ORIGINAL_ARTICLE Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-1 Drug Discovery Background: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy. Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay. http://rbmb.net/article-1-65-en.pdf 2015-10-30 1 9 Drug discovery EGFP Fluorometry HIV-1 Single-round infection Mahdieh Soezi 1 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran - National Institute of Genetic Engineering and Biotechnology, Tehran, Iran AUTHOR Arash Memarnejadian memarnejad@pasteur.ac.ir, mrasadeghi@pasteur.ac.ir 2 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Saeed Aminzadeh 3 AUTHOR Rezvan Zabihollahi 4 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Seyed Mehdi Sadat 5 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Safieh Amini 6 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Soheila Hekmat 7 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Mohammad Reza Aghasadeghi 8 Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR
ORIGINAL_ARTICLE Comparative Analysis of Peripheral Alkaline Phytase Protein Structures Expressed in E. coli Background: Degradation of phytic acid to inorganic phosphate in domestic animals’ diets requires thermostable phytase. Although Basillus subtilis phytase shows a potential to be degraded phytate complex in high temperature, the enzyme activities and yields need to be increased to make them possible for industrial application. Methods: The phytase gene from Bacillus subtilis DR8886 was isolated from Dig Rostam hot mineral spring in Iran and cloned into pET21(+) and pET32(+). Expression was induced with 1.5 mM IPTG and the proteins were purified. Results: The recombinant protein affected by thioredoxin (Trx) from pET32a-PhyC was estimated to constitute about 31% of the total soluble protein in the cells; its concentration was 3.5 µg/ml, and its maximal phytase activity was 15.9 U/ml, whereas the recombinant phytase from pET21a-PhyC was estimated to comprise about 19% of the total soluble protein; its concentration was 2.2 µg/ml, and its maximal phytase activity was 69 U/ml. The molecular masses of recombinant phytase with and without Trx were about 60 kDa and 42 kDa, respectively. Zymography confirmed that the recombinant enzymes were active. Although the concentration of the alkaline phytase expressed by pET32a was approximately 59% greater than that expressed by pET21, its phytase activity was approximately 77% less. Conclusion: This study showed that the peripheral gene (Trx) encoded by the pET32a (+) vector are the principal reason for the decrease in recombinant phytase enzyme activity. http://rbmb.net/article-1-66-en.pdf 2015-10-30 10 18 Bacillus subtilis DR8806 PhyC Gene Thioredoxin Mohammadreza Nassiri Nassiryr@um.ac.ir 1 Institute of Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran - Department of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran AUTHOR Hamid Ariannejad 2 Department of Animal Sciences, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran AUTHOR
ORIGINAL_ARTICLE Refolding Process of Cysteine-Rich Proteins: Chitinase as a Model Background: Recombinant proteins overexpressed in E. coli are usually deposited in inclusion bodies. Cysteines in the protein contribute to this process. Inter- and intra- molecular disulfide bonds in chitinase, a cysteine-rich protein, cause aggregation when the recombinant protein is overexpressed in E. coli. Hence, aggregated proteins should be solubilized and allowed to refold to obtain native- or correctly- folded recombinant proteins. Methods: Dilution method that allows refolding of recombinant proteins, especially at high protein concentrations, is to slowly add the soluble protein to refolding buffer. For this purpose: first, the inclusion bodies containing insoluble proteins were purified; second, the aggregated proteins were solubilized; finally, the soluble proteins were refolded using glutathione redox system, guanidinium chloride, dithiothreitol, sucrose, and glycerol, simultaneously. Results: After protein solubilization and refolding, SDS-PAGE showed a 32 kDa band that was recognized by an anti-chitin antibody on western blots. Conclusion: By this method, cysteine-rich proteins from E. coli inclusion bodies can be solubilized and correctly folded into active proteins. http://rbmb.net/article-1-67-en.pdf 2015-10-30 19 24 Malihe Moghadam 1 Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR Ali Ganji 2 Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR Abdolreza Varasteh 3 Allergy Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR Reza Falak 4 Immunology Research Center, Iran University of Medical Sciences, Tehran, Iran - Department of Immunology, Iran University of Medical Sciences, Tehran, Iran AUTHOR Mojtaba Sankian sankianm@mums.ac.ir 5 Immunology Research Center, Medical School, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR
ORIGINAL_ARTICLE Transcriptional Analysis of VEGF-D and TGFβ Genes in MCF7 Cells Exposed to Saponin Isolated from Holothuria leucospilota (Sea Cucumber) Background: Marine natural products contain a wide range of bioactive compounds with therapeutic properties that have revealed crucial properties in the treatment of some diseases. Some of these compounds have recently received considerable attention for drug discovery. In this study we examined the anti-angiogenic effect of saponin isolated from Holothuria leucospilota (sea cucumber) through evaluation of vascular endothelial growth factor D (VEGF-D) and transforming growth factor-β (TGFβ) expression in a breast cancer cell line. Methods: To investigate the effect of SCS on VEGF-D and TGF-β expression in breast cancer cells, the cells were treated with various concentrations of sample. After 48 h the viability of the cells was evaluated by trypan blue staining, and VEGF-D and TGFβ mRNA expression was were evaluated by real time-PCR. Results: Our results revealed that SCS can suppress cell viability and VEGF-D and TGFβ mRNA expression in breast cancer cells. Sea cucumber saponin at a concentration of 12 μg/ml inhibited VEGF-D and TGFβ expression more than 90% compared with controls. Conclusion: Findings suggest that SCS could inhibit tumor growth via inhibition of angiogenesis. http://rbmb.net/article-1-68-en.pdf 2015-10-30 25 31 Angiogenesis Anticancer Saponin Sea cucumber Mozhgan Soltani 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran AUTHOR Kazem Parivar 2 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran AUTHOR Javad Baharara baharara@yahoo.com 3 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran AUTHOR Mohammad Amin Kerachian 4 Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR Javad Asili 5 Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR
ORIGINAL_ARTICLE In silico Analysis and Molecular Modeling of RNA Polymerase, Sigma S (RpoS) Protein in Pseudomonas aeruginosa PAO1 Background: Sigma factors are proteins that regulate transcription in bacteria. Sigma factors can be activated in response to different environmental conditions. The rpoS (RNA polymerase, sigma S) gene encodes sigma-38 (σ38, or RpoS), a 37.8 kDa protein in Pseudomonas aeruginosa (P. aeruginosa) strains. RpoS is a central regulator of the general stress response and operates in both retroactive and proactive manners; not only does it allow the cell to survive environmental challenges; it also prepares the cell for subsequent stresses (cross-protection). Methods: The significance of RpoS for stress resistance and protein expression in stationary-phase P. aeruginosa cells was assessed. The goal of the current study was to characterize RpoS of P. aeruginosa PAO1 using bioinformatics tools. Results: The results showed that RpoS is an unstable protein that belongs to the sigma-70 factor family. Secondary structure analysis predicted that random coil is the predominant structure followed by extended alpha helix. The three-dimensional (3D) structure was modeled using SWISS-MODEL Workspace. Conclusion: Determination of sequence, function, structure, and predicted epitopes of RpoS is important for modeling of inhibitors that will help in the design of new drugs to combat multi-drug-resistant (MDR) strains. Such information may aid in the development of new diagnostic tools, drugs, and vaccines for treatment in endemic regions. http://rbmb.net/article-1-69-en.pdf 2015-10-30 32 42 Bioinformatics In silico Pseudomonas aeruginosa RpoS Therapy Mansour Sedighi 1 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Mohsen Moghoofei 2 Department of Virology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Ebrahim Kouhsari 3 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Abazar Pournajaf 4 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Behzad Emadi 5 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Masoud Tohidfar 6 Agricultural Biotechnology Research Institute of Iran, Tehran, Iran AUTHOR Mehrdad x Gholami mehrdad_gholami90@yahoo.com 7 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR
ORIGINAL_ARTICLE Association of Htra1 Gene Polymorphisms with the Risk of Developing AMD in Iranian Population Background: Half of the cases of vision loss in people under 60 years of age have been attributed to age-related macular degeneration (AMD). This is a multifactorial disease with late onset. It has been demonstrated that many different genetic loci are implicated in the risk of developing AMD in different populations. In the current study, we investigated the association of high-temperature ‎requirement A-1 (HTRA1) gene polymorphisms with the risk of developing AMD in the Iranian population. Methods: Genomic DNA samples were extracted from 120 patients with AMD and 120 healthy age- and sex-matched controls. A 385 base-pair fragment of the HTRA1 gene promoter region was amplified using the polymerase chain reaction (PCR) technique and sequenced. The frequencies of the alleles were calculated and statistical analysis was performed using SPSS software. Results: Our study demonstrated that the rate of polymorphisms rs11200638 -625 G>A and rs2672598 -487T>C were significantly greater in AMD patients than in healthy controls from the Iranian population. Conclusions: The results of our study indicate that HTRA1 gene promoter region polymorphisms are associated with the risk of developing AMD in the Iranian population. http://rbmb.net/article-1-70-en.pdf 2015-10-30 43 49 HTRA1 Iran Macular Degeneration Single Nucleotide Polymorphisms Mohammad Askari 1 Department of Biotechnology, Pasteur Institute of Iran, Tehran, Iran AUTHOR Amin Reza Nikpoor 2 Department of Immunology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran AUTHOR Fazel Gorjipour 3 Physiology Research Center, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Mohsen Mazidi 4 Biochemistry and Nutrition Research Center, Mashhad University of Medical Sciences, Mashhad, AUTHOR Mohammad Hosein Sanati 5 National Institute for Genetic Engineering and Biotechnology, Tehran, Iran AUTHOR Hajar Aryan 6 Fazeli-Sanati Genetic Laboratory, Tehran, Iran AUTHOR Alireza Irani 7 Eye Research Center, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran AUTHOR Khalil Ghasemi Falavarjani 8 Eye Research Center, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran AUTHOR Hossein Nazari 9 Eye Research Center, Rassoul Akram Hospital, Iran University of Medical Sciences, Tehran, Iran AUTHOR Kazem Mousavizadeh mousavik@gmail.com 10 Cellular and Molecular Research Center, Laboratory of Basic Sciences, Iran University of Medical Sciences, Tehran, Iran AUTHOR
ORIGINAL_ARTICLE In silico Analysis and Modeling of ACP-MIP–PilQ Chimeric Antigen from Neisseria meningitidis Serogroup B Background: Neisseria meningitidis, a life-threatening human pathogen with the potential to cause large epidemics, can be isolated from the nasopharynx of 5–15% of adults. The aim of the current study was to evaluate biophysical and biochemical properties and immunological aspects of chimeric acyl-carrier protein-macrophage infectivity potentiator protein-type IV pilus biogenesis protein antigen (ACP-MIP-PilQ) from N. meningitidis serogroup B strain. Methods: Biochemical properties and multiple alignments were predicted by appropriate web servers. Secondary molecular structures were predicted based on Chou and Fasman, Garnier-Osguthorpe-Robson, and Neural Network methods. Tertiary modeling elucidated conformational properties of the chimeric protein. Proteasome cleavage and transporter associated with antigen processing (TAP) binding sites, and T- and B-cell antigenic epitopes, were predicted using bioinformatic web servers. Results: Based on our in silico and immunoinformatics analyses, the ACP-MIP-PilQ protein (AMP) can induce high-level cross-strain bactericidal activity. In addition, several immune proteasomal cleavage sites were detected. The 22 epitopes associated with MHC class I and class II (DR) alleles were confirmed in the AMP. Thirty linear B-cell epitopes as antigenic regions were predicted from the full-length protein. Conclusion: All predicted properties of the AMP indicate it could be a good candidate for further immunological in vitro and in vivo studies. http://rbmb.net/article-1-71-en.pdf 2015-10-30 50 59 Chimeric protein In silico Neisseria meningitides Serogroup B Vaccine Mehrdad Gholami 1 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Alireza Salimi Chirani Alireza.salimichirani@gmail.com 2 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran AUTHOR Mona Moshiri 3 Department of Pathobiology, Division of Microbiology, Faculty of Public Health, Tehran University of Medical Sciences, Tehran, Iran AUTHOR Mansour Sedighi 4 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Abazar Pournajaf 5 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR Masoud Tohidfar 6 Agricultural Biotechnology Research Institute of Iran, Tehran, Iran AUTHOR Gholamreza Irajian 7 Department of Microbiology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran AUTHOR