Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301Cost-effective Strategies for Depletion of Endogenous Extracellular Vesicles from Fetal Bovine Serum42542976310.22067/jcmr.v11i2.85209ENAzadeh HaghighitalabDepartment of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran0000-0001-6005-4156Maryam M. MatinDepartment of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran0000-0002-7949-7712Fatemeh KhakrahCentral laboratory, Ferdowsi University of Mashhad, Mashhad, IranAhmad AsoodehDepartment of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, IranAhmad Reza BahramiDepartment of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran0000-0001-9122-7216Journal Article20200122 Despite the prominent therapeutic potentials of stem cells, their use in cell therapy has been challenged with some unreproducible and inconsistent outcomes in addition to the risk of rejection and tumorigenesis. Gaining novel insights to the importance of the conditioned medium, secretory factors and extracellular vesicles as the functional components of the cultured stem cells, suggested the idea of substituting the cells with their cell-free counterparts. Biological properties of these products are influenced by the cues received from their microenvironment. Hence, providing optimal and fully defined culture conditions is essential for their preparation. Fetal bovine serum (FBS), one of the most routine supplements of cell culture, is enriched by endogenous extracellular vesicles (EVs). These EVs will affect the yield, purity and functional features of the cell-free products. Here, we endeavored to examine and compare three different methods including ultrasonication, ultrafiltration and polymer-based precipitation, to deplete EVs from FBS. We chose easy to perform and fast methods with the capacity for high-throughput applications. Based on our observations, although all examined methods were able to deplete EVs from FBS to some extent, polymer-based precipitation could be considered as the method of choice with minimal consequences on the biological requirements of FBS to support cell growth and characteristics. Due to similarities between FBS and some other biological solutions, this strategy would be suitable for EV-depletion from other liquids with high concentrations of proteins and nutrients. Moreover, it could be applied for preparation of optimal culture conditions for nanoparticle applications.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301A Rapid Method for Analysis of cDNA Synthesis Using Ion-Pair Reversed-Phase High Performance Liquid Chromatography55582979310.22067/jcmr.v11i2.83212ENMaryam M. MatinDepartment of Biology, Faculty of Science, Ferdowsi University of Mashhad, Mashhad, Iran0000-0002-7949-7712David P. HornbyDepartment of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, United KingdomJournal Article20190925 We have developed a rapid, quantitative method for analysing the outcome of the first strand synthesis step in cDNA library preparation, yield and molecular weight range of the final cDNA products are determined after size fractionation. This method involves conventional cDNA library construction including all enzymatic steps usually required, but replaces radioactive labelling of nucleic acids with fluorescence detection. The separation and quantification steps all involve ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). This quantitative method replaces the use of autoradiography and size exclusion chromatography with combined ion-pair reversed-phase high performance liquid chromatography and in line fluorescence detection. The result of this approach is combination of speed with the generation of reproducible, high quality cDNA libraries.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301Production of Silver Nanoparticles by Marigold Extract59652982110.22067/jcmr.v11i2.84974ENZohreh Sohrabi NezhadBiotechnology and Plant Breeding Department, Ferdowsi University of Mashhad, Mashhad, IranHassan MarashiBiotechnology and Plant Breeding Department, Ferdowsi University of Mashhad, Mashhad, IranNasrin MoshtaghiBiotechnology and Plant Breeding Department, Ferdowsi University of Mashhad, Mashhad, IranJournal Article20200106 Silver nanoparticles are widely used in manufacturing of different products considering their unique physical and chemical properties. Also they have been noticed in medical diagnosis and treatment because of their antibacterial properties. Physical and chemical methods of producing nanoparticles, are expensive and are not safe enough as toxic substances may remain in the final preparations. To solve this problem, biological production of nanoparticles is considered as an efficient alternative method. In present study, synthesis of silver nanoparticles via seeds, petals, roots, and hairy root extracts of Calendula officinalis were performed. These nanoparticles were characterized by means of spectrophotometer, particle size analyzer and transmission electron microscope. Nanoparticles which were synthesized by hairy root extract showed the highest absorption at 430 nm (1/6 a.u) and the smallest size (5/3 nm), in comparison to other examined particles. Results confirmed the better performance of hairy root extracts in the synthesis of silver nanoparticles.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301In silico Analysis of Determinant Factors in Microbial Protease Thermostability66812985310.22067/jcmr.v11i2.84965ENParisa FarrokhSchool of Biology, Damghan University, Damghan, IranFatemeh SalimiSchool of Biology, Damghan University, Damghan, IranJournal Article20200107 Thermostable proteases are one of the pivotal enzymatic groups which play fundamental roles in biotechnologyrelated industries. The identification of bacterial thermostable enzymes through screening programs is a time and cost consuming process. So, extensive bioinformatics and experimental studies have been conducted to reveal thermo stabilizing factors. The current study was aimed to evaluate distinctive indicators among 33 thermostable and 10 mesostable proteolytic enzymes. The frequency of individual amino acids, aliphatic indexes, melting temperatures, isoelectric points, as well as, the frequency of AXXXA and GXXXG motifs were determined and compared among these enzymes. In addition, types of proteolytic enzymes and their active sites were assigned. Moreover, the frequency of alpha helixes, polar surface regions, and packing volumes of these enzymes with the known structures were characterized. Results showed that the frequency of Ala and AXXXA motifs were significantly higher in thermostable proteolytic enzymes, while they possess lower contents of Met, His, Lys and Leu in comparison to mesostable enzymes (P<0.05). According to statistical analysis, thermostable proteolytic enzymes indicated meaningful lower packing volumes than mesostable enzymes (P<0.05). Findings of the current study in addition to more detailed investigations on the thermostability mechanisms of various protein families are essential for designing more efficient industrial enzymes with functional properties at high temperatures.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301Evaluation of IFN-γ and T-bet Expression Levels as Possible Molecular Markers of Schizophrenia82892988410.22067/jcmr.v11i2.85426ENRoghaye LorestaniDepartment of Biology, Faculty of Basic Sciences, Gonbad kavous University, Gonbad kavous, Golestan, IranSohrab BoozarpourDepartment of Biology, Faculty of Basic Sciences, Gonbad kavous University, Gonbad kavous, Golestan, Iran0000-0003-0873-7499Sakineh AlijanpourDepartment of Biology, Faculty of Basic Sciences, Gonbad kavous University, Gonbad kavous, Golestan, IranLeila AhangarDepartment of Plant Production, Collage of Agriculture Science and Natural Resource, Gonbad kavous University, Gonbad kavous, Golestan, IranJournal Article20200212 Schizophrenia is an irritating mental disorder that affects around 1% of the world's population. The immune system contributes to the onset of the disease, particularly through production and secretion of some cytokines. In patients with schizophrenia, the balance of Th1 to Th2 ratio is often altered. In the present study, we investigated these changes by measuring the gene expression levels of IFN-γ and T-bet as Th1 indicators, as well as IL-4 and GATA-3 as representatives for Th2. Blood samples of schizophrenic patients (n=25) and healthy individuals (n=10) were obtained. Total RNA was extracted from leukocytes and cDNA synthesis was performed based on provided protocols. Real-time PCR technique was utilized for the assessment of gene expression levels. Results indicated a significant increase in the expression of IFN-γ and its transcription factor, T-bet, while IL-4 gene expression was reduced significantly. The expression level of GATA-3 gene revealed no meaningful changes. Altogether, results confirmed the relative shift of Th1 to Th2 status in the patient with schizophrenia and re-emphasize the importance of the inflammatory events in the incidence of the disease. Moreover, a new index was introduced based on the IFN-γ and T-bet genes expression, which can determine healthy condition with total accuracy of 79%.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301Immunosuppressive Effects of Human Chorionic Gonadotropin (hCG) on Mesenchymal Stromal Cells90982991310.22067/jcmr.v11i2.85667ENSepideh Sadat HosseiniDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranShadi MehrzadDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranHalimeh HassanzadehStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR) -Khorasan Razavi, Mashhad, IranHamid Reza BidkhoriStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR) -Khorasan Razavi, Mashhad, IranMahdi MirahmadiStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR) -Khorasan Razavi, Mashhad, IranMadjid Momeni-MoghaddamDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, Iran0000-0001-8372-1310Fatemeh SadeghifarDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranMoein FarshchianStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR) -Khorasan Razavi, Mashhad, IranJournal Article20200218 Mesenchymal stem/stromal cells (MSCs) as one of the most important types of adult stem cells secrete a variety of immunomodulatory cytokines. However, their immunomodulatory features strongly depend on the molecular cross-talk between cells and the surrounding microenvironment. Hence, some strategies were proposed to empower their beneficial effects during cell-therapeutic procedures to avoid confusing results. Licensing the cells with chemical compounds could be considered as one of the most applicable methods for induction of anti-inflammatory status in the cells. Human chorionic gonadotropin (hCG) is a pregnancy related hormone which has been shown to be essential for the establishment of a successful pregnancy. HCG supports the implantation of fetus in the maternal endometrium, due to its immunomodulatory effects. Moreover, the regulatory role of hCG has been previously mentioned in case of some autoimmune-based diseases. In the present study, the capacity of this hormone for induction of different immune-encountered genes expression was examined in primary cultures of human adipose tissue derived mesenchymal stem cells (Ad-MSCs). In this regard, Ad-MSCs were exposed to 10 IU of hCG for 72 hours. Molecular studies via quantitative Real-time PCR (qRT-PCR) experiments were performed to detect gene expression modifications based on the application of SYBR Green as the fluorescent dye and in comparison to the RPLP0 as the housekeeping gene. Results confirmed that hCG significantly upregulated TSG-6, TGF-β1, IL-1β and IL-6 expression levels comparing with the control group, while it downregulates COX-2 expression, and had no statistically significant effects on IL-10 andTDO2. In conclusion, priming Ad-MSCs with hCG may enhance the proliferation and immunoregulatory potential of these cells, although it needs further investigations to reveal involved molecular pathways.Ferdowsi University of MashhadJournal of Cell and Molecular Research2008-914711220200301Vitamin E Pretreatment of Mesenchymal Stem Cells: The Interplay of Oxidative Stress and Inflammation991072994410.22067/jcmr.v11i2.85668ENShadi MehrzadDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranSepideh Sadat HosseiniDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranMadjid Momeni-MoghaddamDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranMoien FarshchianStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR)-Khorasan Razavi, Mashhad, IranHalimeh HassanzadehStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR)-Khorasan Razavi, Mashhad, IranMahdi MirahmadiStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR)-Khorasan Razavi, Mashhad, IranFatemeh SadeghifarDepartment of Biology, Faculty of science, Hakim Sabzevari University, Sabzevar, IranHamid Reza BidkhoriStem Cells and Regenerative Medicine Research Department, Academic Center for Education, Culture and Research (ACECR)-Khorasan Razavi, Mashhad, IranJournal Article20200218 Oxidative stress occurs as a result of breaking down the balance between oxidants (e.g., reactive oxygen species (ROS)) and antioxidants in cells. Several studies have shown that there is a close relationship between oxidative stress and inflammation at the sites of injury. Mesenchymal stem cells (MSCs) are exposed to endogenous and exogenous oxidants generated during their ex vivo expansion or following in vivo transplantation. α-tocopherol (vitamin E) is a fat-soluble compound known for its anti-oxidant and anti-inflammatory properties. In many studies, the immunomodulatory effects of vitamin E have been observed in vivo. This study aimed to determine whether pretreatment of MSCs with antioxidants like vitamin E, will enhance the anti-inflammatory and immunomodulatory properties of these cells. For this purpose, adipose-derived MSCs (ASCs) were treated with vitamin E (600 μM) for 48 h. Quantitative PCR (qPCR) experiments were performed to evaluate the expression of genes related to inflammation (IL-1β, IL-6, IL-17, IL-10) or immunomodulation (TSG-6, COX-2, TDO2, TGF-β1). Results indicated that vitamin E significantly increased the expression of COX-2, TSG-6, and IL-1β genes at the mRNA level compared with the control group, while it significantly decreased IL-6 and TGF-β expressions. No effect was observed for IL-17, IL-10, and TDO2 genes. These results suggest that in vitro preconditioning of ASCs with vitamin E may allow the cells to improve their anti-inflammatory and immunoregulatory capacities. Vitamin E pretreatment could lead to the improvement of their therapeutic abilities in conditions that are influenced by oxidative stress.