of M. tuberculosis was detected within 4 hr and no interference was encountered in the amplification and detection of viable M. tuberculosis in the presence of non-target RNA or DNA. Results obtained from the clinical specimens showed 97 and 75% of sensitivity and specificity, respectively. Conclusion: The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity for detection of M. tuberculosis. Furthermore, due to its simplicity and high sensitivity feature, it could be used in limited access laboratories in a cost-effective manner. 169 175 Reihaneh Ramezani Department of Biomedical Sciences, Women Research Center, Alzahra UniversityDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University Department of Biomedical Sciences, Women Research Center, Alzahra UniversityDepartment of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University IranIran Mahdi Forouzandeh Moghadam Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University Iran Mohammad Javad Rasaee Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University Iran Microbial viabilityMycobacterium tuberculosisNASBA-ELISA 10372.pdf Desjardin LE, Perkins MD, Wolski K, Haun S, Teixeira L, Chen Y, et al. Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy. Am J Respir Crit Care Med 1999;160(1):203-10.##Hellyer TJ, DesJardin LE, Hehman GL, Cave MD, Eisenach KD. Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis. J Clin Microbiol 1999;37(2):290-295.##Scuderi G, Golmohammadi M, Cubero J, López MM, Cirvilleri G, Llop P. Development of a simplified NASBA protocol for detecting viable cells of the citrus pathogen Xanthomonas citri subsp. citri under different treatments. Plant Pathology 2010;59(4):764-772.##Global tuberculosis report: World Health Organization; 2014.##Garg SK, Tiwari RP, Tiwari D, Singh R, Malhotra D, Ramnani VK, et al. Diagnosis of tuberculosis: available technologies, limitations, and possibilities. J Clin Lab Anal 2003;17(5):155-163.##Katoch VM. Advances in molecular diagnosis of tuberculosis. Med J Armed Forces India 2003;59(3):182-186.##Bahrmand AR, Velayati AA, Bakayev VV. Treatment monitoring and prevalence of drug resistance in tuberculosis patients in Tehran. Int J Tuberc Lung Dis 2000;4(6):544-549.##Katoch VM. Newer diagnostic techniques for tuberculosis. Indian J Med Res 2004;120(4):418-428.##Fusco V, Quero GM. Nucleic acid-based methods to identify, detect and type pathogenic bacteria occurring in milk and dairy products, structure and function of food engineering Ayman Amer Eissa, IntechOpen. DOI: 10.5772/49937. Available from: https://www.intechopen.com/books/structure-and-function-of-food-engineering/nucleic-acid-based-methods-to-identify-detect-and-type-pathogenic-bacteria-occurring-in-milk-and-dai##van der Vliet GM, Schepers P, Schukkink RA, van Gemen B, Klatser PR. Assessment of mycobacterial viability by RNA amplification. Antimicrob Agents Chemother 1994;38(9):1959-1965. ##Jean J, Blais B, Darveau A, Fliss I. 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Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA). Nucleic Acids Res 1998;26(9):2250-2251. ##Oudshoorn P, Klatser PR, inventors; bioMerieux BV assignee. EF-Tu mRNA as a marker for viability of bacteria. United States patent US 6489110B1. 2002 Dec 03. ##Paul S, Laochumroonvorapong P, Kaplan G. Comparable growth of virulent and avirulent Mycobacterium tuberculosis in human macrophages in vitro. J Infect Dis 1996;174(1):105-112. ##Neonakis IK, Gitti Z, Krambovitis E, Spandidos DA. Molecular diagnostic tools in mycobacteriology. J Microbiol Methods 2008;75(1):1-11. ##Pfyffer GE, Palicova F. Mycobacterium: General Characteristics, Laboratory Detection, and Staining Procedures. In: Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, (eds). Manual of Clinical Microbiology. 10th ed. Washington, D.C.: ASM Press; 2011.##Jacobson GR, Rosenbusch JP. Abundance and membrane association of elongation factor Tu in E. coli. Nature 1976;261(5555):23-26. ##Jean J, Blais B, Darveau A, Fliss I. Rapid detection of human rotavirus using colorimetric nucleic acid sequence-based amplification (NASBA)-enzyme-linked immuno-sorbent assay in sewage treatment effluent. FEMS Microbiol Lett 2002;210(1):143-147. ##Malek L, Sooknanan R, Compton J. Nucleic Acid Sequence-Based Amplification (NASBA™). In: Hilario E, Mackay J, (eds). Protocols for Nucleic Acid Analysis by Nonradioactive Probes. Totowa, NJ: Humana Press; p. 253-260.##Simpkins SA, Chan AB, Hays J, Pöpping B, Cook N. An RNA transcription-based amplification technique (NASBA) for the detection of viable Salmonella enterica. Lett Appl Microbiol 2000;30(1):75-79. ##Uyttendaele M, Debevere J, Lindqvist R. Evaluation of buoyant density centrifugation as a sample preparation method for NASBA-ELGA detection of Campylobacter jejuni in foods. 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Bacteriostatic Potency of Fe2O3 Against Enterococcus faecalis in Synergy with Antibiotics by DDST Method 2 2 Background: In this study, bacteriostatic potency of the Iron oxide nanoparticles against Enterococcus faecalis (E. faecalis) (a clinical sample and the ATCC11700 strain) was investigated. Methods: Nanoparticles’ bacteriostatic concentration was determined and used to appraise the characteristics of the Iron Oxide (Fe2O3) against the isolates. Antimicrobial examinations with 108cfu.ml-1 were performed at the baseline. Due to evaluation level of potency, after performing Minimum Inhibitory Concentration (MIC), the assessment of death kinetic and susceptibility constant of nanoparticles was done by suspension at two MICs in 0 to 360 min treatment time. Results: Fe2O3 nanoparticles in size range of 10-50 nm demonstrated the most effective susceptibility reaction against E. faecalis and ATCC11700 strain in Z=78.125 ml/μg-1 and 39.0625 ml/μg-1, respectively. The kinetic reaction of E. faecalis against Fe2O3 suspension was supposed to be decreased through the elapse of treatment time, whereas increased concentration was along with bacteria growth after a certain time. So, the efficient concentration of nanoparticles was applied with semi-sensitive and antibiotic resistant for both strains. However, synergism of Fe2O3 nanoparticles with Ceftazidime and Clindamycin revealed a higher susceptibility compared with Fe2O3 nanoparticles alone against E. faecalis. Conclusion: The experimental results reveal that Fe2O3 has a strong antimicrobial effect at a certain concentration over the time so could potentially be used for bacterial inhibition and this feature will be strengthened in combination with antibiotics. 176 179 Erfan Shahbazi Department of Microbiology, Shahid Beheshti University Department of Microbiology, Shahid Beheshti University Iran Firouzeh Moreshedzadeh Department of Cell and Molecular Biology, University of Tehran Department of Cell and Molecular Biology, University of Tehran Iran Davood Zaeifi Department of Biology, Tehran North Branch, Islamic Azad University Department of Biology, Tehran North Branch, Islamic Azad University Iran Enterococcus faecalisFerric oxideNanopaticles 10366.pdf Hasani A, Sharifi Y, Ghotaslou R, Naghili B, Hasani A, Aghazadeh M, et al. Molecular screening of virulence genes in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens in Northwest Iran. Indian J Med Microbiol 2012;30(2):175-181.##Sadeghifard N, Kazemian H, Mohebi R, Sekawi Z, Khosravi A, Valizadeh N, et al. Epidemiological alteration in pathogens found in ground meat in Iran: unexpected predominance of vancomycin-resistant Enterococcus faecalis. GMS Hyg Infect Control 2015;10:Doc12.##Feizabadi MM, Maleknejad P, Asgharzadeh A, Asadi S, Shokrzadeh L, Sayadi S. Prevalence of aminoglycoside-modifying enzymes genes among isolates of Enterococcus faecalis and Enterococcus faecium in Iran. Microb Drug Resist 2006;12(4):265-268.##Mohammadi F, Ghafourian S, Mohebi R, Taherikalani M, Pakzad I, Valadbeigi H, et al., Enterococcus faecalis as multidrug resistance strains in clinical isolates in Imam Reza Hospital in Kermanshah, Iran. Br J Biomed Sci 2015;72(4):182-184.##Gajan EB, Abashov R, Aghazadeh M, Eslami H, Oskouei SG, Mohammadnejad D. Vancomycin-resistant Enterococcus faecalis from a wastewater treatment plant in Tabriz, Iran. Pak J Biol Sci 2008;11(20):2443-2446.##Sharifi Y, Hasani A, Ghotaslou R, Varshochi M, Hasani A, Aghazadeh M, et al. Survey of virulence determinants among vancomycin resistant enterococcus faecalis and enterococcus faecium isolated from clinical specimens of hospitalized patients of north west of Iran. Open Microbiol J 2012;6:34-39.##Khani M, Fatollahzade M, Pajavand H, Bakhtiari S, Abiri R. Increasing prevalence of aminoglycoside-resistant enterococcus faecalis isolates due to the aac(6')-aph(2") gene: a therapeutic problem in Kermanshah, Iran. Jundishapur J Microbiol 2016;9(3):e28923.##Heidari H, Hasanpour S, Ebrahim-Saraie HS, Mota-medifar M. Ebrahim-Saraie. High incidence of virulence factors among clinical enterococcus faecalis isolates in southwestern Iran. Infect Chemother 2017;49(1):51-56.##Feizabadi MM, Asadi S, Zohari M, Gharavi S, Etemadi G. Genetic characterization of high-level gentamicin-resistant strains of Enterococcus faecalis in Iran. Can J Microbiol 2004;50(10):869-872.##Gupta AK, Gupta M. Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications. Biomaterials 2005;26(18):3995-4021.##Wu W, Jiang CZ, Roy VA. Designed synthesis and surface engineering strategies of magnetic iron oxide nanoparticles for biomedical applications. Nanoscale 2016;8(47):19421-19474.##Kohler N, Sun C, Wang J, Zhang M. Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir 2005;21(19):8858-8864.##Chertok, B, Moffat BA, David AE, Yu F, Bergemann C, Ross BD, et al. Iron oxide nanoparticles as a drug delivery vehicle for MRI monitored magnetic targeting of brain tumors. Biomaterials 2008;29(4):487-496.##Pareta RA, Taylor E, Webster TJ. 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FDA. 2005.https://www.accessdata.fda.gov/drugsatfda_docs/nda/2005/021821orig1s000micror.pdf.##Fathi Azar Khavarani M, Najafi M, Shakibapour Z, Zaeifi D. kinetics activity of yersinia intermedia against ZnO nanoparticles either synergism antibiotics by double-disc synergy test method. Iran J Biotechnol 2016;14(1):39-44.##Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal 2016;6(2):71-79.##Asakawa S, Bünemann E, Frossard E. Microbially Mediated Processes. In: Huang PM, Li Y, Sumner ME, editors. Handbook of Soil Sciences. Boca Raton, Florida: CRC Press; 2011. p. 1-52.##Limbach LK, Wick P, Manser P, Grass RN, Bruinink A, Stark WJ. Exposure of engineered nanoparticles to human lung epithelial cells: influence of chemical composition and catalytic activity on oxidative stress. Environ Sci Technol 2007;41(11):4158-4163.##Wu B, Huang R, Sahu M, Feng X, Biswas P, Tang YJ. Bacterial responses to Cu-doped TiO2 nanoparticles. 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Immunogenicity of Cork and Loop Domains of Recombinant Baumannii acinetobactin Utilization Protein in Murine Model 2 2 Background: Acinetobacter baumannii (A. baumannii) is a bothersome fatal patho-gen, particularly in healthcare system. Persistence and successful invasion of A. baumannii in vertebrate host cells largely depends on iron acquisition methods. Sidero-phore molecules and Iron-Regulated Outer Membrane Proteins (IROMPs) are the two essential members of iron acquisition system. Siderophores are secreted by bacteria to bind peripheral ferric iron and the IROMPs are expressed at the bacterial outer membrane as the receptor of ferric-siderophore complex. BauA is the corresponding siderophore receptor of A. baumannii. In this study, an attempt was made to assess the immunogenicity of antigenic domains of BauA which could be effective in iron uptake restriction and protection against bacterial invasion of the host cells.  Methods: The antigenic domains of bauA were amplified from A. baumannii ATCC-19606. The PCR products were ligated into pET32a and expressed in Escherichia coli (E. coli) BL21 (DE3). Purification of recombinant domains was done by Nickel-Nitri-lotriacetic Acid (Ni-NTA) affinity chromatography. The recombinant domains were injected into BALB/C mice separately and in combination. Sero-reactivities of the recombinant proteins and mouse challenge tests were carried out.  Results: The antibodies raised in mice could successfully recognize and bind antigenic domains. Passive immunization studies accomplished by immune rabbit serum inhibited the establishment of infection in mice. Conclusion: The results adapted from the present study disclose the protective role of functional domains of BauA, especially the cork domain, suggesting a novel recombinant immunogen candidate. 180 186 Hamid Esmaeilkhani Department of Biology, Shahed UniversityMolecular Microbiology Research Center, Shahed University Department of Biology, Shahed UniversityMolecular Microbiology Research Center, Shahed University IranIran Iraj Rasooli Department of Biology, Shahed UniversityMolecular Microbiology Research Center, Shahed University Department of Biology, Shahed UniversityMolecular Microbiology Research Center, Shahed University IranIran Masoomeh Hashemi Department of Biology, Shahed University Department of Biology, Shahed University Iran Shahram Nazarian Department of Biology, College of Basic Sciences, Imam Hussein University Department of Biology, College of Basic Sciences, Imam Hussein University Iran Fatemeh Sefid Departeman of Biology, Science and Art University Departeman of Biology, Science and Art University Iran Acinetobackr baumanniiImmune seraIronSiderophore 10367.pdf Vergidis P, Falagas ME. Control of Multi-Drug Resistant Acinetobacter Infections. In: Antibiotic Policies, Edition. Springer, 2012, p. 117-125.##Camp C, Tatum OL. A review of Acinetobacter baumanniias a highly successful pathogen in times of war. Lab Med 2015;41(11):649-657.##García-Quintanilla M, Pulido MR, López-Rojas R, Pachón R, McConnell MJ. Emerging therapies for multidrug resistant Acinetobacter baumannii. Trend Microbiol 2013;21(3):157-163.##Dexter C, Murray GL, Paulsen IT, Peleg AY. Community-acquired Acinetobacter baumannii: clinical characteristics, epidemiology and pathogenesis. Expert Rev Anti Infect Ther 2015;13(5):567-573.##De Bonis P, Lofrese G, Scoppettuolo G, Spanu T, Cultrera R, Labonia M,et al. Intraventricular versus intravenous colistin for the treatment of extensively drug resistant Acinetobacter baumannii meningitis. Eur J Neurol 2016;23(1):68-75.##Hsu LY, Apisarnthanarak A, Khan E, Suwantarat N, Ghafur A, Tambyah PA. Carbapenem-resistant Acinetobacter baumannii and Enterobacteriaceae in South and Southeast Asia. Clin Microbiol Rev 2017;30(1):1-22.##Mosqueda N, Espinal P, Cosgaya C, Viota S, Plasensia V, Alvarez-Lerma F, et al. Globally expanding carbapenemase finally appears in Spain: nosocomial outbreak of Acinetobacter baumannii producing plasmid-encoded OXA-23 in Barcelona, Spain. Antimicrob Agents Chemother 2013;57(10):5155-5157.##Romanelli RM, Jesus LA, Clemente WT, Lima SS, Rezende EM, Coutinho RL, et al. Outbreak of resistant Acinetobacter baumannii: measures and proposal for prevention and control. Braz J Infect Dis 2009;13(5):341-347.##Farshadzadeh Z, Hashemi FB, Rahimi S, Pourakbari B, Esmaeili D, Haghighi MA, et al. Wide distribution of carbapenem resistant Acinetobacter baumannii in burns patients in Iran. Front Microbiol 2015;6:1146.##Fernández-Cuenca F, Martínez-Martínez L, Conejo MC, Ayala JA, Perea EJ, Pascual A. Relationship between beta-lactamase production, outer membrane protein and penicillin-binding protein profiles on the activity of carbapenems against clinical isolates Acinetobacter baumannii. J Antimicrob Chemother 2003;51(3):565-574.##Zarrilli R, Pournaras S, Giannouli, Tsakris A. Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages. Int J Antimicrob Agents 2013;41(1):11-19.##Gaddy JA, Arivett BA, McConnell MJ, López-Rojas R, Pachón J, Actis LA. Role of acinetobactn-mediated iron acquisition functions in the interaction of Acinetobacter baumannii strain ATCC 19606T with human lung epithelial cells, Galleria mellonella caterpillars, and mice. Infect Immun 2012;80(3):1015-1024.##Abu Kwaik Y, Bumann D. Microbial quest for food in vivo:‘nutritional virulence’as an emerging paradigm. Cell Microbiol 2013;15(6):882-890.##Olive AJ, Sassetti CM. Metabolic crosstalk between host and pathogen: sensing, adapting and competing. 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Single Nucleotide Polymorphism of TYK2 Gene and Susceptibility to Rheumatoid Arthritis in Iranian Population 2 2 Background: Rheumatoid Arthritis (RA) is a debilitating disorder in which the immune system mainly targets the synovial tissue. Janus kinase family including tyrosine kinase 2 (TYK2) is one of the crucial mediators of the downstream signaling pathway of inflammatory cytokines that further contributes to RA pathogenesis. In this study, the association of TYK2 gene rs34536443 polymorphism, which may affect the function of TYK protein and, hence, the inflammatory settings, with RA susceptibility was investigated. Moreover, its correlation with demographic and serological features of the patients was assessed.  Methods: In the present study, 700 RA patients and 700 sex, age and ethnicity-matched healthy individuals as the control group were included. MGB TaqMan real-time allelic discrimination method was used to determine the rs34536443 polymorphism. Rheumatoid factor, anti-cyclic citrullinated peptide antibody, erythrocyte sedimentation rate and C-reactive protein were also measured. Results: The frequency of rs34536443 minor allele (C allele) was not different between patients and control group [1.7 vs. 2.61 percent, OR (95% CI)=1.35 (0.78-2.33);p=0.27]. There was not a statistically significant association between rs34536443 genotypes and RA susceptibility. Genotypes of rs34536443 polymorphism were associated nor with demographic neither with serological features of RA patients.  Conclusion: In the present study, there was not any association between TYK2 gene rs34536443 polymorphism with either disease susceptibility, demographic and serological features of Iranian RA patients. These findings are not compatible with previous works from other ethnicities, further supporting the role of genetics in disease susceptibility. 187 191 Azadeh Mohamadhosseini Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Iran Reza Mansouri Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Department of Immunology, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences Iran Ali Javinani Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Amir Ashraf-Ganjouei Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Massoumeh Akhlaghi Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Saeed Aslani Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Elham Hamzeh Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Ahmadreza Jamshidi Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Nooshin Ahmadzadeh Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Mahdi Mahmoudi Rheumatology Research Center, Tehran University of Medical Sciences Rheumatology Research Center, Tehran University of Medical Sciences Iran Rheumatoid arthritisSingle-nucleotide polymorphismTYK2 kinase 10374.pdf Grassi W, De Angelis R, Lamanna G, Cervini C. The clinical features of rheumatoid arthritis. Eur J Radiol 1998;27(Suppl 1):S18-24.##Rudan I, Sidhu S, Papana A, Meng SJ, Xin-Wei Y, Wang W, et al. Prevalence of rheumatoid arthritis in low- and middle-income countries: A systematic review and analysis. J Glob Health 2015;5(1):010409.##Cross M, Smith E, Hoy D, Carmona L, Wolfe F, Vos T, et al. The global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study. Ann Rheum Dis 2014;73(7):1316-1322.##Choy E. Understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. Rheumatology (Oxford) 2012;51(Suppl 5):v3-11.##Schwartz DM, Kanno Y, Villarino A, Ward M, Gadina M, O'Shea JJ. JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nat Rev Drug Discov 2017;16(12):843-862.##Yamaoka K, Saharinen P, Pesu M, Holt VET, Silvennoinen O, O'Shea JJ. The Janus kinases (Jaks). Genome Biol 2004;5(12):253.##Krolewski JJ, Lee R, Eddy R, Shows TB, Dalla-Favera R. 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Antibiotic Susceptibility Patterns and Prevalence of Some Extended Spectrum Beta-Lactamases Genes in Gram-Negative Bacteria Isolated from Patients Infected with Urinary Tract Infections in Al-Najaf City, Iraq 2 2 Background: Urinary Tract Infection (UTI) in patients with Chronic Kidney Disease (CKD) caused by multi-drug resistance and Extended Spectrum Beta Lactamase (ESBL)-producing gram-negative bacteria has been increased in different countries. The aim of the present study was to detect the antibiotic susceptibility patterns and the distribution of Bla-TEM, Bla-SHV and Bla-CTX-M genes in gram-negative bacteria isolated from outpatients infected with UTI, with and without CKD in Al-Najaf city, Iraq.  Methods: A total of 120 non-duplicate urine samples were collected from outpatients (37 male and 83 female) infected with UTI in Al-Najaf city, Iraq; 60 samples from patients Without Kidney Disease (WKD) and 60 samples from patients with CKD. The antibiotic susceptibility testing was done according to Kirby-Bauer method. PCR technique was performed to investigate the prevalence of Bla-TEM, Bla-SHV and Bla-CTX-M genes. Results: A total of 126 different gram-negative bacterial strains were isolated. Escherichia coli (E. coli) was the most prevalent bacterium (49 isolates) followed by Idebsiella pneumonia (K. pneumonia) (35 isolates), Pseudomonas aeruginosa (P. aeruginosa) (18 isolates), Citrobacter freundii (C. freundii) (12 isolates), Enterobocter aerogenes (E. aerogenes) (8 isolates) and Proteus mirabilis (P. mirabilis) (4 isolates). All bacterial isolates from UTI patients with CKD were resistant to antibiotics and carried Bla-TEM, Bla-SHV and Bla-CTX-M genes more than isolates from UTI patients with WKD. 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Evaluation of the Effect of Morphine and Imiquimodon Expression of TLR2 and TLR4 from Lesion RNA Extracted from BALB/c Mice Infected with Leishmania major 2 2 Background: Toll-Like Receptors (TLRs) are the cause of phagocytosis activation and destruction of the infection agents. In addition, new evidences support the idea that TLRs play a vital role in starting the acquired immunity reactions. Methods: In this study, it has been attempted to infect the BALB/c mice with Leishmania major (L. major) and treat them using morphine and imiquimod; then the expressions of TLR2,4 from treated lesion were studied by using Real-Time PCR method. Treatment with morphine 1 mg/kg, imiquimod 5% and nalmefene 1 mg/kg began four weeks after the challenge. After treatment period, half of the mice of each group were killed and their lesions were isolated for RNA extraction and making cDNA. For the rest of mice, lesion size was measured weekly. Results: The results showed increase of expression of TLR2 gene among all treated groups relative to the control, and the difference was significant (p<0.05). The expression of TLR4 gene only was reduced in groups under treatment with morphine and morphine plus nalmefene relative to the control group and in the other groups increased. The highest expression of TLR2 was seen in the group treated by glucantime (p<0.0001).  Conclusion: However, in this study it was found that despite decreasing the size of lesion in all treated groups, expression of TLR4  in the morphine, nalmefene, morphine plus nalmefene treated groups compared to the control group was decreased. Therefore, morphine may have a different function mechanism in treatment of the Leishmaniasis with the L. major. 202 205 Parisa Ebrahimisadr Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Iran Fatemeh Ghaffarifar Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Iran John Horton Tropical Projects Tropical Projects United Kingdom Abdolhossein Dalimi Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Department of Parasitology and Entomology, Faculty of Medical Sciences, Tarbiat Modares University Iran Zohreh Sharifi Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine Blood Transfusion Research Center, High Institute for Education and Research in Transfusion Medicine Iran Leishmania majorLesionsMiceMorphineToll-like receptors 10401.pdf Akira S, Takeda K. Toll-like receptor signaling. Nat Rev Immunol 2004;4(7):499-511.##Friedman H, Newton C, Klein TW. Microbial infections, immunomodulation, and drugs of abuse. Clin Microbiol Rev 2003;16(2):209-219.##Hutchinson MR, Coats BD, Lewis SS, Zhang Y, Sprunger DB, Rezvani N, et al. Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia. Brain Behav Immun 2008;22(8):1178-1189.##Rudy SJ. Imiquimod (Aldara): modifying the immune response. Dermatol Nurs 2002;14(4):268-270.##Kropf P, Freudenberg MA, Modolell M, Price HP, Herath S, Antoniazi S, et al. Toll-like receptor 4 contributes to efficient control of infection with the protozoan parasite Leishmania major. Infect Immun 2004;72(4):1920-1928.##Croft SL, Seifert K, Yardley V. Current scenario of drug development for leishmaniasis. Indian J Med Res 2006;123(3):399-410. ##Salimi V, Hennus MP, Mokhtari-Azad T, Shokri F, Janssen R, Hodemaekers HM, et al. Opioid receptors control viral replication in the airways. Crit Care Med 2013;41(1):205-214. ##Kropf P, Freudenberg N, Kalis C, Modolell M, Herath S, Galanos C, et al. Infection of C57BL/10ScCr and C57BL/10ScNCr mice with Leishmania major reveals a role for Toll-like receptor. J Leukoc Biol 2004;76(1):48-57.##Ebrahimisadr P, Ghaffarifar F, Hassan ZM, Sirousazar M, Mohammadnejad F. Effect of polyvinyl alcohol (PVA) containing artemether in treatment of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice. Jundishapur J Microbiol 2014;7(5):e9696.##Sheridan PA, Moynihan JA. Modulation of the innate immune response to HSV-1 following acute administration of morphine: role of hypothalamo-pituitary-adrenal axis. J Neuroimmunology 2005;158(1-2):145-152.##WHO. Working to overcome the global impact of neglected tropical diseases-First WHO report on neglected tropical diseases, WHO/HTM/NTD/2010. 2010.##Zhang Y, Li H, Yi Li, Sun X, Zhu M, Hanley G, et al. Essential role of toll-like receptor 2 in morphine-induced microglia activation in mice. ‎Neurosci Lett 2011;489(1):43-47.##Tolouei S, Hejazi H, Ghaedi K, Khamesipour A, Hasheminia SJ. TLR2 and TLR4 in cutaneous leishmaniasis caused by Leishmania major. Scand J Immunol 2013;78(5):478-484.##