JOURNAL OF BIOTECHNOLOGY
Year:2019 | Volume:10 | Issue:4 |Papers Count:20

The high quality and quantity of extracted DNA are necessary for a variety of molecular biology studies. Low yields and poor quality of genomic DNA extracted from petal due to high levels of secondary metabolites. Carotenoids, anthocyanins, phenolic acids, and flavonoids are the most effective secondary metabolites in petals, which are considered as contaminating compounds and could lead to interfering with DNA during extraction and purification. Considering that the basis of the most molecular research in genetic engineering and genomics is high-quality of DNA, therefore, it seems that finding an efficient method for reducing adverse effects of these contaminating compounds for the extraction is essential. In this regard, iron-magnetic nanoparticles have been used to improve the extraction of high yields and quality of DNA from rose petals in the present work. In the following, to compare the efficiency of DNA extraction, modified Cetyl Trimethyl Ammonium Bromide (CTAB) and rapid detection methods were used. The results showed that petal’ s extracted DNA quantification and qualification by iron-magnetic nanoparticles procedure was much more reliable than two other methods. In addition, this method could extract the optimal amount of DNA with the lowest amounts of samples within few minutes. Due to high qualification and quantification of DNA purification by iron-magnetic nanoparticles, the present procedure could be recommended as an efficient protocol for rose petal DNA extraction.

Aims Significant amounts of waste, including feathers, bones, blood, etc. are yearly produced by the poultry industry. Feathers are composed of 90% keratin protein, and the rest is composed of lipids and water. Keratinases are one of the most diverse and usable enzymes, which can be produced by bacterial and fungal microorganisms. These enzymes show a wide range of application in various fields. Materials and Methods In this study, the keratinolytic activity of the isolated strain from a poultry farm in Mashhad was evaluated and then the medium conditions for keratinase production were optimized. The strains were identified based on the morphological and biochemical methods. 16SrRNA gene of the strain was amplified by PCR and then sequenced. The strain proteolytic activity was examined and compared with its keratinolytic activity. Finally, strain growth ability tested in variety substrate. Findings Using 16SrRNA gene sequencing, morphological and biochemical identification, the strain shared 99. 9% similarity with Bacillus mojavensis. Optimization of various factors, including temperature, pH, incubation time, carbon and nitrogen sources, aeration and inoculum size showed that the isolated strain has the highest keratinolytic activity at 37° C, 48 hour incubation period, pH=9. 5, sucrose 1%, 3% substrate, aeration 75% and 6% (v/v) inoculum amount. None of the nitrogen sources had a positive effect. Conclusion The FUM-1 keratinolytic activity was increased approximately 3. 38 fold by condition optimization of the medium, indicating the importance of environmental conditions. In the study, the strain with high keratinolytic activity was suggesting its potential use in biotechnological.

The use of enzymes in organic solvents represents an important area of industrial and biotechnological development. However, organic solvents often cause protein denaturation, thereby reducing the activity and stability of enzymes. Use of stabilizing additives, protein engineering and chemical modification of enzymes are common strategies to overcome this problem. In this study, a cysteine protease from the latex of Ficus johannis was purified and the activity and stability of the protease were investigated in the presence of different organic solvents. The effect of trehalose, sorbitol, and sucrose on the enzyme activity was also studied in the presence of organic solvents. The results showed that the enzyme activity was elevated in the presence of low concentrations of organic solvents increased, while it was decreased with increasing concentration of organic solvents. However, the enzyme still retained 60% of its activity at 30% organic solvent concentration. The enzyme was considerably stable in the presence of organic solvents, maintaining almost 90% of its stability in the presence of 50% of all solvents. As stabilizing additives, sugars enhanced the catalytic activity and stability of the enzyme, and trehalose was the most effective sugar. The easy purification procedure and considerable activity and stability of the protease in the presence of organic solvents could suggest this enzyme as a good candidate for peptide synthesis industry.

Gene expression, flow of information from DNA to proteins, is a fundamental biological process. Expression of one gene can be regulated by the product of another gene. These regulatory relationships are usually modeled as a network; genes are modeled as nodes and their relationships are shown as edges. There are many efforts for discovering how genes regulate expression of themselves. This paper presents a new method that employs expression data and ontological data to infer co-expression networks, networks made by connecting genes with similar expression patterns. In brief, the method begins by learning associations between the available ontological information and the provided co-expression data. Later, the method is able to find both known and novel co-expressed pairs of genes. Finally, the method uses a self-organizing map to adjust estimation made by the previous step and to form the GCN for the input genes. The results show that the proposed method works well on the biological data and its predictions are accurate; consequently, co-expression networks generated by the proposed method are very similar to the biological networks or those that constructed with no missing data. The method is written in C++ language and is available upon request from the corresponding author.

Hordeum vulgare is a one-year-old herb of the Poaceae family. It is an important cereal used by humans which has been applied in many cases instead of wheat. The limitation of experimental methods is one of the important problems for identifying protein-protein interactions. So, in recent years, computational methods have played an important role in predicting and identifying protein-protein interactions. In this study, for constructing protein-protein interaction (PPI) network, the experimental PPI information of six model organisms includes Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Oryza sativa, and Arabidopsis thalian were extracted from the Intact database. Inparanoid was used for identifying barley orthologous proteins with model organisms. The Interolog method which was used in this study can predict protein-protein interactions by mapping protein interactions of the model organisms on orthologous proteins. After removing repetitive interactions, the final predicted barley PPI network contained 235966 interactions between 7350 proteins. This study is the first report presented on protein-protein interaction prediction in barley.

Aims The objective of this research was to develop a novel method for the synthesis of colloidal solutions of titanium dioxide nanoparticles with high stability and life span. Materials & Methods Based on mentioned points, the issue of this study is the synthesis of nanoparticles via chemical reduction process. The morphologies, compositions, and physicochemical properties of the prepared samples were characterized by TEM, XRD and DLS. Also, the cytotoxic effect of fabricated NPs against human white blood cells (WBCs) was investigated via MTT assay. In addition, antibacterial activity was investigated. Findings The results of this study indicate that the diameter of the synthesized nanoparticles is about 50nm and contains the anatase phase, in the range of 2θ from 25-80° C, and the hydrodynamic radius of nanoparticles is about 95. 8± 12. 78nm and the zeta potential of nanoparticles is about-34. 87± 4. 78mV. Also, the effect of toxicity of titanium dioxide nanoparticles on the white blood cell line showed that these nanoparticles cause the toxicity of cells at concentrations above 200μ g/ml, but in lower concentrations, normal cells can survive. Also, these nanoparticles at the same low concentrations. Conclusion In conclusion, colloidal solutions with high stability were successfully synthesized, which, in addition to increasing the antibacterial properties due to diminished dimensions.

In view of the constant increase of nanotechnology and nanomaterials applications in our daily life, to determine whether they are safe, “ in vitro” and “ in vivo” screening methods are needed. Obviously, application of models that are similar to the physiological tissues process of the human body could be a better candidate. The three-dimensional spheroid method, spheroid were generated using commercial microplates, has many benefits (in comparison with traditional methods or monolayer cell culture) such as the growth of the cells in 3D, similar to the body’ s physiological tissue, an alternative for animal models, cell-to-cell interactions, and better cell signaling. In this study, the toxicity of silver nanoparticles by using three factors such as metabolic activity, live/dead assay, and spheroid surface area was evaluated using two different methods (2D vs 3D) under treatment with various concentrations of silver nanoparticles at different times. The results showed that different cells types, cancer and/or normal lung cells, have significant differences. In addition, it was observed that distinct differences in terms of cytotoxicity of silver nanoparticles between 2D and 3D culture systems and also the rate of growth/non-growth of spheroids are highly depended on cell type and various concentrations have fundamental importance in such studies. The present study provides evidence that cellular dimensions (3D vs 2D) play a pivotal role in the results and outcomes of inflammation and cytotoxicity with nanoparticles due to the spatial-temporal structure.

Diatoms biosilica shell, frustule, is substitute biostructures to mesoporous silica particles, which possesses their wide surfaces, nano-diameter porosity, mechanical strength, and thermal stability, optical capabilities, and the ability to bind to biomolecules can be used in biosensing applications. In this study, diatom species called Chaetoceros muelleri, was used for the fabrication of the Fe2O3-Au-Biosilica magnetic package. After micro-algae cultivation, the synthesis of gold nanoparticles (AuNPs) on silica walls was carried out using the bio-synthesis method which evaluations have demonstrated the continuous formation of spherical AuNPs on the walls and its surfaces. After this step, the magnetic iron oxide nanoparticles were attached to the silica surface of the diatom, this, in turn, leads to system guiding using a magnetic field. Surface modification of diatoms magnetic complex, by using the APTES, allowed the attachment of fluorescence Rhodamine and the Herceptin antibody (Trastuzumab) to the structure. As well as the attachment of the fabricated system to target cells (SKBR3) was confirmed by fluorescence microscopic analysis. The results of this study indicate the ability and specificity of the diatom silicone shell as a “ multipurpose” package for diagnostic and therapeutic activities.

Introduction Since the gastrointestinal system plays an important role in the function of the immune system, its role in the control or treatment of autoimmune diseases cannot be ignored. Therefore, the intestinal strengthening, which much of gastrointestinal function depends on it, can be effective in this direction. Also, because the intestine plays an important role in the immune system in addition to digestion, it can help maintain the immune system’ s function by keeping its bacteria balanced. In this regard, probiotics and prebiotics can be useful, which this issue was investigated in the present study. Conclusion Probiotics have an important role in the prevention and control of multiple sclerosis.

Aims One of the most important regenerative medical purposes is the production of alternative tissues with proper function. Fibroblast cells are one of the most important types of cells in the repair process that also play a role in the formation of blood vessels. Stimulation of fibroblastic cells requires the appearance of external signals to begin the proliferation and recall of other cells, as well as angiogenesis. The aim of this study was to investigate the effects of M13 in combination with RGD peptide on fibroblastic cells. Materials and Methods For this study, M13 bacteriophage was first amplified and isolated. Then RGD peptide was synthesized and purified. Then, isolated mouse fibroblastic cells were culture on surfaces coated with M13 bacteriophage, bacteriophage M13 and RGD, gelatin, and surfaces without coated as a control for 48 hours. MTT assay was used to measure the proliferation and survival of cells, and then the expression of FGF-2, TGF-β 1 and VEGF-A genes was measured by real-time PCR. Findings The results of this study showed that the M13 and RGD bacteriophage increased cell proliferation and the fibroblast cell survival rate. In addition, expression of FGF-2, TGF-β 1 and VEGF-A genes in cultured fibroblasts on the M13 and RGD bacteriophages surface increased significantly. Conclusion Our research showed that scaffolds of M13 bacteriophage and RGD peptide are nontoxic and bio-compatible so they can be a suitable candidate for induction of repair and angiogenesis in tissue engineering.

Aims Hematopoietic stem cells are responsible for the production of blood cells in the bone marrow. During the process of differentiation, these cells commitment to two precursor cell lines include myeloid and lymphoid cells. Various blood cells, excluded lymphocytes, generates from myeloid cells. Some patients with severe anemia or thrombocytopenia receive hematopoietic stem cell through transplantation. Finding a potential component for inducing differentiation of hematopoietic stem cells before transplantation, could be an appropriate strategy for the acceleration of blood cells production in recipient persons. Various studies indicate the ability of Curcumin for inducing of cell differentiation. This component can alter many of cellular mechanisms. Material and methods The aim of this project was to evaluate the effects of Nanocurcumin on mRNA expression levels of GATA1, GATA2, c-Myb and Hhex genes and alteration of cellular ROS in umbilical cord blood-derived hematopoietic stem cells. Nanocurcumin was synthesized from Curcumin, Oleic acid, and PEG400. The rate of Nanocurcumin delivery into the cells was also evaluated. Findings Our results show that intracellular ROS and expression levels of GATA1, c-Myb, and Hhex transcription factors were significantly increased after treatment with Nanocurcumin (p<0. 05). These transcription factors involve in myeloid differentiation. Conclusion Enhancement of these transcription factors expression making Nanocurcumin a potential candidate for applying in myeloid differentiation media and basic and clinical studies.

The SPTBN4 gene, a part of the spectrin protein family, plays important roles in various cellular processes, including cell cycle, nerve cell development, and so on. Recently, a new miRNA has been found in this SPTBN4 gene, which was registered at the NCBI database. The aim of the present study was to investigate the expression of this miRNA, called SPTBN4-miR1, in the process of differentiation of human embryonal carcinoma cell line NT2 and also the overexpression effect of this miRNA on the differentiation of these cells. RT-qPCR results indicate that SPTBN4-miR1-5p and SPTBN4-miR1-3p show a significant increase in expression in the process of neural differentiation from day three until the 8th and 14th day of differentiation. Then, after overexpressing the SPTBN4-miR1 precursor in NT2 cells and retinoic acid treatment, the expression of pluripotent and differentiation revealed the role of SPTBN4-miR1-5p and SPTBN4-miR1-3p in promoting differentiation and exclusion from the pluripotent state. It seems that by making further studies and finding out the possible targets of these miRNAs, a distinctive marker can be achieved and used to improve the differentiation process.

Revealing DNA sequences is vital for all branches of biological sciences. Next-Generation Sequencing (NGS) is a different approach in this area so that it has created a great evolution in biology science and covers various aspects of genome, transcriptome, epigenome and metagenome-level studies. NGS is considered as a high-performance method for genomic and transcriptomic information analysis in comparison with traditional methods due to providing good genomic coverage, determining each single pairs of bases and eliminating the first generation sequencing disadvantages (Sanger sequencing). Use of NGS has begun since 2005 and 2006, after the commercialization of various apparatus companies such as ABI/SOLiD Illumina, Science Roch/454Life, and Solexa to study the transcriptome of the model and nonmodel organisms. Recently, RNA sequencing is used widely to identify genes associated with growth and development processes and their expression patterns in response to a variety of biological and non-biological stresses, in various organs and growth stages in different organisms. It helps scientists to determine the amounts of gene expression, differentiation of different isoforms of genes, detection of gene fusions and characterization of small RNA as well as alternative splicing events, duplicate elements, exon of genes, new transcripts, UTRs, SNPs, and somatic mutations. The RNA-seq method typically consists of providing suitable biological samples, isolation of total RNA, enrichment of non-ribosomal RNAs, conversion of RNA to cDNA, construction of a fragment library, selecting size and adding linkers and sequencing on high-throughput sequencing platform, alignment, and assembly of the reads and downstream analysis.

Aims Aequorin as a bioluminescence protein due to ease of use, non-toxic, and high capability of detecting has long been the interest of researchers. The aim of this study was to design a method for accurate and simple detection of important therapeutic agents using a bioluminescence inhibition based assay by using aequorin. Materials & Methods In this study, important drugs in therapeutic monitoring with structural similarity to Coelenterazine, were selected and their interaction with aequorin was investigated. Further, the conditions of the bioluminescence assay were optimized to achieve the lowest detection limit. Findings Among the drugs whose effects have been tested on aequorin, the only benserazide resulted in inhibition of the bioluminescence activity. This analyte can significantly reduce the bioluminescence of aequorin in a concentration-dependent manner. The best dose-response curve was obtained and IC50 of 0. 26μ M was calculated. The linear calibration curve was obtained in a range of about 100 to 1500nM with LOD and LOQ of 79 and 260nM, respectively. Furthermore, we demonstrated the application of the approach in human serum samples with a recovery of 97%. Guddem-Schild graph was plotted to determine the mechanism of inhibition which indicated that the IC50 of benserazide changed in the presence of different concentrations of Coelenterazine. Conclusion The proposed method can be used for measuring benserazide which can easily be applicable for real samples. Also, the results show that benserazide inhibits the bioluminescence activity of aequorin by competitive inhibition.

Aims In bone tissue engineering, the scaffold as a supportive structure, plays a vital role. Putting the scaffold in dynamic cell culture, such as perfusion bioreactor, makes the role of mechanical parameters such as shear stress and hydrodynamic pressure more important. On the other hand, these mechanical parameters are influenced by scaffold architecture. In this study, the effects of bone scaffold architecture on mechanical stimuli have been analyzed and their effects on the mesenchymal stem cell fate have been predicted. Material and Methods Using the tools of computer simulation, five bone scaffolds (Gyroid, high porous Gyroid, Diamond, IWP, and gradient architecture Gyroid) based on mathematical functions of minimal surfaces were designed and exposed in a simulated dynamic cell culture under the inlet velocities of 1, 10, 25, 50, and 100μ m/s. Cell accumulation on the inner part of the scaffold was considered as an 8. 5-micron layer. This layer was designed for Gyroid and IWP scaffolds. Findings Based on the results, Diamond scaffold showed the most efficient performance from the homogeneity of stresses point of view. In the presence of the cell layer, the von Mises stress was reported as 60 and 50 mPa on the Gyroid and IWP scaffolds, respectively which eases osteogenic differentiation. Conclusion In gradient architecture scaffolds under dynamic conditions, there is a gradient in shear stress that causes various signaling in different positions of theses scaffold and facilitates multi-differentiation of the cells on the same scaffold.

Aims Curcumin is a natural molecule that due to its various curative effects including antibacterial properties, it can be used as a medicine, albeit after reducing its disadvantages. The aim of the present study is to develop a method for preparation of nanoparticles of curcumin using PAA, PVA, and PEI polymers with a view to improve its stability, increasing bioavailability and aqueous solubility as well as study its effectiveness against methicillinresistant to Staphylococcus aureus. Materials & Methods In order to synthesize polymeric nanoparticles including curcumin with the nano-precipitation method, optimizing the effective concentration of polymer, curcumin, and water were determined by using the Response Surface Method (RSM). Synthesized nanoparticles were characterized by Scanning Electron Microscope (SEM), Dynamic Light Scattering (DLS) and zeta potential measurement methods. Furthermore, minimal concentration inhibitory of synthesized nanoparticles against the Staphylococcus aureus resistant to methicillin was measured. Findings The created nanoparticles were round, discrete and smooth in surface morphology and the average particle size for PAA, PVA, and PEI were 149± 7nm, 175± 8nm, and 184± 9nm respectively. The minimum inhibitory concentration for PAA, PVA and PEI nanoparticles against the Staphylococcus aureus were 0. 480± 0. 024, 0. 390± 0. 019 and 0. 340± 0. 017mg/ml. The concentration of solvent, polymer, and curcumin was important to obtain small size particles. Conclusion The results indicated that the water solubility of curcumin significantly improved by particle size reduction up to the nano range. The inhibitory property of curcumin nanoparticles has greatly increased due to the smaller particle size and their increased penetration into the bacteria and nanoparticles loaded with curcumin could be a promising drug carrier for the treatment of cancer, infections and other diseases.

Introduction Nowadays, bone tissue repair with increasing bone disorders and injuries have special importance. Bone tissue engineering provided specific solutions to these problems. The present study was conducted with the aim of purification of recombinant fusion peptide containing hydroxyapatite affinity tag using the ceramic chromatography column. Material & methods In this study, a fusion peptide was designed which at one side comprised the heparin-binding domain sequence, which can be attached to various types of growth factors involved in tissue repair and entrap these factors at the site of the lesion. On the other side, it contained a tag, which included a sequence derived from a laboratory study based on phage expression. The reason for keeping the sequence of this tag is to attach the peptide to the scaffold containing hydroxyapatite and purifying the recombinant peptide by the hydroxyapatite column. Therefore, the gene sequence was optimized and synthesized for expression in the prokaryotic host of E. coli strain BL21. Then the gene sequence was subcloned by double digestion with the SacI and BamHI enzymes into the expression vector of pET-21a(+). The expression of the recombinant peptide was investigated by SDS-PAGE and western blot. In order to optimize the purification conditions, two-step purification was carried out by applying fundamental changes in the main work method of the manufacturer company and was purified with acceptable purity. Finally, the existence of peptide assemblies was investigated by the SLD method. Finding The results of PCR cloning, enzymatic digestion using SacI and BamHI enzymes and sequencing indicated the accuracy of the cloning process. On the other hand, expression of the fusion peptide was confirmed by SDS-PAGE and Western blot techniques, and its migration onto the gel resulted in a band cleavage of about 12 kDa. Changes made to the manufacturer’ s workflow allowed the purification process to be optimized and the results of the DLS method showed the purity of the purified peptide. Conclusion The results indicate the desirable expression and remarkable purity of the fusion peptide designed in this study.

The stimulants are materials that increase alertness and reduce physical and mental fatigue. These drugs increase the activity of excitatory receptors and reduce the activity of inhibitory receptors in the central nervous system. Methamphetamine, also known as crystal, is a psychoactive substance. This drug is stimulating nerves and by a direct effect on the brain, mechanisms cause joy and excitement in people. Methamphetamine in low to moderate doses (5 to 30 mg) causes euphoria; excitement, increased heart rate, and blood pressure, mydriasis, increased body temperature and decrease appetite. High but non-lethal doses of methamphetamine cause mental disorders and psychotic symptoms, seizures, and rhabdomyolysis. Cardiovascular toxicity of methamphetamine-induced hypertension, arrhythmia, acute coronary syndrome and ischemic ventricular. The most important cellular mechanisms involved in the damage caused by Methamphetamine are oxidative stress, excitotoxicity, and mitochondrial damage. The synthesis of methamphetamine in illegal workshops mainly of six methods which are based on the raw materials are divided into two groups. Raw materials in the synthesis of methamphetamine are ephedrine and phenyl propanol. In reduction, methods involve Birch reduction, Nagai and hydrogenation Rosenmund ephedrine and pseudoephedrine are used as raw material, in Lockhart methods and amination reduction methods based on phenyl propanol as raw material.

Recent researches on the application of nanoparticles have been focused on nanostructures of gold with rod morphology, due to having outstanding optical properties for diagnostics and therapeutics of the diseases. The rod morphology of the nanostructures enables strong and sensitive absorption of surface plasmon in the infrared region. In the present research, based on the sensitivity of surface plasmon resonance of gold nanorods to trace changes in the local environment, as well as the importance of rapid detection of trace amounts of albumin in urine, functionalization, and stability of these nanostructures with anti-albumin antibody has been investigated in different concentrations, volumes, time and pH changes. The results of spectroscopic studies of different samples in the visible spectrum near-infrared waves showed that gold nanorods have desirable stability, and their rod morphology characteristic is maintained. The study of the temporal stability of samples showed that the complex samples were stable up to 48 hours for sensing applications. Primary monitoring of the function of the nanobiosensor in the presence of albumin with two normal and abnormal levels of concentration revealed remarkable changes in interparticle distance, size, and morphology of the nanostructures. According to this research, the rod nanostructures can be used to design simple nanobiosensors.

With the approval of the law for supporting knowledge-based firms in 2010, a new wave in Iran’ s science, technology and innovation system began with a focus on the knowledge based economy and innovation-based. Currently, there are more than 4, 000 knowledge-based firms in Iran that nearly 5% of them are active in biotechnology. The aim of the present study is to design an empirical model of the relationship between financial and tax incentives of this law on some of the performance indicators of biotechnology knowledge-based firms. For this purpose, after analyzing the content of related documents and designing the study model, for evaluating the direct and interacting effects between policy tools, identifying the important empirical factors and their level, “ 23 factorial design” was used. Study target community includes 113 manufacturing knowledge-based firms in the field of biotechnology. The findings of this study on input additionality indicators show the positive effect of the threefold interaction of factors on the R&D expenditure and the positive effects of commercialization financing and technology financing and their interactions on R&D employee. In the present study, there was no relationship between the effectiveness of policy tools on output additionality indicators.