Modares Journal of Biotechnology

Abstract Designing new drug delivery systems is important; therefore, in the present study the interaction between an anti-cancer drug, bicalutamide, and an amide/acid hydrogel was studied. Analyzing was done by using docking and molecular dynamics simulation methods. Molecular dynamics simulations were performed at 37 and 42 °C. The results showed that the binding free energies of the drug to the hydrogel system at two temperatures were similar, and altering the temperature did not affect the stability of the system. The van der Waals interaction is the most crucial interaction between the drug and the hydrogel, which depends on the distance between the drug and hydrogel. Intra- and intermolecular hydrogen bonds and van der Waals interactions, are the major factors in the stability of the hydrogel system. Due to the stability of the studied system, it can be used as a drug carrier.

Abstract Vitamins D and E are two common medicines for diabetes treatment. Among the main issues in this field is the release of insulin into the circulatory system. Increasing the stability of insulin hexamer is an evolving strategy in improving insulin secretion efficiency. Insulin protein is commonly found in three forms: monomer, dimer, and hexamer. In this study, for the first time, computational approaches were used to investigate the effect of vitamins D3 and E on the stability of insulin hexamer. The molecular docking results indicate six specific binding sites for these vitamins. These bind to the hydrophobic sites of insulin subunits due to their structural rings and hydrophobic properties. The G-mmpbsa analysis indicates the stabilizing role of both vitamins. The binding of these vitamins to the hexamer has significantly increased the binding energy between insulin subunits. Also, the number of hydrogen bonds between monomeric subunits of each insulin homodimer increased in the presence of the vitamins. It also significantly increases the number of internal hydrogen bonds of hexamer protein. Accordingly, vitamins D3 and E bind to and stabilize the insulin hexamer, resulting in a slower and more balanced insulin release as well as a longer half-life for the dimer in the bloodstream. These findings will pave the way to design a new strategy to regulate insulin release and increase its half-life in the blood for type II diabetes treatment. Besides, hexamer stabilization can be an effective treatment strategy for type I diabetes through slow release from an implanted biosensor system.

Abstract Background and Objectives: Alzheimer’s disease is the most common neurodegenerative disease and the memory impairment is the main prominent symptom of this disease. The hippocampus of the brain, is the first region that undergoes changes in Alzheimer’s. Systems biology tools such as high-throughput techniques, enable us to explore signature genes involved in disease initiation and advancement which can be considered as new therapeutic and diagnostic candidates in complex diseases like Alzheimer’s.

Methods: A total of 85 samples obtained from the hippocampus of the brain of healthy individuals and individuals with Alzheimer’s were selected from two datasets. Differential expression analysis was performed independently for both datasets and the results were integrated. Genes with the same expression pattern in the two datasets were used to construct a gene-gene network using the STRING database. The obtained network analysis was performed to detect key genes associated with the disease.

Results: In this study, 73 genes with the same expression pattern were found in the two datasets. The obtained network analysis led to the identification of SNAP25, UNC13A, SYN2 and AMPH as key genes connected with Alzheimer’s disease.

Conclusion: The role of the reported key genes in endocytosis, neurotransmitters release and synaptic vesicle cycle facilitate proper functioning of memory. Expressional changes and mutations in each of these genes effect other pathways and lead to Alzheimer’s. Thus, the key genes reported in this study, can be considered as potential markers in developing diagnostic and therapeutic methods for Alzheimer’s.

Abstract Immunotoxins are an attractive way to treat cancer; in this method, high-cytotoxic protein toxins target cancer cells specifically. An immunotoxin consists of a targeting component (an antibody, cytokine, or other protein that binds to the cell), that is chemically conjugated or fused in DNA level to a cytotoxic cargo (a bacterium, plant or cytotoxic human protein). Immunotoxin, with the help of specific receptors, recognizes the target cell and enters the cell by endocytosis. After entering the cytocell, it kills the target cancer cell with the help of a toxic component. Although various immunotoxins with different structures have been studied and tested in recent decades, only three immunotoxins Denileukin Diftitox, Tagraxofusp and Moxestumomab Pasudotox - have been clinically approved for the treatment of leukemia. In this article, we review important research and two challenges in production and development of immunotoxins that have limited their clinical success. Further, we highlight methods to overcome these obstacles. These challenges include target and non-target cell toxicity and immunization.

Abstract Multiple sclerosis (MS) is one of the most common autoimmune diseases in Iran and the world. To date, many drugs have been developed to control the progression of MS as a chronic inflammatory disease of the central nervous system. Rituximab is a chimeric mouse-human monoclonal antibody that binds to the CD20 receptor on the surface of B cells and induces apoptosis. Today, Numerous studies have confirmed the increasing role of non-coding RNAs in regulating the expression of genes and molecular processes, including apoptosis. Furthermore, bioinformatic analysis results indicate that TUG1 LncRNA is differentially expressed in MS patients. Thus, In the present study the possible role of TUG1 in regulating rituximab mechanism of action and apoptosis induction was experimentally investigated. To do this, specific DNAzyme against TUG1 was designed and transfected into Raji cells in the presence or absence of the drug. After transfection, RNA extraction and cDNA synthesis were performed. Then, the expression of target genes was examined by Real-Time PCR technique. The results showed an increase in CD20 expression and a decrease in SMAD2 expression levels. Furthermore, decreased TUG1 gene expression led to an increase in apoptosis and cell accumulation in the G1 phase. It seems that TUG1 expression level can play a significant role in CD20 expression in B cells and therefore on the therapeutic efficacy of rituximab.

Abstract Inflammatory bowel disease is a chronic inflammatory disease of the gastrointestinal tract. Despite numerous endeavors over the past few years, as well as an increase within the number of patients with the disease, there are currently limited medications available to manage intestinal inflammation. Designing a new biological treatment using natural bioactive medications with fewer side effects and more secure transmission than chemical compounds could be advantageous. In this study, a new strategy for the controlled release of Gallic acid as a bioactive polyphenol with anti-inflammatory impacts was proposed. This bioactive compound was loaded on a Cerosome nanocarrier and its stability was investigated. Cerosome-forming lipid (CFL) was synthesized through a two-step chemical reaction and then the Cerosomes were prepared by thin layer hydration by distinctive proportions of DPPC: CFL mole ratio. Cerosome with a mean diameter of 335 nm and zeta potential of -23 mV were homogeneous. The optimal formulation of the Cerosomal gallic acid system shows 34% loading and controlled release of the medication in gastrointestinal fluid environments. Structural stability was systematically evaluated by physicochemical characterization methods, and Cerasomes showed greater stability than liposomes and could be present longer in the bloodstream. These results indicate that Cerasomes can be a better medication delivery system for long-term storage and controllable release of gallic acid and have remarkable applications as carriers of intestinal inflammation drug delivery.

Abstract Wound healing and skin remodeling occur directly after skin damage, so the use of platelet rich growth factors (PRGF) and probiotics is important to accelerate this process because of their positive effects on wound healing and antibacterial activities. Combination of above biomaterials with tissue engineering techniques led to the production of a new wound dressing. Therefore, in this study, PRGF was obtained from platelet-rich plasma and a multi-layered scaffold was fabricated by electerospining method using polyurethane (PU) fibers, PRGF and gelatin fibers. Scanning electron microscopy (SEM), tensile and water contact angle tests were performed to assess the characteristics of the scaffolds. The human Adipose Mesenchymal Stem Cells (hAMSCs) were extracted and cultured with the fibroblast cells (HU-02) as co-culture cells and Lactobacillus plantarum was cultured on scaffolds with or without PRGF to evaluate cell viability, toxicity and proliferation, then antibacterial activities of L.plantarum were examined. The result of MTT assay after 14 days indicated that PRFG and L.plantarum had significant positive effect on viability and proliferation of co-culture cells. SEM photograph illustrated adhesion and proliferation of cells and bacteria on scaffolds up to 21 days. The Agar-well diffusion test confirmed the antibacterial effect of L.plantarum on Pseudomonas aeruginosa, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli with strong inhibition zone. The current multi-layered scaffold provides the appropriate wound dressing for cell adhesion, proliferation and prevents wound infection.

Abstract Stem cells are characterized by their capacity for self-renewal and their ability to differentiate into specific cell types under the influence of their microenvironment. It is known that matrix chemistry controls stem cell differentiation. Single cell encapsulations of the Mesenchymal stem cells into a semi-permeable microgel, allows a greater control of the stem cell fate. In this study, a chip for single-cell encapsulation was designed and fabricated using microfluidic technology. By using microfluidic chip, human bone marrow mesenchymal stem cells (hBMSCs) are encapsulated inside alginate and alginate-poly-l lysine (PLL) microgels. The results of long-term viability of MSCs inside alginate-PLL microgels, shows a significant increase compared to alginate microgels. Mesenchymal stem cell proliferation in alginate-PLL microgels also increased significantly on days 14 and 21. It seems that PLL improves cell adhesion and function by creating a positively charged matrix. Microscopic studies indicate that the morphology of the cells inside the microgels is spherical. However, the average diameter and volume of cells in microgels containing PLL are smaller than others, which indicates more proliferation and space limitation inside the microgels. Therefore, single cell alginate-PLL microgels provide a suitable substrate in clinical studies for tissue engineering, organ transplantation and cell therapy.

Abstract This study aimed to isolate and identify bacteria from soils contaminated with copper and have access to a capable bacterial strain for producing copper nanoparticles (CuNPs). The present study showed the extracellular production of copper nanoparticles using strain Ta-31. The effect of various factors such as substrate, supernatant volume, enzyme inducer, and electron donor was investigated on the production process. The properties of synthesized nanoparticles were identified by using UV-Vis, FTIR, XRD, SEM, and EDS analysis.

Moreover, the growth curve of strain Ta-31 was plotted in the presence and absence of an enzyme inducer (concentration of 0.1 mM copper sulfate). After the phylogenetic analysis, 16S rDNA gene sequences were determined, and their phylogenetic tree of the selected strain was plotted. The results showed that the best conditions for producing CuNPs, glucose 1% as an electron donor, 2 mM copper sulfate, and 20 ml supernatant had the best production. Strain Ta-31 arrived at the end of the log phase and the beginning of the stationary phase after 15 h. CuNPs were spherical and irregular, and the size of CuNPs was more in the range of 30-40 nm. According to the results, strain Ta-31 belonged to Staphylococcus pasteuri sp. with 99.88% similarity.

Abstract

Background: Shigella and Enterohemorrhagic Escherichia coli are among the most common causes of bacterial diarrhea, and no effective vaccine candidate for these bacteria have approved yet. Due to the role of IpaD protein and Shigella enterotoxin B subunit (StxB) in Shigella and E. coli O157: H7 pathogenicity, STX1B-IpaD chimeric protein can be used as a suitable molecule to produce a recombinant vaccine candidate. This study aimed to clone, express, and purify STX1B-IpaD chimeric protein to develop an effective vaccine candidate against Shigella and E. coli O157: H7 species. Materials and Methods: IpaD gene with NdeI and BamHI restriction enzyme sites was isolated from a recombinant vector and subcloned into the pET28a -STX1B expression vector. Vector was transferred to E.coli strain Rosetta (DE3) and confirmed by PCR and restriction enzyme digestion. SDS-PAGE and western blotting were used to confirm the recombinant protein. The recombinant STX1B-IpaD protein was purified by affinity chromatography, and its concentration was measured by the Bradford method. Results: The PCR and restriction enzyme digestion showed the accuracy of the gene cloning. The protein electrophoresis showed the proper expression and correct molecular weight (27 kDa) of STX1B-IpaD. The western blot analysis confirmed the recombinant protein. The recombinant protein concentration was estimated at more than 0.3 gr/L. Conclusion: An effective method for the production of recombinant proteins is codon optimization and effective expression in heterologous hosts. After the immunogenicity in the animal model, this recombinant protein can be used as a chimeric vaccine candidate against EHEC and Shigella bacteria.

Abstract Evaluating the response of the stem cells to different mechanical stimulation is an important issue to obtain control over cell behavior in the culture environment. One of the effective parameters in the mechanoregulation of stem cells is the microstructure of scaffolds. Evaluating the effect of microstructure of scaffold in the lab environment is very complicated. Therefore, in this study, the effect of scaffold architecture on mechanical factors in the scaffold was investigated under oscillatory fluid flow by using numerical modeling. In this study, distribution of shear stress and fluid velocity in three types of scaffolds with spherical, cubical and regular hexagonal pores with length of 300, 350, 400, 450 and 500 micrometers were investigated by using computational fluid dynamics method. The results of the computational fluid dynamics model showed that the scaffold with spherical and cubic pores shape with length of 500 micrometers and scaffold with hexagonal pores with length of 450 micrometers experienced shear stress in the range of 0.1-10 mPa. This range of the shear stress is suitable for differentiation of the stem cell to bone cells. Moreover, the result of exerting oscillatory fluid flow to these scaffolds indicated that dead zones of the scaffold, where isn’t suitable for cell seeding, was decreased due to the access of fluid flow to the different area of scaffold. The results of this study can be used in a laboratory to achieve optimal stem cell culture to provide suitable environment culture for differentiation of stem cells toward the bone cell.

Abstract Aflatoxin B1 is a type of mycotoxin produced by Aspergillus fungi during food production and storage. Aflatoxins have many toxic effects on the body that cause mutagens, teratogens and have high carcinogenic properties that cause cancer in the liver and other organs. Although conventional device methods for measuring aflatoxin B1 in food are sensitive and accurate, they have disadvantages such as high diagnostic time, high cost, the need for a trained user, and the creation of false positive results. Therefore, the development of new measuring methods has been prioritized by researchers. Among these measurement methods is the use of biosensors, which are fast, simple and more affordable and are used in the food industry today. In this work, a colorimetric optical aptasensor using gold nanoparticles with appropriate sensitivity and high selectivity was used to detect aflatoxin B1 in serum and buffer. For this purpose, gold nanoparticles were synthesized by reducing HAuCl₄ by sodium citrate (with a size of 14.40 nm and a zeta potential of -27.5). In this method, the protective effect of DNA sequence on the surface of gold nanoparticles has been used in the presence or absence of aflatoxin with the intervention of salt and the characteristic of visual color change. The detection limit of this method was estimated to be 50 ng/L and its linear range was 200-28000 ng/L. As a result, the designed aptasensor can be used for quick identification and screening of this toxin in contaminated food.