Iranian Journal of Biotechnology
Year:2017 | Volume:15 | Issue:2|Papers Count:9

Background: Rice seed proteins are lacking essential amino acids (EAAs). Genetic engineering offers a fast and sustainable method to solve this problem as it allows the specific expression of heterologous EAA-rich proteins. The use of selectable marker gene is essential for generation of transgenic crops, but might also lead to potential environmental and food safety problems. Therefore, the production of marker-free transgenic crops is becoming an extremely attractive alternative and could contribute to the public acceptance of transgenic crops.Objectives: The present study was conducted to examine whether AmA1 can be expressed specifically in rice seeds, and generate marker-free transgenic rice with improved nutritive value.Materials and Methods: AmA1 was transferred into rice using Agrobacterium -mediated co-transformation system with a twin T-DNA binary vector and its integration in rice genome was confirmed by southern blot. Transcription ofAmA1 was analyzed by Real-Time PCR and its expression was verified by western analysis. Protein and amino acid content were measured by the Kjeldahl method and the high-speed amino acid analyzer, respectively.Results: Five selectable marker-free homozygous transgenic lines were obtained from the progeny. The expression of recombinant AmA1 was confirmed by the observation of a 35 kDa band in SDS-PAGE and western blot. Compared to the wild-type control, the total protein contents in the seeds of fi vehomozygous lines were increased by 1.06~12.87%.In addition, the content of several EAAs, including lysine, threonine, and valine was increased significantly in the best expressing line.Conclusions: The results indicated that the amino acid composition of rice grain could be improved by seed-specific expression ofAmA1.

Background: In recent years, nanomaterials have been widely used in large quantities which make people be more frequently exposed to the chemically synthesized nanoparticles (NPs). When NPs are introduced into an organism, they may interact with a variety of cellular components with yet largely unknown pathological consequences.Objective: It was found that NPs enhance the rate of protein fi brillation in the brain by decreasing the lag time for nucleation. Protein fibrillation is implicated in the pathogenesis of the several neurodegenerative diseases such as Parkinson’s disease (PD). a-Synuclein (aS) is natively an unfolded protein which is involved in the pathogenesis of PD. In the present study, we have analyzed the effects of three different NPs on αS fi brillation.Materials and Methods: αS protein expression and purification was done and fibrils formation was induced in the absence or presence of the three types of NPs (i. e., TiO2, SiO2, and SnO2). The enhancement of the fluorescence emission of Thioflavin T (ThT) and transmission electron microscopy (TEM) were used to monitor the appearance and growth of the fibrils. The adsorption of αS monomers on the surface of NPs was investigated by tyrosine fluorescence emission measurements.Results: We found that TiO2-NPs enhances αS fibril formation even at a concentration of 5 mg.mL-1, while the two other NPs show no significant effect on the kinetics of the fi brillation. Intrinsic tyrosine emission measurement has confirmed that the TiO2-NPs interact with αS fi brillation products. It is suggested that TiO2-NPs may enhance the nucleation of aS protein that leads to protein fi bril formation.Conclusion: The fi brillization process of aS protein is profoundly aff ected by the presence of TiO2-NPs. This finding unveils the neurotoxicity potential of the TiO2-NPs, which may be considered as a probable risk for PD.

Background: Aptamers are single stranded DNA (ssDNA) or RNA molecules. The potential of aptamers for binding to the diff erent targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment (SELEX) method is much dependent on the successful conversion of double stranded DNA (dsDNA) to ssDNA.Objective: There are diff erent methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 diff erent sequences. This study was designed to evaluate the effi ciency of another technique for confi rming the ssDNA generation in comparison to the polyacrylamide electrophoresis (PAGE) analysis.Materials and Methods: Real-time PCR was employed in the present study for PCR amplifi cation of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis.Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a signifi cant reduction of Tm from 73.8 to 41.5 °C. Applying PAGE analysis, it was not eff ectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was effi cient enough to confi rm ssDNA generation in accordance with the increasing the number of SELEX rounds.Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confi rmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation.

Background: Colorectal cancer is the third most common type of aggressive cancers. Chemotherapy, surgery, and radiotherapy are the common therapeutic options for treating this cancer. Due to the adverse side-effects of these methods, immunotherapy is considered as an appropriate alternative therapeutic option. Treatment through the application of monoclonal antibodies is considered as a novel alternative therapeutic method for cancers. The variable fragments of the antibodies’ heavy chain or VHHs have a wide application in molecular biology and biotechnology. VHHs are compatible with the phage display technology which allows rapid and high throughput screening for antibodies isolation.Objectives: We aimed to use naive VHH phage library to isolate a specific nanobody against colorectal tumor associated antigen; the AgSK1.Materials and Methods: In this research, naive VHH phage library was panned against two colorectal cell lines; Ls174T and HT29 expressing different levels of AgSK1 tumor associated marker. The high affinity binders were selected and subcloned for higher expression levels of the VHH. The affinity and specificity of the isolated VHH were tested using ELISA. The reactivity of the VHH toward cancer cells was analyzed by competitive ELISA applying sera isolated from colorectal cancer patients.Results: Results show that the isolated VHH recognizes and binds to the colorectal cancer cells with a high affinity. Moreover, the isolated nanobody is able to compete with the antibodies in the patient sera for the binding to the cancer cells.Conclusions: Results suggest that this nanobody has a specific reaction toward colorectal cells and can be used for further investigation on the tumor associated antigens or production of mimotopes useful for immunotherapy.

Background: The presence of pharmaceuticals at low concentrations (ng to mg) in the environment has become a hot spot for researchers in the past decades due to the unknown environmental impact and the possible damages they might have to the plantae and fauna present in the aquatic systems, as well as to the other living organisms.Objectives: The aim of the present investigation was to develop a bacterial consortium isolated from diff erent origins to evaluate the ability of such a consortium to remove a mixture of pharmaceuticals in the batch system at lab scale, as well as assessment of its resistance to the other micropollutants present in the environment.Material and Methods: Using a closed bottle test, biodegradation of the mixed pharmaceuticals including Diclofenac (DCF), Ibuprofen (IBU), and Sulfamethoxazole (SMX) (at a concentration of 3 mg.L-1 of each drug) by the bacterial consortium was investigated. The test was carried out under metabolic (pharmaceutical was used as the sole source of carbon) and co-metabolic condition (in the presence of glucose). Finally, the ability of the bacterial consortium to resist other micropollutants like antibiotics and heavy metals was investigated.Results: Under the metabolic condition, the mixed bacteria (i.e., consortium) were able to metabolize 23.08% and 9.12% of IBU, and DCF at a concentration of 3 mg.L-1 of each drug, respectively. Whereas, in co-metabolic conditions, IBU was eliminated totally, in addition, 56% of the total concentration of DCF was removed, as well. In both metabolic and cometabolic conditions, removal of SMX was not observed. The selected bacteria were able to resist to most of the applied antibiotics and the used heavy metals, except mercury, where only one strain (S4) was resistant to the later heavy metal.Conclusion: Results suggest that the developed consortium might be an excellent candidate for the application in the bioremediation process for treating ecosystems contaminated with the pharmaceutical.

Background: A number of microorganisms and their enzymes have been reported as xanthan depolymerizers. Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp.XD are the only species in the genus which are now known to degrade xanthan.Objectives: Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms.The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under diff erent environmental conditions.Materials and Methods: The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identifi ed using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at different temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC (thin-layer chromatography), respectively.Results: Six isolates from different Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to beP. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed diff erent levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55oC and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl (>0.1 mol.L-1). Furthermore, the isolated Paenibacillusspp. could degrade several other polysaccharides including xylan, CMC (carboxymethyl cellulose), starch, alginate, and pectin.Conclusion: Novel strains of the six diff erent Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes.

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression inE. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid.Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized.Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F’. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG (0.5 mM) and incubation with 0, 100, 200 and 300 μg.mL-1 ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve.Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 mg.mL-1 ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07×109, 3.21×109, 2.32×1010, 8.11×108, respectively. The plasmid yields were 55 ng.mL-1, 69 ng.μL-1, 164 ng. mL-1 and 41 ng. mL-1, respectively.Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration (200 mg.mL-1) was signifi cantly (p<0.01) higher than other doses.

Background: Nanoparticles have been applied to medicine, hygiene, pharmacy and dentistry, and will bring significant advances in the prevention, diagnosis, drug delivery and treatment of disease. Green synthesis of metal nanoparticles has a very important role in nanobiotechnology, allowing production of non-toxic and eco-friendly particles.Objectives: Green synthesis of silver nanoparticles (AgNPs) was studied using pine pollen as a novel, cost-effective, simple and non-hazardous bioresource. The antifungal activity of the synthesized AgNPs was investigatedin vitro.Materials and Methods: Biosynthesis of AgNPs was conducted using pollen of pine (as a novel bioresource) acting as both reducing and capping agents. AgNPs were characterized using UV-visible spectroscopy, X-ray diffraction and transmission electron microscopy. In evaluation for antifungal properties, the synthesized AgNPs represented significantin vitro inhibitory eff ects onNeofusicoccum parvum cultures.Results: Pine pollen can mediate biosynthesis of colloidal AgNPs with an average size of 12 nm. AgNPs were formed at 22oC and observed to be highly stable up to three months without precipitation or decreased antifungal property. AgNPs showed significant inhibitory effects against Neofusicoccum parvum.Conclusion: The first report for a low-cost, simple, well feasible and eco-friendly procedure for biosynthesis of AgNPs was presented. The synthesized AgNPs by pine pollen were nontoxic and eco-friendly, and can be employed for large-scale production. The nanoparticles showed strong eff ect on quantitative inhibition and disruption of antifungal growth.

Background: The resistance of the bacteria and fungi to the innumerous antimicrobial agents is a major challenge in the treatment of the infections demands to the necessity for searching and fi nding new sources of substances with antimicrobial properties. The incorporation of the essential oils (EOs) in chitosan fi lm forming solution may enhance antimicrobial properties. However, its use as the feeding additive in the poultry nutrition needs to clarify the product’s activity against both pathogen and the useful microbes in the gastrointestinal tract.Objectives: In the present study, we carried out an in vitro investigation and evaluated the antimicrobial activity of chitosan fi lm forming solution incorporated with essential oils (CFs+EOs) against microbial strains includingStaphylococcus aureus, Escherichia coli, Enterococcus faecium, Lactobacillus rahmnosus, Aspergillus niger and Alternaria alternate.Material and Methods: In three replicates, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of diff erent treatments including: 1- essential oils (EOs), 2- chitosan fi lm solution (CFs), and 3-chitosan fi lm solution enriched with EOs (CFs+EOs) were determined against above mentioned microbes.Results: The results indicated that the chitosan solution enriched with essential oils (CFs+EOs) is capable of inhibiting the bacterial and fungal growth even at the lowest concentrations. The MIC and MBC for all the antimicrobial agents against Escherichia coliand Staphylococcus aureus were very low compared to the concentrations needed to inhibit the growth of useful bacteria, Lactobacillus rahmnosus and Enterococcus faecium. The antifungal activity of chitosan was enhanced as the concentration of EOs increased in the fi lm solution.Conclusion: Chitosan-EOs complexes are the promising candidate for novel contact antimicrobial agents that can be used in animal feeds.