Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study
Mohammad
Taghizadeh
Laboratory of Biophysics and Molecular Biology, Institute of Biochemistry and Biophysics, University of Tehran, Tehran,
Iran
author
Bahram
Goliaei
Laboratory of Biophysics and Molecular Biology, Institute of Biochemistry and Biophysics, University of Tehran, Tehran,
Iran
author
Armin
Madadkar Sobhani
Institute of Biochemistry and Biophysics (IBB), Tehran University, Tehran, Iran.
author
text
article
2016
eng
Background: Protein flexibility, which has been referred as a dynamic behavior has various roles in proteins’ functions. Furthermore, for some developed tools in bioinformatics, such as protein-protein docking software, considering the protein flexibility, causes a higher degree of accuracy. Through undertaking the present work, we have accomplished the quantification plus analysis of the variations in the human Cyclin Dependent Kinase 2 (hCDK2) protein flexibility without affecting a significant change in its initial environment or the protein per se.Objectives: The main goal of the present research was to calculate variations in the flexibility for each residue of the hCDK2, analysis of their flexibility variations through clustering, and to investigate the functional aspects of the residues with high flexibility variations.Materials and Methods: Using Gromacs package (version 4.5.4), three independent molecular dynamics (MD) simulations of the hCDK2 protein (PDB ID: 1HCL) was accomplished with no significant changes in their initial environments, structures, or conformations, followed by Root Mean Square Fluctuations (RMSF) calculation of these MD trajectories. The amount of variations in these three curves of RMSF was calculated using two formulas.Results: More than 50% of the variation in the flexibility (the distance between the maximum and the minimum amount of the RMSF) was found at the region of Val-154. As well, there are other major flexibility fluctuations in other residues. These residues were mostly positioned in the vicinity of the functional residues. The subsequent works were done, as followed by clustering all hCDK2 residues into four groups considering the amount of their variability with respect to flexibility and theirposition in the RMSF curves.Conclusions: This work has introduced a new class of flexibility aspect of the proteins’ residues. It could also help designing and engineering proteins, with introducing a new dynamic aspect of hCDK2, and accordingly, for the other similar globular proteins. In addition, it could provide a better computational calculation of the protein flexibility, which is, especially important in the comparative studies of the proteins’ flexibility.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
1
12
https://www.ijbiotech.com/article_81323_363a4c664563afcfe0f6166c93dda2c3.pdf
dx.doi.org/10.15171/ijb.1419
Optimization of Carbon and Nitrogen Sources for Extracellular Polymeric Substances Production by Chryseobacterium indologenes MUT.2
Mojtaba
khani
Department of Bioscience and Biotechnology, Malek Ashtar University, Tehran, Iran.
author
Ali
Bahrami
Department of Bioscience and Biotechnology, Malek Ashtar University, Tehran, Iran.
author
Asma
Chegeni
Department of Bioscience and Biotechnology, Malek Ashtar University, Tehran, Iran.
author
Mohammad Davoud
Ghafari
Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran.
author
Ali
Mansouran zadeh
Department of Bioscience and Biotechnology, Malek Ashtar University, Tehran, Iran.
author
text
article
2016
eng
Background: Bacterial Extracellular Polymeric Substances (EPS) are environmental friendly and versatile polymeric materials that are used in a wide range of industries such as: food, textile, cosmetics, and pharmaceuticals. To make the production process of the EPS cost-effective, improvements in the production yield is required which could be implemented through application of processes such as optimized culture conditions, and development of the strains with higher yield (e.g. through genetic manipulation), or using low-cost substrates. Objectives: In this work, the effects of carbon and nitrogen sources were studied in order to improve the EPS production by the submerged cultivation of Chryseobacterium indologenes MUT.2. Materials and Methods: The mesophilic microorganism Chryseobacterium indologenes MUT.2, was grown and maintained in the Luria Bertani agar. The initial basal medium contained: glucose (20 g.L-1), yeast extracts (5 g.L-1), K2HPO4 (6 g.L-1), NaH2PO4 (7 g.L-1), NH4CL (0.7 g.L-1), and MgSO4 (0.5 g.L-1). For evaluating the carbon and nitrogen sources’ effect on the fermentation performance, cultures were prepared in 500 mL flasks filled with 300 mL of the medium. The single-factor experiments based on statistics was employed to evaluate and optimize the carbon and nitrogen sources for EPS production in the liquid culture medium of Chryseobacterium indologenes MUT.2. Results: The preferred carbon-sources, sucrose and glucose, commonly gave the highest EPS production of 8.32 and 6.37 g.L-1, respectively, and the maximum EPS production of 8.87 g.L-1 was achieved when glutamic acid (5 g.L-1) was employed as the nitrogen source. Conclusions: In this work, the culture medium for production of EPS by Chryseobacterium indologenes MUT.2 was optimized. Compared to the basal culture medium in shake-flasks and stirred tank bioreactor, the use of optimized culture medium has resulted in a 53% and 73% increase in the EPS production, respectively.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
13
18
https://www.ijbiotech.com/article_14132_0c9a98e8683b813ca91b5244080c4603.pdf
dx.doi.org/10.15171/ijb.1266
VIT-CMJ2: Endophyte of Agaricus bisporus in Production of Bioactive Compounds
Chandan
Gautam
Department of Biomolecules Lab, School of Bio Sciences and Technology, VIT University, Vellore, India
author
Mukund
Madhav
Department of Biomolecules Lab, School of Bio Sciences and Technology, VIT University, Vellore, India
author
Astha
Sinha
Department of Biomolecules Lab, School of Bio Sciences and Technology, VIT University, Vellore, India
author
William
Osborne
Department of Biomolecules Lab, School of Bio Sciences and Technology, VIT University, Vellore, India
author
text
article
2016
eng
Background: Agaricus bisporus is an edible basidiomycete fungus. Both the body and the mycelium contain compounds comprising a wide range of antimicrobial molecules, contributing in improvement of immunity and tumor-retardation. Objectives: The presence of endophytes capable of producing bioactive compounds was investigated in Agaricus bisporus. Materials and Methods: Endophytes from Agaricus bisporus was isolated on LB agar. The obtained isolates were characterized morphologically and biochemically. Further 16S rRNA sequencing was implemented for molecular analysis of isolates. The isolate was mass produced and the bioactive compounds were extracted using ethyl acetate, chloroform and hexane. Agar well diffusion method was carried out to seek the potential of any antimicrobial activity of the crude bioactive compounds against known pathogens. GC-MS and FT-IR analysis were performed for the identification of bioactive compounds. Results: VIT-CMJ2 was identified as Enterobacter sp. as revealed by 16S rRNA sequencing. Chloroform extract of VIT-CMJ2 showed a maximum zone of inhibition of 19 mm against Salmonella typhi followed by hexane and ethyl acetate extracts. The GC-MS analysis revealed the presence of several bioactive compounds having effective antimicrobial activity like butyl ester, Behenicalcohol, S , S-dioxide derivatives and some others which were later confirmed by FT-IR spectral stretches. Conclusions: The present study shows the insight on the way endophytes interact with Agaricus bisporus; thereby improving the nutritional profile.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
19
24
https://www.ijbiotech.com/article_14133_21a0dba1c214d1170ed71ded6ef5db69.pdf
dx.doi.org/10.15171/ijb.1287
Green Extracellular Synthesis of the Silver Nanoparticles Using Thermophilic Bacillus Sp. AZ1 and its Antimicrobial Activity Against Several Human Pathogenetic Bacteria
Ali
Deljou
Department of Biotechnology, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran
author
samad
Goudarzi
Buali sina
author
text
article
2016
eng
Background: Silver nanoparticles (AgNPs) are among the most effective antimicrobial agents that are used in the medicine and pharmaceutics. During the past decades, metal nanoparticles synthesis through application of the biological methods has increasingly been used, as the biologically synthesized particles are mostly non-toxic as well as effective. Objectives: The main goal for undertaking the present investigation was to evaluate the extracellular synthesis of the AgNPs by a native thermophilic Bacillus Sp. AZ1 that was isolated from a hot spring in Ardebil province. Subsequently the antimicrobial potentials of the nanoparticle was evaluated against several human pathogenic organisms. Materials and Methods: The biosynthesized AgNPs were confirmed visually by appearance of a dark brown color formation in the mixture as well as silver surface plasmon resonance band by using UV-Visible spectroscopy. The AgNPs were further characterized by SEM, EDX and TEM. The antimicrobial activity of the AgNPs was investigated using Salmonella typhi, Escherichia coli, Staphylococcus epidermis, and Staphylococcus aureus, by applying disk diffusion method. Results: Identification of the strain AZ1 by the 16S rRNA sequence analysis showed 99% sequence homology between this strain and B. licheniformis. The obtained UV-Visible spectrum of the aqueous medium containing silver ion, showed a peak at 425 nm which indicates a correspondence to the plasmon absorbance of the silver nanoparticles. The biosynthesized AgNPs were found to be in the size range of ~7-31 nm with spherical the shape. Studies regarding the antibacterial effect of the particles showed the highest inhibitory effect against the two strains; E. coli, and S. typhi, respectively. Conclusions: Our study presents a simple green synthesis process for the production of an extracellular nanoparticles which is environmental friendly. Biosynthesis of the AgNPs by a thermophilic bacillus from the hot spring (Qeynarjeh, Ardebil) in Iran with the highest similarity to Bacillus licheniformis is reported for the first time.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
25
32
https://www.ijbiotech.com/article_14130_f84bd584aa6275924b43faeb389471d6.pdf
dx.doi.org/10.15171/ijb.1259
Optimization of RGD-modified Nano-liposomes Encapsulating Eptifibatide
Hassan
Bardania
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
author
Seyed Abbas
Shojaosadati
Department of Biotechnology Group Chemical Engineering, Tarbiat Modares University, Tehran, Iran
author
Farzad
Kobarfard
Department of Medical Chemistry, Faculty of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
author
Farid
Dorkoosh
Department of Pharmaceutics, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
author
text
article
2016
eng
Background: Eptifibatide (Integrilin) is an intravenous (IV) peptide drug that selectively inhibits ligand binding to the platelet GP IIb/IIIa receptor. It is an efficient peptide drug, however has a short half-life. Therefore, antithrombotic agents like eptifibatide are required to become improved with a protected and targeted delivery system such as using nano-liposomes to the site of thrombus. Objectives: The goal in the present report was to optimize encapsulation efficiency of the eptifibatide into Arg-Gly-Asp (RGD)-modified nano-liposomes (RMNL). As well, it was intended to evaluate the effect of sodium lauryl sulfate (SLS) on drug release. Materials and Methods: The effect of five independent variables including number of freeze/thawing cycles, concentration of eptifibatide, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and dipalmitoyl-GRGDSPA peptide on drug entrapment efficiency (DEE) was investigated using response surface methodology (RSM). The effect of different concentrations of SLS on encapsulation and drug release from RMNL was also investigated. The size and morphology of RMNL were characterized using transmission electron microscopy (TEM). Results: The maximum DEE (38%) was obtained with 7 freeze/thawing cycles, 3.65 mmoL eptifibatide, 7 mM DSPC, 3 mM cholesterol, and 1 mM dipalmitoyl- GRGDSPA peptide. SLS has significantly increased the drug release from RMNL, although its effect on encapsulation efficiency was not significant. Conclusions: The optimization of the formulations for valuable and expensive peptide drugs is essential to have the maximum encapsulation efficiency and the minimum experiments.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
33
40
https://www.ijbiotech.com/article_14135_82926dc54152825191a76d6b552ab2e7.pdf
dx.doi.org/10.15171/ijb.1399
Osteogenic Differentiation and Mineralization on Compact Multilayer nHA-PCL Electrospun Scaffolds in a Perfusion Bioreactor
Maliheh
Yaghoobi
Biomedical Engineering Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
author
Sameereh
Hashemi-Najafabadi
Biomedical Engineering Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
author
Masoud
Soleimani
Hematology Group, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
author
Ebrahim
Vasheghani-Farahani
Biomedical Engineering Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
author
Seyyed Mohammad
Mousavi
Biotechnology Group, Faculty of Chemical Engineering, Tarbiat Modares University, Tehran, Iran
author
text
article
2016
eng
Background: Monolayer electrospun scaffolds have already been used in bone tissue engineering due to their high surface-to-volume ratio, interconnectivity, similarity to natural bone extracellular matrix (ECM), and simple production. Objectives: The aim of this study was to evaluate the dynamic culture effect on osteogenic differentiation and mineralizationi into a compact cellular multilayer nHA-PCL electrospun construct. The dynamic culture was compared with static culture. Materials and Methods: The calcium content, alkaline phosphatase (ALP) activity and cell viability were investigated on days 3 and 7. Results: When the dynamic culture compared to static culture, the mineralization and ALP activity were increased in dynamic culture. After 7 days, calcium contents were 41.24 and 20.44 mg.(cm3)-1, and also normalized ALP activity were 0.32 and 0.19 U.mg-1 in dynamic and static culture, respectively. Despite decreasing the cell viability until day 7, the scanning electron microscopy (SEM) results showed that, due to higher mineralization, a larger area of the construct was covered with calcium deposition in dynamic culture. Conclusions: The dynamic flow could improve ALP activity and mineralization into the compact cellular multilayer construct cultured in the perfusion bioreactor after 7 days. Fluid flow of media helped to facilitate the nutrients transportation into the construct and created uniform cellular construct with high mineralization. This construct can be applied for bone tissue engineering.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
41
49
https://www.ijbiotech.com/article_14138_8bce3668921ec632085be849dbd5ffe5.pdf
dx.doi.org/10.15171/ijb.1382
Higher Expression Level and Lower Toxicity of Genetically Spliced Rotavirus NSP4 in Comparison to the Full-Length Protein in E. coli
Mehdi
Sahmani
Department of Clinical Biochemistry and Genetics, Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
author
Siavash
Azari
Department of Biotechnology, School of Paramedical Sciences, Qazvin University of Medical Sciences, Qazvin, Iran
author
Majid
Tebianian
Department of Biotechnology, Razi Vaccine and Serum Research Institute, Karaj, Iran
author
Nematollah
Gheibi
Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran
author
Farzaneh
Pourasgari
Department of Biotechnology, Razi Vaccine and Serum Research Institute, Karaj, Iran
author
text
article
2016
eng
Background: Rotavirus group A (RVA) is recognized as a major cause of severe gastroenteritis in children and new-born animals. Nonstructural protein 4 (NSP4) is responsible for the enterotoxic activity of these viruses in the villus epithelial cells. Amino acids 114-135 of NSP4 are known to form the diarrhea-inducing region of this viral enterotoxin. Therefore, developing an NSP4 lacking the enterotoxin domain could result in the introduction of a new subunit vaccine against rotaviruses in both humans and animals. Objectives: The aim of this study is the evaluation of rotavirus A NSP4 expression in E. coli expression system before and after removal of the diarrhea-inducing domain, which is the first step towards further immunological studies of the resulting protein. Materials and Methods: Splicing by overlap extension (SOEing) PCR was used to remove the diarrhea-inducing sequence from the NSP4 cDNA. Both the full-length (FL-NSP4) and the spliced (S-NSP4) cDNA amplicons were cloned into pET-32c and pGEX-6P-2. Expression levels of the recombinant proteins were evaluated in E. coli BL21 (DE3) by Western blot analysis. In addition, the toxicity of pET plasmids bearing the S-NSP4 and FL-NSP4 fragments was investigated by plasmid stability test. Results: For FL-NSP4, protein expression was detected for the strain containing the pGEX:FL-NSP4 plasmid, but not for the strain carrying pET:FL-NSP4. Hourly sampling up to 3 h showed that the protein production decreased by time. In contrast, expression of S-NSP4 was detected for pET:S-NSP4 strain, but not for pGEX:S-NSP4. Plasmid stability test showed that pET:S-NSP4 recombinant plasmid was almost stable, while pET:FL-NSP4 was unstable. Conclusions: This is the first report of production of rotavirus NSP4 lacking the diarrhea-inducing domain (S-NSP4). S-NSP4 shows less toxicity in this expression system and potentially could be a promising goal for rotavirus immunological and vaccine studies in the future.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
50
57
https://www.ijbiotech.com/article_14131_65e0497ffe7341d2b06ce5bdb29b4acb.pdf
dx.doi.org/10.15171/ijb.1233
Phylogenetic Analysis of Aedes aegypti Based on Mitochondrial ND4 Gene Sequences in Almadinah, Saudi Arabia
Khalil
AL ALI
Department of Medical Laboratory Technology, College of Applied Medical Sciences, Taibah University, Almadinah Almanwra, Kingdom of Saudi Arabic
author
Ayman
El-Badry
Department of Medical Parasitology, Kasr Al-Ainy School of Medicine, Cairo University, Cairo, Egypt
author
Mouhanad
AL ALI
Department of Institut Supérieur de la Santé et des Bioproduits d’Angers, Université d’Angers, Angers, France
author
Wael
El-Sayed
Department of Microbiology, Faculty of Science, Ain Shams University, Cairo 11566, Egypt
Department of Biology, Faculty of Science, Taibah University, Almadinah Almunawarah 344, Saudi Arabia
author
Hesham
El-Beshbishy
Department of Medical Laboratory Technology, College of Applied Medical Sciences, Taibah University, Almadinah Almanwra, Kingdom of Saudi Arabic
Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Nasr City, Cairo, Egypt
author
text
article
2016
eng
Background: Aedes aegypti is the main vector of the yellow fever and dengue virus. This mosquito has become the major indirect cause of morbidity and mortality of the human worldwide. Dengue virus activity has been reported recently in the western areas of Saudi Arabia. There is no vaccine for dengue virus until now, and the control of the disease depends on the control of the vector. Objectives: The present study has aimed to perform phylogenetic analysis of Aedes aegypti based on mitochondrial NADH dehydrogenase subunit 4 (ND4) gene at Almadinah, Saudi Arabia in order to get further insight into the epidemiology and transmission of this vector. Materials and Methods: Mitochondrial ND4 gene was sequenced in the eight isolated Aedes aegypti mosquitoes from Almadinah, Saudi Arabia, sequences were aligned, and phylogenetic analysis were performed and compared with 54 sequences of Aedes reported in the previous studies from Mexico, Thailand, Brazil, and Africa. Results: Our results suggest that increased gene flow among Aedes aegypti populations occurs between Africa and Saudi Arabia. Conclusions: Phylogenetic relationship analysis showed two genetically distinct Aedes aegypti in Saudi Arabia derived from dual African ancestor.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
58
62
https://www.ijbiotech.com/article_14134_c352342aadcaab1564b8d833431517dd.pdf
dx.doi.org/10.15171/ijb.1329
Cloning, Codon Optimization, and Expression of Yersinia intermedia Phytase Gene in E. coli
Maryam
Mirzaei
Department of Biology, Master of Science, Faculty of Science, Shahrekord University, Shahrekord, Iran
author
Behnaz
Saffar
Department of Genetics, Faculty of Sciences, Shahrekord University, Shahrekord, Iran
author
Behzad
Shareghi
Department of Biology, Faculty of Science, Shahrekord University, Shahrekord, Iran
author
text
article
2016
eng
Background: Phytate is an anti-nutritional factor in plants, which catches the most phosphorus contents and some vital minerals. Therefore, Phytase is added mainly as an additive to the monogastric animals’ foods to hydrolyze phytate and increase absorption of phosphorus. Objectives: Y. intermedia phytase is a new phytase with special characteristics such as high specific activity, pH stability, and thermostability. Our aim was to clone, express, and characterizea codon optimized Y. intermedia phytase gene in E. coli. Materials and Methods: The Y. intermedia phytase gene was optimized according to the codon usage in E. coli. The sequence was synthesized and sub-cloned in pET-22b (+) vector and transformed into E. coli Bl21 (DE3). The protein was expressed in the presence of IPTG at a final concentration of 1 mM at 30°C. The purification of recombinant protein was performed by Ni2+ affinity chromatography. Phytase activity and stability were determined in various pH and temperatures. Results: The codon optimized Y. intermedia phytase gene was sub-cloned successfully.The expression was confirmed by SDS-PAGE and Western blot analysis. The recombinant enzyme (approximately 45 kDa) was purified. Specific activity of enzyme was 3849 (U.mg-1) with optimal pH 5 and optimal temperature of 55°C. Thermostability (80°C for 15 min) and pH stability (3-6) of the enzyme were 56 and more than 80%, respectively. Conclusions: The results of the expression and enzyme characterization revealed that the optimized Y. intermedia phytase gene has a good potential to be produced commercially andto be applied in animals’ foodsindustry.
Iranian Journal of Biotechnology
National Institute of Genetic Engineering and Biotechnology of Iran
1728-3043
14
v.
2
no.
2016
63
69
https://www.ijbiotech.com/article_14136_ef6f9f044a1b735f7804bc45da7d693f.pdf
dx.doi.org/10.15171/ijb.1412