Modares Journal of Biotechnology

Abstract The CD80 protein, a member of the super-family of immunoglobulin, is a transmembrane protein expressed on the surface of the antigen-presenting cells (APC). This protein has two receptors on the surface of T cells (CTLA-4 and CD28), due to the binding of this protein to these receptors, the inhibitory and stimulatory pathway in the T cells begin, respectively. Naturally, CD80 proteins tend to have more binding affinity to CTLA-4 than CD28, and this is a factor in the extinction of T cells in the immune system in order to prevent autoimmunity. The aim of the present study is to create a variant of the CD80 protein that has an increased binding affinity to CD28 to bind to this receptor more strongly and induce more simulate pathways than the wild type of this protein (primary CD80 protein) in T cells. To identify this variant, first, the ancestral sequence was mutated by R software at positions 31 and 92 with amino acids that play an important role in the formation of hydrogen bonds. The R software output sequences were modeled with the SWISS-MODEL server. Then, each output model was docked with the HADDOCK server, and finally, the electrostatic and van der Waals energies between the receptors and the ligands were calculated. Among all the built-in models, the mutated K31Y, R92F has the best electrostatic and van der Waals energies and has the ability to have a much better connection to its CD28 receptor compared to the ancestral type of CD80.

Abstract Aims: Breast cancer is the most common malignancy in women throughout the world. Among the various methods for preventing and treating cancer, plant natural compounds have more benefits than chemical drugs and have less side effects. Recently, many studies have been carried out on the antioxidant, anticancer, antiproliferation, and antiinflammatory properties of plant lignans, indicating the importance of these compounds in the prevention and treatment of various diseases. In the present study, the cytotoxic effects and inducing apoptosis of pinoresinol and lariciresinol lignans were investigated on SKBr3 breast cancer cell line.
Materials & Methods: SKBr3 cells were treated with different concentrations of both pinoresinol and lariciresinol, separately for 72 hours. Then, cell viability and cells morphological changes were determined using MTT assay and inverted light microscope, respectively. Also, apoptosis induction was analyzed by flow cytometry using an Annexin V-FITC apoptosis detection kit.
Findings: Both pinoresinol and lariciresinol treatments induced morphological changes, decreased cell growth, survival, proliferation and a significant increase in apoptosis induction in SKBr3 cell line compared to control cells in a concentration-dependent manner.
Conclusion: Inducing apoptosis and preventing the growth and proliferation of cancer cells are important mechanisms in the treatment of cancer. Pinoresinol and lariciresinol can be used to reduce cell proliferation and increase apoptosis induction in prevention and treatment of breast cancer.

Abstract Biosurfactants are valuable microbial metabolites that have considerable applications in different industries. They offer so many advantages over their synthetic counterparts such as biodegradability, low toxicity, activity at extreme conditions, ability to be produced from renewable wastes and by-products. In the present study, biosurfactant production of Halomonas sp. MM93 in nutrient broth medium at 30^°C after 72h was investigated using oil spreading and hemolysis tests. The emulsification capacity of the biosurfactant was also evaluated in a defined production medium during 96h. Effect of olive oil, n-Hexan, and kerosene as hydrophobic carbon sources to induction of biosurfactant production by the strain MM93 was also investigated. Due to the importance of stability in the case of industrial use, the effect of extreme temperature, pH and salinity on the stability of bacterial culture supernatant was evaluated. This strain created a clear zone of 2.5cm diameter in an oil-spreading test and its E24 index was 45%. Halomonas sp. MM93 could reduce the surface tension of the culture medium from 70 to 40 mN/m. Also, the produced biosurfactant showed remarkable stability at high temperature (100°C), extreme acidic and alkaline conditions (pH=2-12), and high salinity (20g/L). According to obtained data, native isolated moderately halophilic bacterium, Halomonas sp. MM93 could be considered as a potent strain in terms of producing stable biosurfactants for various industries especially the processes of increasing microbial recovery of oil that need Compounds with High surface activity and high stability.

Abstract The waste produced by households, industry and factories to use as wash water and cooling systems and equipment, is caused environmental complications. Waste created a suitable environment for the growth of odor and pathogenic bacteria. This study aimed to isolate microorganisms in the wastewater and use them for the reduction of BOD (biochemical oxygen demand) and COD (chemical oxygen demand).The samples were collected aseptically from different parts of wastewater of herbal distillation industry. The bacterial strains were isolated from the samples in LB media. The isolates were distinguished based on the morphology and biochemical characteristics. Among the total 69 isolates, four isolates were selected to measure the ability to reduce BOD and COD and added to the waste water. Reduction of BOD was measured using BOD meter. Also, COD was determined by titration method. The isolates were identified by biochemical tests. The amount of BOD and COD reduction after adding selected strains to wastewater was 47.43 - 71.82% and 44.79 - 56.5% respectively. Also, the consortium of bacterial strains showed better ability to reduce BOD and COD (38.32-57.29%and 76.6-83.21% respectively). The results determined that wastewater contains bacterial strains which have shown significant reduction in BOD and COD and organic matter decomposition in wastewater and reuse it in agricultural and industrial sectors. Therefore, we can use these bacteria for wastewater treatment.

Abstract Electrical Kindling is one of the most popular epileptic model techniques that cause seizures such as temporal lobe epilepsy. So far, various therapies have been used to treatment of epilepsy. Among these treatments, low-frequency stimulation (LFS) has been widely considered for improving effect on drug-resistant epilepsy, but its mechanism is not well understood. Since calcium entering to the cytoplasm and increasing its concentration is one of the reasons for seizure, metabotropic glutamate receptor (mGluR1), dopamine receptor (D1) and ADPR cyclase (CD38), which increased calcium in the cytoplasm from different pathways, were selected. With this aim that by examining the change in the expression of these genes, we help to clarify the LFS improvement effect. In this study, the hippocampus of rats was used and the changes in genes expression were investigated using real-time PCR technique. The results showed that the expression of all selected genes increased significantly after kindling and then after the LFS the expression of all was returned to sham value. Hence, one of the ways in which LFS interferes may be related to the pathway for calcium entering to the cytoplasm.

Abstract Aims: Peroxidases are used in a wide range of biotechnological processes, most of which are carried out at high temperatures and high pH levels. Since most of the commonly used peroxidases are unstable and inactive in alkaline conditions and high temprature, it is necessary to find thermoalkalophilic peroxidases for practical purposes.
Materials and Methods: In this study, extracellular production of peroxidase in the native strain Bacillus tequilensis was studied. for this purpose, Enzyme activity was evaluated using two substrates 2,4-DCP and pyrogallol in bacterial liquid culture and the effect of culture time on enzyme production, as well as the effect of parameters such as pH and temperature on enzyme activity investigated. The relative purification of the enzyme was performed using ion exchange chromatography with sephadex DEAE A50 and the kinetic parameters of enzyme activity were evaluated. In this study, kinetic parameters such as Km and Vmax were calculated.
Results: Measurement of enzyme activity at different times of culture indicated that the highest amount of peroxidase production was obtained 72 h after bacterial culture.

Abstract This study was conducted to assess genetic diversity among the 32 tall fescue half-sib families using a randomized complete block design (RCBD) experiment in the four replicates. Based on analysis of variance, significant differences were observed among studied genotypes at the probability of 1% for plant height, canopy diameter, days to heading, days to pollination, crown diameter, fresh forage yield, dry forage yield, number of stem and seed yield in first harvest and in canopy diameter, crown diameter, fresh forage yield and dry forage yield in second harvest. Based on the results of mean comparisons, highest dry forage yield in the first harvest was obtained in genotype 32 by 758.5 grams. Principal component analysis by considering eigenvalues greater than one, caused to introduction of three components which determined 80.5% of the variation among the samples. In cluster analysis, the greatest of distinction between the groups was achieved with three clusters, and by cutting the dendrogram genotypes in three groups. According to the results, the third cluster was superior to other two clusters in terms of most traits. The genotypes of third cluster, according to the value of this cluster in terms of forage yield and seed yield will be of particular importance in breeding programs. In the breeding of cross-pollinated forage crops, success in selection depends on creating diversity by genetic recombination and achievement of heterosis. Due to the distance between groups 1 and 3, probably the most successful crosses will be achieved among genotypes of these two groups.

Abstract Since one of the main problems in bone tissue repair is the bacterial infections, recently the development of drug-eluting nanocomposite scaffolds for bone regenerative medicine applications has attracted significant attention. In this study Polycaprolactone (PCL)-based composite scaffolds containing 10vol% of titanium dioxide nanoparticles (~21nm), and bioactive glass particles (~6µm), were prepared without drug and also loaded by Tetracycline hydrochloride (TCH) antibiotic (0.57, 1.15 mg/mL) through solvent casting method for bone tissue engineering applications. Structural characterizations based on Scanning Electron Microscopy (SEM), and FTIR analysis were utilized to study the chemical bonds of glass/ceramic particles, and antibiotic crystals on the surface. In addition, in vitro cytotoxicity, and antibacterial analysis were performed by MTT, and Agar well-diffusion assays, respectively. In this study polymeric and composite scaffolds were fabricated with TCH clusters decorated on the surface. It was shown that the bioactive glass/PCL scaffolds loaded by 0.57 mg/mL of TCH revealed significant antibacterial effect, despite the acceptable cell viability. These scaffolds seem to be of interest as a potential candidate in drug-eluting scaffolds for bone tissue engineering applications.

Abstract Graphene quantum dots (GQDs) have attracted increasing attention due to their unique properties such as high water solubility, photoluminescence activity, good biocompatibility, physical, chemical and electrical properties which makes them appropriate candidates for use in a variety of bio-applications, sensors and photocatalysts. The objective of this study is synthesis of GQDs and improving their surface properties via chemical modification.
Here, urea and citric acid as carbon precursor were used. Citric acid was self-assembled into graphene framework via hydrothermal method at 160 °C for 4 h. Then, the synthesized GQDs were carbonized and chemically activated by KOH treatment. The surface area and pore structures of GQDs were analyzed by nitrogen adsorption/desorption isotherms. The results showed that the specific surface area of carbonized-activated graphene quantum dots (CA-GQDs) have been increased from 0.06 to 1204.0 m²/g and pore structures have been enhanced significantly. The XRD pattern of GQDs confirmed the basic structure of graphite layer. The TEM images indicated the unique morphology of GQDs and the sizes of GQDs were less than 5 nm. Thus, our applied method is an effective approach in the formation of GQDs with large BET surface area and narrow pore structures which reveals their potential applicability in biomedical field.

Abstract Mercury is one of the most harmful pollutants in the environment, which in the event of ingestion into the human body, it is not metabolized and severe nervous, respiratory and metabolic disorders will occur due to the formation of stable complexes with biological molecules. Therefore, the development of precise, fast and inexpensive methods for mercury detection in the environment is of great importance. Carbon quantum dot is a new fluorescent substance with unique physical and chemical properties which is taken into consideration for diagnostic applications, especially for heavy metals detection at low concentrations. In this study, carbon dots with blue fluorescent emission were synthesized using the pyrolysis method and characterized. Fluorescent emission intensity of synthesized carbon dots was decreased significantly in interacting with mercury. Then for providing an easy-to-use mercury detection method, carbon dot was encapsulated in sodium alginate hydrogel. Quenching fluorescent intensity of encapsulated carbon dots in the hydrogel structure was evaluated in the presence of mercury and 1micromolar detection limit was obtained. Encapsulating carbon dots in hydrogel structure not only extend the use of carbon dot as a mercury sensor but also can be used as an adsorbent for mercury pollutant from the environment.

Abstract Fungi are the most important agents of biodeterioration in museums, libraries, and repositories. The objectives of the paper were to evaluate the microbial diversity in biodeteriorated manuscripts located in a repository of the central library of Astan Quds Razavi (AQR) and to estimate the fungal occurrence of the repository air. The sterile cotton swabs and nitrocellulose membranes were used for sampling the manuscripts, while the sedimentation method was used for the microbial sampling of air. To evaluate the biodeteriorative impacts of fungi, fungal spore’s suspension inoculated on paper strips. Fourteen and six fungal isolates were collected from the three different deteriorated substrates and the repository air samples, respectively. Among the fungi isolates, Aspergillus sp. was isolated in high frequency (36%), followed by Penicillium sp. (21/5%) and Altelnania sp. (14%). Fungi species including P. chrysogenum, Cladosporium cladosporioides, Talaromyces diversus, and Aspergillus sp. were isolated from B1 sample as a parchment. The most fungal isolates (53%) in the air repository including Purpureocillium lilacinum, Talaromyces diversus, Cladosporium sp., and Aspergillus sp were achieved from MEA medium. The low number of isolated fungi from repository air can be attributed to the efficiently controlled environment factors of AQR repository. The combination of finding provides some support for the conceptual premise that it could be a direct relationship between the isolated microorganisms from air and those isolated from the manuscripts. The presence of color spots on paper strips can approve the biodeterioration of paper via fungal activities.

Abstract Abnormal angiogenesis is associated with various diseases such as solid tumors and metastasis, retinopathies, and rheumatoid arthritis. VEGF-A is the most important mediator of angiogenesis among all growth factors. The bioactivity of VEGF is mediated by two tyrosine kinase receptors VEGFR-1 and VEGFR-2 present on endothelial cells. VEGF signaling through VEGFR-2 is the major angiogenic pathway that leads to stimulation of endothelial cell ingrowths into the tumor. It comprised of an extracellular portion, a cytoplasmic portion, and a short transmembrane domain. The extracellular portion consists of seven Ig-like domains (D1–D7), of which the 1st to 3rd domains function as ligand-binding sites. In the present work, a soluble recombinant extracellular domain 1-3 of VEGFR-2 was expressed in Pichia pastoris to inhibit the VEGF-induced angiogenesis. The 975 bp DNA fragment containing extracellular domain 1-3 kdr, was designed according to the nucleotide sequence at GenBank and protein sequence at Swiss-Prot. The recombinant secretory expression vector (pPinkαHC/KDR1-3) was constructed and transferred into yeast by electroporation. The high expression transformants were identified through complementation of adenine auxotrophy and cultured. KDR1-3 was expressed under the induction of %1 methanol and confirmed by using SDS-PAGE and western blot techniques. After being purified by affinity chromatography using Ni-NTA resins, binding of expressed product to hVEGF165 was proved by two direct ELISA and ELISA receptor binding assays. The data showed that human VEGFR-2 extracellular domain 1-3 with eukaryotic protein structure, that there is no reported paper about, was successfully expressed.

Abstract The interactions between carbon nanotubes (CNTs) and proteins were considered much attention. Advanced CNT applied biomolecules require mutual understanding of their interactions with biological molecules. Enhanced biomedical applications of CNTs have necessitated the need for the understanding their interaction with biomolecules. Non-covalent interactions of blood peptides, such as hepcidin, with carbon nanotubes, have important effects in a wide range of biological applications that are detected by analyzing the thermodynamic parameters of the interaction between CNTs and peptides. In addition, the effects of different parameters in order to evaluate how the interaction of CNTs with peptide affects and structural changes and stability of peptides were studied. In this study, based on molecular dynamics (MD) simulation, the structural changes of hepcidin 20 in interaction with multi walled carbon nanotubes (MWCNTs-COOH ) were investigated. The simulation results revealed that carbon nanotubes cause to loose the hepcidin structure and make structural changes in this peptide. On the other hand, the loose of the hepcidin structure may lead to a change in its activity. The results indicated that significant changes were made in the structure of hepcidin 20 in the presence of carbon nanotubes. The difference of parameter amounts calculated in heptidine 20 is related to their N-terminal, and loop regions.

Abstract Escherichia coli is a Gram-negative bacterium, the second most common bacteria in the intestine and the main indicator of urban water pollution, is the most common cause of urinary tract infection and also is one of the main factors in food poisoning and diarrhea. Drug resistance of this bacterium to antibiotics is a global challenge. Horizontal gene transfer (HGT) is the movement of genetic material between unicellular and other gene transfer pathways which is an important factor in the evolution of many organisms, antibiotic resistance in bacteria, gene function. Antibiotic resistance in E. coli can be transferred to another species of bacteria through HGT mechanisms. Today, Bioinformatics methods have been used to understand of gene transfer from HGT mechanism. In this study, we used bioinformatics tools such as PredictBias, ACLAME, Mobil Genetic Elements (MGEs) PAI-ID, and Alien_Hunter in order to genes analysis that related from antibiotic resistance in E. coli. Bioinformatics and MIC assays result show that from 26 to 30 genes have been identified in all safthwers. Most of genes that identified show over 50 percent of GC content. put P gene with 178, blaCMY with 62, BlaTEM with 43, and aac-6 with 66 homology in the PredictBias website identified. Also in the ACLAME website, mob (A-C) and rep (A-C) gene family are highest number of horizontal gene transfer from infection bacteria strain. Those cluster genes are the highest resistant of laboratory tests which carries resistant genes such as blaSHV and blaCHV on the blaCMY plasmid.

Abstract Breast cancer is a serious health concern for women. It contributes to about 23% of the cancer cases, and accounts for the second largest number of deaths among all cancers. Expensive and time consuming recognition methods currently available for recognition of breast cancer potentiates the need for improvement of novel, specific and ultrasensitive strategies. Biosensors are sensitive, specific and cost effective procedures. These also display the benefit of quick response due to direct calculation in physiological fluids (saliva, blood, serum, milk, urine etc.) in a non-invasive way. Aptamer-based biosensors for cancer cell recognition have shown advantages of rapidness, simplicity and cost-efficiency over traditional approaches. In this study, by linking DNAzyme and aptamer together, we established colorimetric biosensor for the detection of MCF7 breast cancer cells. MUC1 and PTK7 aptamers used as specific aptamers to binding to the breast cancer cells. This manner may also evade the modification of DNA and the use of labels, which can intensely rise the cost-efficiency and simplicity of cancer cell discovery. Our results indicated that these aptamers showed good activity for breast cancer cells detection in which in the control cells no activity was observed. Results also indicated that there is a good linear relationship between the cancer cell values and colorimetric signal. Finally, the obtained results indicated a cost effective and conveniently operated approach for cancer diagnosis in future.