Iranian Journal of Biotechnology
Volume:19 Issue: 1, Winter 2021

Salinity is one of the most important environmental stresses which reduces the nutrient uptake, growth and yield of crops including sunflower. The aim of this study was evaluating the expression pattern of telomerase gene, TERT, in sunflower plants under salinity stress. Sunflower plants of both sensitive and resistant lines were grown in greenhouse and treated with different levels of NaCl (2, 5 and 8 dSm-1). The expression pattern of TERT gene was evaluated at 8th leaf stage 6, 12 and 24 hours post salt treatment using real time-PCR, since the effects of salt stress are eventually manifested in the leaves. In both lines, salt-subjected plants showed reduced size and dried leaves, due to breakthrough of the growth. Compared to the control group, treated groups tended to indicate downregulated pattern of TERT gene expression. This study offers TERT as a new gene affected by salt stress when growth is arrested.

The details of molecular mechanisms underlying the differentiation of Mesenchymal Stem Cells (MSCs) into specific lineages are not well understood. We aimed to construct the interactome network and topology analysis of bone marrow mesenchymal stem cell of CAGE data. Applying the enrichment results, we wanted to introduce the common genes and hub-microRNA and hubgenes of these giant network. In this study, we constructed gene regulatory networks for each non-mesenchymal cell lineage according to their gene expression profiles obtained from FANTOM5 database. The putative interactions of TF-gene and protein-protein were determined using TRED, STRING, HPRD and GeneMANIA servers. In parallel, a regulatory network including corresponding miRNAs and total differentially expressed genes (DEGs) was constructed for each cell lineage. The results indicated that analysis of networks’ topology can significantly distinguish the hub regulatory genes and miRNAs involved in the differentiation of MSCs. The functional annotation of identified hub genes and miRNAs revealed that several signal transduction pathways i.e. AKT, WNT and TGFβ and cell proliferation related pathways play a pivotal role in the regulation of MSCs differentiation. We also classified cell lineages into two groups based on their predicted miRNA profiles. In conclusion, we found a number of hub genes and miRNAs which seem to have key regulatory functions during differentiation of MSCs. Our results also introduce a number of new regulatory genes and miRNAs which can be considered as the new candidates for genetic manipulation of MSCs in vitro.

Abiotic environmental stresses, especially drought stress, is one of the most important problems in arid and semi-arid regions. Like other major crops, Brassica napus is vulnerable to drought stress.

The present study was conducted to evaluate efficacy of Sargassum angustifolium extract on mitigating adverse effects of drought stress on B. napus seedlings during vegetative growth under greenhouse conditions.

Seedlings were periodically sprayed with the seaweed extract until they reached 7-leaf stage. Then water deficit stress was imposed and measurements were performed at morphological, biochemical and molecular levels on three phases: 80% field capacity for 20 days (Phase I), 60% field capacity for 20 days (Phase II) and 40% field capacity for 20 days (Phase III). Real-Time PCR assay was carried out to monitor the changes in expression of the genes involved in proline biosynthesis.

Morphological measurements revealed that seaweed treatment improved shoot height and dry weight compared to control (p<0.05). Biochemical analyses indicated that foliar application of seaweed extract significantly enhanced the photosynthetic pigments’ content, free radical scavenging and superoxide dismutase activity (p<0.05). Moreover, proline content was significantly increased in plant tissues treated with seaweed extract (p<0.05). The results of Real-Time PCR assay showed that the increase in proline content is due to enhanced expression of P5CS which is involved in biosynthesis of proline, and to decreased expression of PRODH which catalyzes proline degradation.

Overall, the results obtained in this research suggest that application of S. angustifolium extract as a biostimulant is able to protect canola seedlings against deteriorating effects of drought stress.

Barley (Hordeum vulgar L.) is a valuable platform for producing recombinant proteins. Before using different barley cultivars as an efficient platform for molecular farming, optimization of cultural conditions and studying the effective factors on the tissue culture are critical. In this study, we evaluated callus induction, plant regeneration and changes in the levels of total antioxidant, total phenol and endogenous hormones of three Iranian barley cultivars (Reyhan, Yousef and Bahman) and Golden Promise cultivar. We used immature embryos as explants on MS-based medium containing 3 mg.L-1 2,4-D for callus induction. Calluses were transferred to regeneration media with 2 mg.L-1 BAP. The levels of endogenous hormones were measured using High-Performance Liquid Chromatography system and total antioxidant and total phenols were determined using a spectrophotometer. We demonstrated that callus formation was very high in all cultivars (about 91%) and all immature embryo explants had the potential to produce embryogenic calluses. The present study also showed that the regeneration rates among the studied cultivars were very different and the Iranian cultivars showed lower regeneration percentages (about 1.4%) compared to Golden Promise cultivar (about 72.5%). The levels of endogenous hormones in Iranian cultivars and Golden Promise varied distinctly and significant differences in terms of total antioxidants and total phenols were found in the two groups. Accumulated evidence suggests that for successful regeneration of recalcitrant cultivars, external treatments should be done in a way to reduce the inhibitory effects of internal factors.

Brucella spp. are intracellular pathogens, therefore cell-mediated immunity is the main response to inhibit survival and growth of the bacteria in vertebrate host. Many eukaryotic plasmid vectors are being used in setting up DNA vaccines which may show different efficiencies in same conditions. This is important in designing the vaccines and immunization strategies. We looked into the probable differences of immune responses induced by different eukaryotic DNA plasmid vectors (pcDNA3.1 and pVAX1) harboring the same Omp31 gene of B. melitensis. Female BALB/c mice were immunized with pcDNA-omp31 and pVAX-omp31 and further boosted with recombinant Omp31. Subclasses of specific serum IgG against the rOmp31 were measured by ELISA. Cytokines responses to rOmp31 in Splenocyte cultures of the immunized mice were evaluated by measuring the production of IL4, IL-10, IL-12 and IFN-γ. Protective responses of the immunized mice were evaluated by intraperitoneal challenge with pathogenic Brucella melitensis 16M and Brucella ovis PA76250. Both DNA vaccine candidates conferred potent Th1-type responses with higher levels of cytokines and immunoglobulins observed in mice immunized with pVAX-omp31. Although pcDNA-omp31 and pVAX-omp31 both elicited protective immunity, mice immunized with the latter showed a higher protection against both B. melitensis and B. ovis PA76250. The results of this study highlight the significant differences between efficiency of diverse plasmid backbones in DNA vaccines which code for an identical antigen. Comparing various plasmid vectors should be considered as an essential part of the studies aiming construction of DNA vaccines for intracellular pathogens.

Osteoporosis is a bone disease alters the amount and variety of proteins in bone tissue and increases the potential of bone fracture. Antiresorptive therapy is one of the most popular treatment methods for osteoporosis. To reduce side effects and enhance the bioavailability of drug agents, the controlled delivery of drug is commonly utilized. We investigated the controlled release of Alendronate in different composites of layered double hydroxide (LDH) using poly (ε-caprolactone) (PCL) as a matrix. We prepared different microsphere composites of ALD intercalated in various amounts of LDH, using PCL as a matrix. The controlled release of ALD from these composites is subsequently investigated. Samples are characterized and in vitro cell cytotoxicity, attachment, osteogenic activity including alkaline phosphatase activity and mineralization are examined using MG-63 human osteosarcoma cells. The results showed that the release of ALD is more desirable and controlled in the samples having a higher amount of LDH incorporated into the PCL matrix. MG63 cells show a significant increase in viability, attachment, and mineralization while alkaline phosphatase activity remains almost at a constant level after 3 weeks. Overall, the findings showed that by incorporation of 15 wt% of LDH, the composite microsphere is capable of holding the antiresorptive drug longer and release it in a more controlled manner. This is an advantageous and promising characteristic for a carrier that could be used as a potential candidate for osteoporosis treatment.

Baculovirus expression system, introduced more than 20 years ago, is considered as a useful tool for large and complex eukaryotic recombinant protein production. A baculovirus expression vector is a recombinant virus which desired foreign protein coding sequences is under control of the virus gene promoter. Baculovirus only infects insect cells and do not normally infect vertebrates therefore, they possess no risk of biological risks for human.

The aim of this study was to recombinant expression of vascular endothelial growth factor (VEGF) reseptor-2 specific Nanobody in the baculovirus expression system.

Gene of specific Nanobody against the VEGF reseptor-2 that called 3VGR19 was cloned and expressed in baculovirus system.

3VGR19 Nanobody gene was amplified by Polymerase Chain Reaction (PCR) using the specific primers, and was cloned in pFastBac HTA plasmid. DH10Bac bacteria was transformed with resulted donor plasmid. The cultured Sf9 insect cell line was transfected with recombinant bacmid, and finally, the expression and purification of 3VGR19 was confirmed in insect cells.

In conclusion, Transient infection of insect cells with baculovirus can be a promising technology for expression of antibody fragments.

The immune system function depends on the coordination activity of the components of system and communications between them which leads to the formation of a complex communication network between immune cells. In this network, cytokines have an important role in the communication between immune cells through the interaction to their specific receptors. These molecules cause to cellular communications and normal function of a tissue. Reconstruction of such a complex network can be a way to provide a better understanding of cytokines’ function. Our main goal from reconstructing such a network was investigation of expressed cytokines and cytokines receptors in various lineage and tissues of immune cells and identifying the lineage and tissue with the highest expression of cytokines and their receptors In this study, gene expression data related to part of the Immunological Genome Project (ImmGen) and receptor-ligand interactions dataset were used to reconstruct the immune network in mouse. In next step, the topological properties of reconstructed network, expression specificity of cytokines and their receptors and interactions specificity were analyzed. The results of the network analysis were indicated that non- hematopoietic stromal cells have the highest expression of cytokines and cytokine receptors and interactions specificity is very high. Our results show that chemokine receptor of Ccr1 receives the largest number of signals between receptors and only expressed in three hematopoietic lineages. The most of the network communications belonged to non-hematopoietic stromal and macrophage cells. The relationships between stromal cells and macrophages are necessary to create an appropriate environment for differentiation of immune cells. Studying the cellular expression specificity of receptor and ligand genes reveal the high degree of specificity of these genes that indicate non-random transfer of information between cells in multicellular organisms.

Ankylosing spondylitis (AS) is a type of arthritis which can cause inflammation in the vertebrae and joints between the spine and pelvis. However, our understanding of the exact genetic mechanisms of AS is still far from being clear. To study and find the mechanisms and possible biomarkers related to AS by surveying inter-gene correlations of networks. A weighted gene co-expression network was constructed among genes identified by microarray analysis, gene co-expression network analysis, and network clustering. Then receiver operating characteristic (ROC) curves were conducted to identify a significant module with the genes implicated in the AS pathogenesis. Real-time PCR was performed to validate the results of microarray analysis. In the significant module obtained from the network analysis there were eight AS related genes (LSM3, MRPS11, NSMCE2, PSMA4, UBL5, RPL17, MRPL22 and RPS17) which have been reported in previous studies as hub genes. Further, in this module, eight significant enriched pathways were found with adjusted p-values < 0.001 consisting of oxidative phosphorylation, ribosome, nonalcoholic fatty liver disease, Alzheimer's, Huntington's, and Parkinson's diseases, spliceosome, and cardiac muscle contraction pathways which have been linked to AS. Furthermore, we identified nine AS related genes (UQCRB, UQCRH, UQCRHL, UQCRQ, COX7B, COX5B, COX6C, COX6A1 and COX7C) in these pathways which can play essential roles in controlling mitochondrial activity and pathogenesis of autoimmune diseases. Real-time PCR results showed that three genes including UQCRH, MRPS11, and NSMCE2 in AS patients were significantly differentially expressed compared with normal controls. The results of the present study may contribute to understanding of AS molecular pathogenesis, thereby aiding the early prognosis, diagnosis, and effective therapies of the disease.

ZnO nanoparticles (ZnO-NPs) are one of the most popular metal oxide nanoparticles, which exhibit significant antibacterial properties against various pathogens. Among nanoparticle synthesis methods, the green synthesis using plant extract is considered as an eco-friendly and cost-effective method for ZnO-NPs production, compared to the chemical procedures

This study aimed to evaluate the green synthesis of ZnO-NPs loaded on silica gel matrix (ZnO/SG nanocomposite) by using methanol leaf extract of Daphne oleoides as a new extract and a cost-effective method. Furthermore, the antibacterial activity of the synthesized structure is evaluated against some pathogenic bacteria and the results are compared with unsupported ZnO-NPs.

For ZnO/SG nanocomposite synthesis, a solution of Zn (NO3 )2 was stirred with silica gel. Then the Daphne oleoides extract was added and stirred continuously until white precipitate was formed. The precipitate was heated at 200 ˚C for calcination, and ZnO/SG nanocomposite was obtained. The phytochemical constituents of leaf extract were then analyzed by gas chromatography–mass spectrometry (GC-MS). Afterwards, the structure of ZnO-NPs on SiO2 matrix (ZnO/ SG nanocomposite) was characterized by field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction analysis (XRD), and Fourier transform infrared spectroscopy (FTIR). Surface area measurement was also determined by Brunauer-Emmett-Teller (BET) techniques. Furthermore, the antibacterial activity of ZnO/SG nanocomposites against pathogenic bacteria was evaluated using agar-based disk diffusion method standardized by clinical and laboratory guidelines.

The leaf extract of Daphne oleoides encompassed five major polyphenolic components. The results of the nanocomposite structure showed that ZnO-NPs with an average particle size of 38 nm were obtained and stabilized on the silica gel matrix. The BET surface area measurement of ZnO/SG nanocomposite was compared with unsupported ZnO-NPs, and the results indicated that the surface area of ZnO/SG nanocomposite was increased. Furthermore, the structure showed more powerful antibacterial activity against pathogens than unsupported ZnO-NPs.

Green synthesis of ZnO-NPs supported on the silica gel matrix with the leaf extract of Daphne oleoides is a benign and effective procedure for ZnO/SG nanocomposite synthesis. Embedding ZnO-NPs in silica gel matrix prevents the agglomeration of nanoparticles and prepare homodispersed nanoparticles. This structure revealed great antibacterial activity against many pathogens.

Cyanobacteria have been the focus of extensive researches because of their high potential for the development of new generations of useful natural compounds with vast applications. For the entire last ten years, a lot of attention has been dedicated to the cyanobacterial lipids as a main source of valuable materials for clean energy production. As there is a direct relationship between biofuel properties and compositional characteristics of fatty acids, a selected lipid-producing cyanobacterial strain was examined and analyzed in terms of fatty acid composition. The biodiesel quality parameters were carefully examined as well. A cyanobacterial strain was isolated from waterfalls in the northern part of Iran and identified as Synechocystis sp. MH01. The fatty acids profile of the selected strain, as tested in various culture conditions, was analyzed by gas chromatography (GC) and compared with control subjects to further validating the biodiesel quality parameters. The autotrophic cultivation of Synechocystis sp. MH01 resulted in biomass and lipid productivity of 109 mg.L-1 day-1 and 22.89 mg.L-1day-1, respectively. The mixotrophic cultivation of MH01 strain in sucrose-containing medium led to an approximately 1.8 and 1.22 fold increase in biomass and lipid productivity compared with the autotrophic condition. The addition of glycine to BG11 medium caused up to ~1.3 and ~1.18 fold increase in biomass and lipid productivity compared with control subjects. The analysis of qualitative parameters of the biodiesel, as derived from the lipids, indicated that Synechocystis sp. MH01 has a high ability for lipid production under optimal culture conditions. It seems feasible to evolve the Synechocystis sp. MH01 further particularly for more lipid production as a promising primary raw material for biofuel production through fine-tuning of medium composition.

Soil salinity is a major abiotic stress that limits plant growth and yield worldwide. To better understand the mechanism of salt stress adaptation in maize (Zea may), proteomic analysis of maize responses to salt stress were analyzed in seedling. Taking maize seedlings untreated and treated with NaCl for 24 h as material, isobaric tags for relative and absolute quantitation (iTRAQ) were used to analyze the protein expression profile of maize seedlings after salt stress. The result showed that 270 differentially expression proteins (DEPs) were identified in maize seedlings after salt stress.The majority proteins had functions related to translation, ribosomal structure and biogenesis (15%), posttranslational modification, protein turnover, chaperones (14%) and others metabolism. Quantitative real-time PCR analysis showed that the EF-Tu, peroxiredoxin, FoF1-type ATP synthase, glutamate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, Acetyl-CoA acetyltransferase and nucleoside diphosphate kinase genes were up-regulated in the adaptation of maize to salt stress. The coped with salt stress of maize seedlings might be included nitrogen and glutamate (Glu) metabolism and energy homeostasis, nucleotide transport and metabolism, soluble sugar, fatty acid and nucleoside triphosphates synthesis. Moreover, the enhancement of plant to scavenge ROS, such as peroxiredoxin, might play significant roles in the adaptation of maize to salt stress.Taken together, these proteins might have important roles in defense mechanisms against salt stress in maize.We hope that this study provides valuable information for the further utilization and study on the molecular mechanisms of defense mechanisms in maize.

Cipangopaludina cahayensis contains active fibrinolytic proteins and has been considered a potential anti-cancer agent. However, its anti-cancer characteristics and functions have yet to be elucidated. To study the fibrinolytic activity and anticancer activity of crude protein extracts from Cipangopaludina cahayensis. Crude proteases were separated and extracted from the Cipangopaludina cahayensis through homogenization, desalting, ammonium sulfate fractionation, dialysis, and ion exchange chromatography. The fibrinolytic activity of extracted proteins was assessed using the fiber plate method. Total protein concentrations of the crude proteases were determined via BCA assay. Molecular weights (MWs) were determined through SDS-PAGE electrophoresis. The crude extract had a MW of ~ 50 kDa, and the highest protein concentration was 3.026 mg.mL-1. The optimum pH for fibrinolytic activity was 7.0. Cell culture assays demonstrated that the addition of the crude enzyme extracts to the human ovary cancer cell line Ovcar-3 resulted in significant growth defects. Our data showed that crude proteins purified from Cipangopaludina cahayensis are novel fibrinolytic proteases and have potential anti-cancer properties

Acetate accumulation in the culture medium is known as an inhibitor in recombinant protein production in Escherichia coli. Various approaches have been proposed and evaluated to overcome this challenge and reduce the concentration of acetate. In this study, we examined the effect of acetate kinase A antisense on acetate production rate in E. coli We also used PAMAM dendrimers as a suitable delivery agent for antisense transformation into E. coli host cell. This study aimed to decrease acetate production as a by-product using an antisense-dendrimer complex to increase mass cell and subsequently recombinant Albumin production in E. coli. Here, to study the effect of this treatment on recombinant protein production, we used pET22b/ HAS construct. The ackA gene expression was inhibited by designed antisense to reduce acetate concentration in culture medium. AckA antisense was transferred to E. coli by PAMAM dendrimer. Finally, ackA expression and recombinant Albumin production were evaluated Real-Time PCR and densitometry, respectively. Our data showed, designed antisense lead to reduction of acetate kinase gene expression and subsequently acetate concentration in the culture medium. Finally, acetate concentration reduction and cell mass increase result in enhanced recombinant Alb production in the treated group (1.25 mg.mL-1) compare to the control group ( 0.59 mg.mL-1). Reduction of acetate in E. coli fermentation process decreased the recombinant Alb production following cell growth and cell mass increase. In the current study, we showed that an antisense can be a useful tool for ackA gene expression reduction. Also, we noted that PAMAM dendrimer could be a proper delivery agent for oligonucleotide antisense transformation into bacterial cells.