فهرست مطالب
Iranian Journal of Biotechnology
Volume:16 Issue: 3, Summer 2018
- تاریخ انتشار: 1397/06/04
- تعداد عناوین: 9
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Pages 164-172BackgroundA genome-scale metabolic network model (GEM) is a mathematical representation of an organisms metabolism. Today, GEMs are popular tools for computationally simulating the biotechnological processes and for predicting biochemical properties of (engineered) strains.ObjectivesIn the present study, we have evaluated the predictive power of two GEMs, namely iBsu1103 (for Bacillus subtilis 168) and iMZ1055 (for Bacillus megaterium WSH002).Materials And MethodsFor comparing the predictive power of Bacillus subtilis and Bacillus megaterium GEMs, experimental data were obtained from previous wet-lab studies included in PubMed. By using these data, we set the environmental, stoichiometric and thermodynamic constraints on the models, and FBA is performed to predict the biomass production rate, and the values of other fluxes. For simulating experimental conditions in this study, COBRA toolbox was used.ResultsBy using the wealth of data in the literature, we evaluated the accuracy of in silico simulations of these GEMs. Our results suggest that there are some errors in these two models which make them unreliable for predicting the biochemical capabilities of these species. The inconsistencies between experimental and computational data are even greater where B. subtilis and B. megaterium do not have similar phenotypes.ConclusionsOur analysis suggests that literature-based improvement of genome-scale metabolic network models of the two Bacillus species is essential if these models are to be successfully applied in biotechnology and metabolic engineering.Keywords: Biochemical capability, Bacillus Species, Computational biotechnology, Model validation, Systems biology
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Pages 173-184BackgroundPrediction of the protein localization is among the most important issues in the bioinformatics that is used for the prediction of the proteins in the cells and organelles such as mitochondria. In this study, several machine learning algorithms are applied for the prediction of the intracellular protein locations. These algorithms use the features extracted from protein sequences. In contrast, protein interactions have been less investigated.ObjectivesAs protein interactions usually occur in the same or adjacent places, using this feature to find the location would be efficient and impressive. This study did not aim at increasing the total accuracy of the conducted research. The study has focused on the features of the proteins interaction and their employment which lead to a higher accuracy.Materials And MethodsIn this study, we have examined the protein interaction network as one of the features for prediction of the protein localization and its effects on the prediction results. In this regards, we have gathered some of the most common features including Amino Acid Composition, Dipeptide Compositions, Pseudo Amino Acid Compositions (PseAAC), Position Specific Scoring Matrix (PSSM), Functional Domain, Gene Ontology information, and the Pair-wise sequence alignment. The results of the classification are compared to the ones using protein interactions. For achieving this goal different machine learning algorithms were tested.ResultsThe best-obtained results of using single feature set obtained using SVM classifier for PseAAC feature. The accuracy of combining all features with PPI data, using the Decision Tree and Random Forest classifiers, was 82.49% and 83.35%, respectively. In another experiment, using just protein interaction data with the different cutting points resulted in obtaining an accuracy of 93.035% for the protein location prediction.ConclusionIn total, it was shown that protein(s) interaction has a significant impact on the prediction of the mitochondrial proteins location. This feature can separately distinguish the locations well. Using this feature the accuracy of the results is raised up to 5%.Keywords: Machine learning, Mitochondria, Protein localization, Protein-Protein Interaction (PPI)
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Pages 185-192BackgroundConsidering natural thermal stability, Geobacillus stearothermophilus amylase and Cel5E from Clostridium thermocellum are good candidates for industrial applications. To be compatible with the industrial applications, this enzyme should be stable in the high temperatures, so any improvement in their thermal stability is valuable.ObjectivesUsing in silico approach and identifying point mutations in the structure amylase of G. stearothermophilus and Cel5E from C. termocellum we tried to increase thermal stability of the enzymes along with their catalytic activity to reach a new industrial amylase with higher thermostability and an improved function.Materials And MethodsIn this study we predicted the 3D structure of the enzymes, then simulated the molecular docking study using MolDock, PLANTS, and Lamarkian genetic algorithm as scoring functions for the docking and in silico engineering of the protein aiming to increase the thermal stability and catalytic activity.ResultsA series of thermal stability increasing point mutations were exerted around the active site of the enzyme, then by docking procedure, the binding affinity was measured and finally a list of mutations which theoretically improved the increased thermal stability as well as catalytic activity were proposed.ConclusionsBased on the in silico results obtained the modified enzymes seems to be suitable candidates for considering in both laboratory and industrial scales.Keywords: Amylase, Catalytic activity, Cel5E, Geobacillus stearothermophilus, Industry, Thermal stability
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Pages 193-199BackgroundPetroleum drilling and exploration operations generate a significant amount of oily wastes. Comparing with physical and chemical remediation methods, bioremediation is cost-effective, highly efficient, and environment-friendly technology.ObjectivesThe aim of this study was to evaluate biological treatment efficiency for an oil-contaminated soil prepared from an abandoned drilling mud pit located in the Khangiran region , Khorasan Razavi Provinvce, Iran.Materials And MethodsFollowing chemical analysis of the contamination, isolation and phylogenetic analysis of the predominant hydrocarbon-utilizing bacteria were performed in order to elucidate microbial diversity. Afterwards, the full factorial design was used to optimize the concentration of nitrogen (0, 100, 250, and 500 ppm) and phosphate (0, 10, 25, and 50 ppm) in the soil. The prepared microcosms were incubated at 30 °C for a 60-day period. The total heterotrophic bacterial content was enumerated during bioremediation process. The total petroleum hydrocarbon concentration was analyzed after 60 days to evaluate biodegradation extent.ResultsThe mud pit was mainly contaminated with the weathered diesel compounds ranging from C12 to C30. Investigation of the indigenous microflora showed that a variety of hydrocarbon-utilizing bacteria were present in the untreated soil dominated by Actinobacteria including the genera Gordonia, Nocardia, Nocardioides, and Rhodococcus. The full-factorial soil microcosms nutrient optimization indicated that nitrogen amendment could be the most effective at 250 ppm, while excess nitrogen would lead to a diminished microbial activity. The main effect statistical analysis demonstrated that addition of phosphate did not cause a significant improvement in the remediation process. In the optimal nutrient conditions, the total microbial count increased from 1.76 × 107 CFU.g-1 to 1.24 × 109 CFU.g-1 and led into 75.4 % w/w biodegradation in two months, while in the control microcosm without nutrient amendment only 36.6 % of the depletion was observed.ConclusionsThese results indicate that biostimulation could be a promising candidate for a successful field-scale bioremediation process in the studied drilling mud pit.Keywords: Bioremediation, Diesel, Mud pit, Microflora, Nutrient optimization, Oil-based drilling
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Pages 200-212BackgroundPlant biomass and agricultural waste products disposal is a serious problem in agriculture based countries. These wastes, usually rich in xylan can be satisfactorily converted to industrially important and useful products by efficient biotechnological application of potent xylanase producing bacteria which generally have high temperature and pH optima.ObjectiveThe aim was to isolate and identify xylanolytic bacterial isolates from Bhilai-Durg region of Chhattisgarh, India which was otherwise unexplored for isolation of thermoalkalophilic xylanase producing bacteria. Partial scale up of process development was performed.Materials And MethodsXyalanse producing bacteria were isolated from probable samples following three stages of screening procedures. The potent isolates were identified and various parameters affecting xylanase production were optimized using the conventional one-factor-at-a-time approach.ResultsTwo potent indigenous bacterial isolates belonged to genus Bacillus and designated as Bacillus sp. MCC2728 and Bacillus sp. MCC2727 were isolated from forest soils with the ability to degrade xylan. Significant differences were observed in their morphology and phenotypic characters amongst themselves and with its closest type strains implying the novelty of the two isolates. After optimization, maximum xylanase levels were obtained at pH 9.0, 55 °C for Bacillus sp. MCC2728 and 50 °C for Bacillus sp. MCC2727, 5% inoculum, agitation speed (150 rpm). Yeast extract and peptone are best nitrogen sources and wheat bran, the best carbon source. The GenBank/EMBL/DDBJ accession numbers of strains Bacillus sp. MCC2728 and Bacillus sp. MCC2727 are KP742971 and KT444621 respectively. Wheat bran, Yeast extract and peptone proved to be the best carbon and nitrogen sources respectively and xylose as an additive was found to be contributing to maximize the xylanase yields.ConclusionTwo potent thermoalkalophilic novel bacterial isolates were successfully isolated with xylan degrading ability which may be used as promising xylanase producing candidates for various industrial purposes using agricultural based waste residues.Keywords: Bacillus sp, Optimization, Phylogenetic analysis, Thermoalkalophilic bacteria, Xylanase
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In Vitro Cytotoxic Activity of a Lactococcus lactis Antimicrobial Peptide Against Breast Cancer CellsPages 213-220BackgroundNisin, an effective natural food preservative, is an antimicrobial peptide produced by Lactococcus lactis. Although it has been mainly studied and developed as a potential alternative for antibiotics, other pharmacological effects of the nisin including cytotoxic and anti-tumor activity have been attracted many attentions.ObjectivesHere, we aimed to evaluate in vitro cytotoxic activity of the nisin against breast cancer cells.Materials And MethodsThe effect of temperature, pH, and chemical composition of the medium on the yield of nisin production was evaluated. As well, the anti-proliferative effect of nisin against a breast cancer cell line (i. e., MCF-7) and a non-cancerous cell line (i.e, HUVEC) was determined using MTT assay. Furthermore, the potential of the synergistic effect of the nisin on the doxorubicin cytotoxicity was evaluated.ResultsThe optimum culture condition for the nisin production by L. lactis was found to be MRS medium (pH 6.3) supplemented with the tryptone and incubation at 30 °C. MTT assay results indicate that nisin exhibits a high and selective cytotoxicity against MCF-7 cell line with IC50 value of 5 μM. Furthermore, a combination of nisin and doxorubicin at sub-inhibitory concentrations were more cytotoxic compared to either of drugs alone.ConclusionIt could be suggested that nisin, either alone or in combination with other chemotherapeutic agents, could be a potential therapeutic for the breast cancer cells.Keywords: Antimicrobial peptide, Breast Cancer, Cytotoxicity, Nisin, Lactococcus lactis
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Pages 221-226BackgroundTransgenic mice are being considered as invaluable tool in biological sciences towards comprehension of the cause of the genetic diseases. Manipulated embryonic stem (ES) cells are used to produce loss-of-function mutant mice. Microinjection of manipulated ES cells into blastocoel cavity, and morula fusion are the two main techniques in producing transgenic mice. So far, no reports have dealt with the comparison of these two methodologies provide.ObjectiveThe object of this study was to determine advantages and disadvantages of knockout mouse creation protocols.Materials And MethodsBoth blastocyst microinjection and morula aggregation were implemented to produce chimeric mice and the advantages and disadvantages of each technique were evaluated. For this, embryonic stem cells were transfected with a GFP-expression vector. In blastocyst microinjection technique, first transfected ES cell were cultured and appropriate colonies were selected. The cells were microinjected to blastocoel cavity of the expanded blastocyst. In morula aggregation technique, the transfected ES cell colonies were sandwiched between two naked morulas. After 16 h incubation in a 5% CO2 at 37 °C the morulas and infected ES cell were aggregated to produce a new morula. All the injected blastocyst and aggregated morulas were transferred to uterus of foster mice. The new born mice were analyzed for chimera confirmation.ResultsFive chimeric mice (21.75%) from morula aggregation and eight chimeric mice (63%) from blastocyst microinjection were born. The results indicated that both techniques can be used to generate chimeric mouse, however the success rate was higher in blastocyst microinjection.ConclusionMorula fusion stands out where the required instrumentations are in place. Furthermore, the quality of ES cells plays a prominent role in the success rate. When the cell quality is low the blastocoel microinjection is recommended. The microinjection technique is more effective than morula aggregation.Keywords: Blastocoel microinjection, Embryonic stem cell, Loss-of-function mice mutants, Morula fusion
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Pages 227-234BackgroundGroundnut bud necrosis virus (GBNV) (Tospovirus genus, Bunyaviridae family) infects the major crops of solanaceae, leguminosae and cucurbitaceae in India. Temperature is an important factor which influences the plant growth and development under diseased conditions.ObjectiveIn the present study, we evaluated the effect of four different temperatures on the symptoms expression and viral RNA accumulation in the GBNV inoculated cowpea plants.
Material andMethodsFor the evaluation of viral symptoms and RNA accumulation, we used 2-3 leaf stage cowpea plants grown in the controlled conditions. GBNV was mechanically inoculated by sap method to the cowpea plants and inoculated plants were incubated at four different temperatures (30, 25, 20 and 15 °C).ResultsThe first visible symptom of GBNV infection at the inoculated site was observed in the form of chlorotic spots which were converted into the necrotic spots as the infections succeeded. Some yellow mosaic symptoms were also observed at the systemic site during viral infection cycle. Plants incubated at higher (30 and 25 °C) temperatures showed a severe necrosis and a higher viral RNA accumulation at the inoculated site and facilitated the viral spread at the systemic site. However, viral RNA accumulation was less at the systemic site than the inoculated site. In contrast, symptoms expression and viral RNA accumulation were decreased at the inoculated site at low (20 and 15 °C) temperatures, no viral symptoms were observed at the systemic site (15 °C); in addition to viral RNA accumulation suppression at this site. GBNV infection at the inoculated site induced the higher accumulation of H2O2 followed by the induction of cell death at higher temperatures (30 and 25 °C) than the lower (20 and 15 °C) temperatures.ConclusionThis study suggests that viral RNA accumulation parallels with the H2O2 production and induction of cell death by GBNV infection in cowpea plants is temperature dependent.Keywords: Chlorosis, H2O2, Hypersensitive response (HR), Programmed cell death (PCD), Necrosis -
Pages 235-240BackgroundRecently, the incidence of allergic diseases has been on the rise; Dust mite is the major indoor allergen which needs a special consideration.ObjectivesThis study was carried out to identify and investigate the molecular properties of a new allergen named Hsp60 and to afford a foundation for future research of the allergic diseases caused by Dermatophagoides farinae.Materials And MethodsUsing polymerase chain reaction (PCR) with degenerate primer, the cDNA of Dermatophagoides farinae Hsp60 was amplified and sequenced. Next, the cDNA fragment was cloned into the prokaryotic expression vector pET-32a for the expression of the Hsp60. Then, it was further characterized by Elisa and Western Blot analysis.ResultsThe partial cDNA sequence of the Dermatophagoides farinae Hsp60 was determined, and the recombinant Hsp60 was successfully expressed in Escherichia coli BL21. ELISA and Western blot analysis showed that the recombinant protein could be specifically recognized by SIgE from sera of the Dermatophagoides farina-allergic patients.ConclusionsOur group has, for the first time, demonstrated the fact that there is an Hsp60 family of Dermatophagoides farinae and analyzed the allergenicity of the Hsp60 with immunological method. These results provide a foundation for further allergological research of the Dermatophagoides farinae Hsp60.Keywords: Dermatophagoides farinae, Expression, Hsp60