Modares Journal of Biotechnology

Abstract The combustion of fossil fuels containing sulfur results in the release of sulfur dioxide into the atmosphere and environmental pollution. Hence, the researchers focused on the biological desulfurization method. Dibenzothiophene is used as the model molecule to study the ability of the desulfurization of microorganisms. The most suitable sources of carbon, nitrogen and sulfur concentration optimized by response surface method to obtain the highest cell growth and biological desulfurization activity. The performance of iron nanoparticles on the growth and biodesulfurization activity of thermophilic bacterium Bacillus thermoamylovorans strain EAMYO was investigated. Characterization of starch-modified iron nanoparticles was performed by TEM, SEM. The images of TEM and SEM of starch / Iron nanoparticles showed that the Fe₃O₄ and Fe0 nanoparticles were 20 and 30 nm, respectively. The investigating the growth of microorganism in the presence of iron nanoparticles showed that these nanoparticles not only did not have a toxic effect on microorganism growth, but also increased the growth of microorganism in 96 h (OD 660 = 1.864, 1.896 respectively in the presence of nanoparticles Fe₀ and Fe₃O₄), while the highest rate of growth in the absence of nanoparticles in 96 h (OD660 = 1.51). Also, the activity of desulfurization in the presence of starch/Fe₀ nanoparticles and starch/Fe₃O₄ / starch increased by 26.52% and 10.75%, respectively, compared to the cells without the coating of iron nanoparticles.

Abstract Integrin inhibitors may change conformational and dynamical properties of integrin, but its molecular properties in this process is not clearly understood. Tumstatin is an anti-angiogenesis protein derived from collagen XVIII, but little is known about how tumstatin applies its antiangiogenic and antitumor effects. It has been reported that 18 amino acids fragment of tumstatin has anti-tumor activities similar to tumstatin. We used molecular docking and molecular dynamic simulations to describe inhibitor activity of peptide in molecular level. We described the binding of this peptide to Hybrid/EGF-4 interface and that these interactions might contribute to improved hydrophobic interactions at these regions and also fixed the mobile domains of integrin. In the complex, we recognized a novel binding site on integrin for integrin inhibitors that may have critical role in integrin inhibition. These results support the idea that hydrophobic interactions between Hybrid/EGF-4 domain and peptide-anti tumor might contribute to stability of bended state and therefore inhibit integrin activation.  Our finding can be applied to understand the mechanism of out-in pathway integrin signaling and development of integrin targeted drug.

Abstract The bioluminescence process is a widespread phenomenon in Nature. These enzymes are identified in some domains of life, but the luciferases from the lampyridea genus are considered for biological applications. The molecular cloning of a new type of Iranian firefly luciferase from Lampyroidea maculata was reported, previously. In this study, we analyzed the rare codons of the Iranian insect luciferase gene using the computational databases as ATGme, RACC, LaTcOm, and Sherlocc. Also, the structural modeling process of this enzyme was performed. Next, the status of these rare codons in this structural model was studied using SPDBV and PyMOL software. In the following, the substrate binding site was studied using the AutoDock Vina. By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. AutoDock Vina was used in the molecular docking that recognizes Asp⁵³¹ that yield closely related to luciferin and AMP binding site.. This bioinformatics analyzes play an important role in the design of new drugs.

Abstract α-crystallin is a member of small heat shock protein family (sHSP) which shows both structural and chaperone functions. This protein plays important role in eye lens transparency and indicates protective function in the other tissues. The lenticular levels of copper ions significantly enhance in diabetic patients, aged and cataractous lenses. In eye lenses, the free copper ions induce ascorbic acid auto-oxidation, leading to formation of dehydroascorbic acid and other oxidative products as well as reactive oxygen species. The oxidized forms of ascorbic acid along with the reducing sugars enter into pathological reactions with the eye lens proteins, forming toxic advanced glycation end-products (AGEs). As one of the main components of eye lens antioxidant defense mechanism, glutathione could scavenge the copper ions, inhibiting the formation of reactive oxygen species in eye lenses.
In the current study, the structural and functional properties of human αB-crystallin were assessed using different spectroscopic methods. In the presence of copper ions, αB-crystallin exhibited important alterations in both structure and chaperone activity which upturned in the presence of glutathione. Moreover, incubation of human αB-crystallin with copper resulted in significant increase in the protein oligomeric size distribution which largely prevented upon simultaneous incubation with glutathione.
Overall, glutathione may scavenge free copper ions in the lenticular tissue, inhibiting their damaging effects on crystallin proteins and other redox-sensitive molecular targets such as ascorbic acid. Our results may introduce a new protective role for glutathione which is highly important in diabetic and aged lenses showing increased levels of copper ions.  

Abstract Iron oxide nanoparticles are one of the nano carriers that are suitable for novel drug delivery systems due to low toxicity, biocompatibility, loading capacity and controlled drug delivery to cancer cells. The purpose of this study is the synthesis of coated iron oxide nanoparticles for delivery of Doxorubicin (DOX) and its effects on cancer cells.
In this study, Fe₃O₄ magnetic nanoparticles were synthesized by Polyol method, and then doxorubicin was loaded onto PEGylated iron oxide nanoparticles. FT-IR was used to ensure PEG binding to nanoparticles and loading the drug onto nanoshell. Comparison of the mean size and the crystalline structure of nanoparticles were performed by TEM and X-ray diffraction pattern. Then, the effect of cytotoxicity was evaluated on AGS and MCF-7 cancer cells by MTT assay.
According to FT-IR results, the presence of O-H and C-H bands at 2927 cm^-1 and 3392 cm^-1 peaks correlated with PEG binding to nanoparticles. XRD pattern showed the cubic spinel structure of trapped magnetite nanoparticles carrying medium with a mean size of 14 nm. 21.67% of Doxorubicin was loaded into Fe₃O₄-PEG nanoparticles, which the highest drug release recorded during the first 24 hours. MTT assay at 24, 48 and 72 h treatments showed that with increasing concentrations of doxorubicin loaded Fe₃O₄-PEG nanoparticles from 0 to 50 μm, the cytotoxic effects of the drug gradually increase.
This study showed that PEGylation of iron oxide nanoparticles and using them in drug delivery process to increase the effect of Doxorubicin on AGS and MCF-7 cancer cells

Abstract Diabetes mellitus is a major health problem in the worldwide. Inhibition of DPP-IV is one of the methods to control diabetes type 2. Inhibition of this enzyme may improve glycemic control in diabetics by preventing the rapid breakdown and there by prolonging the physiological action of incretin hormones. Furthermore, improving the antioxidant system in diabetic patients can prevent the occurrence of secondary disease caused by oxidative stress. Therefore, the head of skipjack tuna was hydrolyzed with alcalase enzyme (1/5% of raw material weight) for 4 hours, in order to produce a product with antidiabetic and antioxidant activities. The DPP-IV inhibition activity, DPPH radical scavenging activity and reducing power of hydrolysate were measured. The results showed that the skipjack tuna head protein hydrolysate possess bioactive properties in a concentration dependent manner and increasing the protein concentration leads to a significant increase in bioactive properties of hydrolyzed product (p≤0.05). The IC50 of protein hydrolysate in DPP-IV inhibition and DPPH radical scavenging activities were obtained 1.016±0.02 mg/ml and 0.297±0.015 mg/ml, respectively. Also the reducing power of hydrolysate was 0.176±0.002 in 2.5 mg/ml protein concentration. Overall, according to the obtained results, it can be concluded that protein hydrolysate of skipjack tuna head possess high antioxidant and antidiabetic activities in vitro, and can be used as a food additive to enhance health level if additional research be conducted.

Abstract In stevia (Stevia rebaudiana), breeding programs are mainly aimed at developing plants with high Rebaudioside-A (RA) content. To this end, in order to screen stevia plants and selection of varieties with the highest amount of desired sweeteners (RA) using molecular markers, the present study was conducted on RNA-seq data of varieties having different amounts of RA. We took advantage of CLC to make de novo transcriptome assembly for each variety with k-mer and contig length values of 20 and 200bp, respectively. The assembly was annotated using the latest Arabidopsis proteome release. To identify signatures of candidate polymorphic SSRs among the stevia varieties, the assembled sequences were used as an input for CandiSSR, followed by designing primer pairs for identified polymorphic SSRs. We identified 368 potential polymorphic SSRs based on the stevia transcriptome analysis, among which 360 were qualified for primer design. Almost 89% of the contig sequences possessing polymorphic SSRs had the best blast hit against Arabidopsis proteome. We found contigs similar to the UDP-Glycosyltransferase protein family and Deoxyxylulose-5-phosphate synthase which are involved in biosynthesis pathway of steviol glycosides. Also, gene set enrichment analysis using PlantGSE through Hypergeometric test (FDR<0.05) identified enriched metabolic pathways in the sequences contained polymorphic SSRs; It is therefore most likely that such connections exist between the SSRs and biosynthesis of steviol glycosides. Hence, it could conceivably be hypothesized that the SSR markers developed in this study would be reliable in molecular breeding of stevia toward selection of varieties with high RA content.

Abstract In this study, the effect of red and blue illumination on continuous culture of two different species of microalgae and cynicocytosis was studied. Comparison of blue and red lights in the cyanobacteria, Synechocystis sp. PCC6803, showed that this specie grows very fast under red light illumination, but it has very slow growth rate under blue light exposure. In spite of huge difference in growth rate, the lipid content and the fatty acid composition of Synechocystis was approximately the same for red and blue light illumination. For microalgae, Chlorella Sorokiniana, the blue light resulted to slightly higher growth rate than the red light. The C18:3 unsaturated fatty acid content was significantly higher for red light illumination compare to blue light illumination. Overall, considering the lower energy requirement for illumination of red, this light is more efficient than blue light for cultivation of Chlorella Sorokiniana.

Abstract Aptamers, DNA or RNA single-stranded sequences, have different applications in biological investigations, such as apatsensors, due to their many advantages including high specificity and affinity, cost-effectiveness and easy synthesis. In this study, an aptasensor was designed based on the changes in the SPR spectra of gold nanoparticle, in order to detect carcinoembryonic antigen (CEA) cancer indicator as a marker for breast cancer. In the presence of aptamer, gold nanoparticles were stable, SPR spectrum of gold nanoparticle was unchanged after adding NaCl. However, in the presence of CEA as a cancer marker, aptamer binds to the target molecule and by adding NaCl consequently the SPR spectrum of gold nanoparticles is changed. The results of this study showed that the designed aptasensor enables the detection of CEA over a range of 50 ng ml^-1. The limit of detection was about 22.75 ng ml^-1. It seems this aptasensor can be used in detection of carcinoembryonic antigen cancer marker.

Abstract Abstract. Phycocyanine (PC) belongs to a group of protein receptor proteins called phycobiliprotein. All of the phycobiliprotein are multi-chain proteins made up of apoproteins. Which are covalently attached to the phyclobilins. This experimental study was carried out on strain of native Anabaena doliolum, Isolated from soils and waters of south Iran were Masjed Soleyman area. The cyanobacteria were grown and stored in BG11 medium. Then, the amount of phycocyanin produced under different light treatment and the amount of phycocyanin extracted using different ratios of multi-buffer and at two different temperatures were evaluated. The results of this study showed that the highest growth rate is when the sample is exposed to green light for three to five days. The best amount of extraction for distilled water and at a refrigerator temperature (0^◦C) with a ratio of 3:1 biomass/solvent is equal to 0.03 ± 15 µg/ml. Also, at the environment temperature, phosphate buffer is a more suitable solvent for extracting phycocyanine at a ratio of one to two with a value of 0.05 ± 8 µg/ml. In general, it can be said that the growth rate, pigment production and optimum extraction conditions for each species are quite different, and the optimal extraction of phycocyanin in a species is also dependent on various factors such as time, temperature, solvent and the ratio of biomass to solvent.

Abstract The present study was accomplished to purify and biochemically characterize the phenol-degrading enzyme from the bacteria existed in petroleum-contaminated soils. The catechol 1, 2 dioxygenase was extracted from Aneurinibacillus migulanus Isolate ZNU05 and purified using Q-Sepharose ion exchange chromatography column. The enzyme activity was examined under different pHs (ranged from 4 to 9), at different temperatures (ranged from 20 to 70˚C), in the presence of various metal ions chloride salts (Ca²⁺, K⁺, Mn²⁺, Co²⁺, Zn²⁺, Mg²⁺, Cu²⁺ and Na⁺), and with various solvents (ethanol, ethyl acetate, petroleum ether, acetonitrile, N-amyl alcohol, N-hexane, and toluene). In addition, the enzyme activity was investigated using different substrates such as phenol, catechol, benzoic acid, pyrogallol and α-naphtol. SDS-PAGE analysis indicated that there was a single-band protein with a molecular weight of approximately 40 kDa. The catechol 1, 2 dioxygenase had a maximum activity at temperature 30˚C at pH 8.5. Moreover, the catalytic activity of the enzyme was increased in the presence of cobalt and zinc ions as well as organic solvent of amyl alcohol, while it was decreased or inhibited in the presence of the other metal ions and organic solvents used. Among different substrates on enzyme activity, catechol was the most favorable for the enzyme, so that, the V_max and K_m were 8.959 U/mg and 4.992 µg/mL for the substrate, respectively.

Abstract Today, nanosilver is one of the most commercialized nanomaterials. The demand for synthesis of Nanosilver through biocompatible routs due to wide biomedical application has increased. Use of plants and plant products as sustainable and renewable resources in the synthesis of nanoparticles is more advantageous over other biological routes. In this study, biosynthesis of silver nanoparticles (AgNPs) using aqueous extract of Withania somnifera as reducing agent is reported. Effect of parameters such as AgNO3 concentration, aqueous extract, pH and formation time were investigated and optimized by UV-visible spectroscopy in the synthesis of nanoparticles. At room temperature, the solution color started to change from pale yellow to dark brown due to the reduction of silver ion. The transmission electron microscopy (TEM) was applied for size and morphological analysis of nanoparticles. TEM result shows a spherical structure with an average size ranging from 24-35 nm for silver nanoparticles.

Abstract
Objectives: In this study, the effect of different concentrations (1-50 mg/L) of colloidal Ag-NPs investigated on the growth, fatty acids profile and biodiesel indices of N. oculata, after estimating EC50 (20.88 mg/L).
Materials and methods: In this research, N. oculata was selected owing to fast growth and its ability to synthesize lipids for biodiesel production. This microalga exposed to colloidal silver nanoparticles under marine conditions for 72 h. The optical density (OD) and fatty acid profiles were investigated using spectrophotometric analysis and gas chromatography, respectively. Statistical analysis growth data was performed using ANOVA and Duncan's multiple test at 2% probability level.
Results: The algal growth significantly decreased in N. oculata cells treated with the 5-50 mg/L of Ag-NPs. The increase of saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) as well as the decrease of monounsaturated fatty acids (MUFAs) contents were also observed in response to 25 mg/L of Ag-NPs in compared to the control. The important indicators of biodiesel oxidative stability containing LCSF, CFPP and CP increased in N. oculata exposed to Ag-NPs, while the level of DU decreased. The results of this study showed that despite the toxicity of silver nanoparticles, this nanoparticle can increase the biodiesel stability produced from N. oculata.

Abstract Foot and Mouth Disease (FMD) is a highly contagious and devastating disease that spreads rapidly and causes many economic damages. One of the important methods for detection of FMD and particularly differentiation of vaccinated from infected animals, is the use of non-structural proteins as antigens in ELISA kits. The purpose of this study was cloning of the gene sequence and expression of the antigenic regions of 3D nonstructural protein as one of the diagnostic options. For amplification of the antigenic regions of FMD virus 3D protein, specific primers containing NdeI and EcoRI restriction sites were designed and the polymerase chain reaction was performed. The sequences cut by these two enzymes, were inserted into PET21a+ vectors. The recombinant plasmids were then transformed into E. coli (DH5α). Colony-PCR tests and enzymatic digestions were performed on the resulting colonies and the presence of the target gene was confirmed. The gene sequence was further confirmed after sequencing. For production of recombinant antigens, the recombinant vector was transferred to the expression host of E. coli-BL21. The bacteria containing the recombinant gene were induced with IPTG and the expression of the recombinant protein was confirmed using the SDS-PAGE method. The molecular weight of the recombinant protein was about 24 kDa, and it can be used in the design of ELISA diagnostic kit.

Abstract Abstract: Hyperglycemia is a major cause of diabetes. Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications such as retinopathy, nephropathy and cardiovascular diseases. The most important molecules in endothelial cells that can sense elevated level of glucose and transmit signals into the cell are G protein-coupled receptors (GPCRs).
In the present study, according to bioinformatics analysis of genomic sequences between healthy and patient individuals, two G proteins GPR182 and CALCRL were selected and their expression level were examined in hyperglycemic and normal conditions in HUVEC as a model of vascular endothelial cells at different glucose concentrations and various time intervals. In addition, the effects of hyperglycemia on cell viability and cell cytotoxicity were assessed by MTT and LDH assay respectively and also morphological changes by immunohistochemistry.
Overall our data reveal a probable role for GPR182 and CALCRL in hyperglycemia-induced endothelial dysfunction. Thus, they could be developed as a potential molecular targets for the endothelial dysfunction therapy.