Search

Abstract Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10–15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1–2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Extracellular vesicles (EVs) are enclosed by a lipid-bilayer membrane and secreted by all types of cells. They are classified into three groups: apoptotic bodies, microvesicles, and exosomes. Exosomes play a number of important roles in the intercellular communication and crosstalk between tissues in the body. In this study, we use three common methods based on different principles for exosome isolation, namely ultrafiltration, precipitation, and ultracentrifugation. We use field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) analyses for characterization of exosomes. The functionality and effect of isolated exosomes on the viability of hypoxic cells was investigated by alamarBlue and Flow-cytometry. The results of the FESEM study show that the ultrafiltration method isolates vesicles with higher variability of shapes and sizes when compared to the precipitation and ultracentrifugation methods. DLS results show that mean size of exosomes isolated by ultrafiltration, precipitation, and ultracentrifugation methods are 122, 89, and 60 nm respectively. AlamarBlue analysis show that isolated exosomes increase the viability of damaged cells by 11%, 15%, and 22%, respectively. Flow-cytometry analysis of damaged cells also show that these vesicles increase the content of live cells by 9%, 15%, and 20%, respectively. This study shows that exosomes isolated by the ultracentrifugation method are characterized by smaller size and narrow size distribution. Furthermore, more homogenous particles isolated by this method show increased efficiency of the protection of hypoxic cells in comparison with the exosomes isolated by the two other methods.

Abstract The immunotherapeutic application of interleukin-2 (IL-2) in cancer treatment is limited by its off-target effects on different cell populations and insufficient activation of anti-tumor effector cells at the site of the tumor upon tolerated doses. Targeting IL-2 to the tumor microenvironment by generating antibody-cytokine fusion proteins (immunocytokine) would be a promising approach to increase efficacy without associated toxicity. In this study, a novel nanobody-based immunocytokine is developed by the fusion of a mutant (m) IL-2 with a decreased affinity toward CD25 to an anti-vascular endothelial growth factor receptor-2 (VEGFR2) specific nanobody, denoted as VGRmIL2-IC. The antigen binding, cell proliferation, IFN-γ-secretion, and cytotoxicity of this new immunocytokine are evaluated and compared to mIL-2 alone. Furthermore, the pharmacokinetic properties are analyzed. Flow cytometry analysis shows that the VGRmIL2-IC molecule can selectively target VEGFR2-positive cells. The results reveal that the immunocytokine is comparable to mIL-2 alone in the stimulation of Primary Peripheral Blood Mononuclear Cells (PBMCs) and cytotoxicity in in vitro conditions. In vivo studies demonstrate improved pharmacokinetic properties of VGRmIL2-IC in comparison to the wild or mutant IL-2 proteins. The results presented here suggest VGRmIL2-IC could be considered a candidate for the treatment of VEGFR2-positive tumors.

IntroductionAntibiotic resistance and weak bioavailability of antibiotics in the skin due to systemic administration leads to failure in eradication of vancomycin- and methicillin-resistant Staphylococcus aureus (VRSA and MRSA)-associated wound infections and subsequent septicemia and even death. Accordingly, this study aimed at designing a photocrosslinkable methacrylated chitosan (MECs) hydrogel coated by melittin-derived peptide 1 (MDP1) that integrated the antibacterial activity with the promising skin regenerative capacity of the hydrogel to eradicate bacteria by burst release strategy.MethodsThe MECs was coated with MDP1 (MECs-MDP1), characterized, and the hydrogel-peptide interaction was evaluated by molecular docking. Antibacterial activities of MECs-MDP1 were evaluated against VRSA and MRSA bacteria and compared to MECs-vancomycin (MECs-vanco). Antibiofilm activity of MECs-MDP1 was studied by our novel ‘in situ biofilm inhibition zone (IBIZ)’ assay, and SEM. Biocompatibility with human dermal fibroblast cells (HDFs) was also evaluated.Results and DiscussionMolecular docking showed hydrogen bonds as the most interactions between MDP1 and MECs at a reasonable affinity. MECs-MDP1 eradicated the bacteria rapidly by burst release strategy whereas MECs-vanco failed to eradicate them at the same time intervals. Antibiofilm activity of MECs-MDP1 were also proved successfully. As a novel report, molecular docking analysis has demonstrated that MDP1 covers the structure of MECs and also binds to lysozyme with a reasonable affinity, which may explain the inhibition of lysozyme. MECs-MDP1 was also biocompatible with human dermal fibroblast skin cells, which indicates its safe future application. The antibacterial properties of a photocrosslinkable methacrylated chitosan-based hydrogel coated with MDP1 antimicrobial peptide were successfully proved against the most challenging antibiotic-resistant bacteria causing nosocomial wound infections; VRSA and MRSA. Molecular docking analysis revealed that MDP1 interacts with MECs mainly through hydrogen bonds with reasonable binding affinity. MECs-MDP1 hydrogels eradicated the planktonic state of bacteria by burst release of MDP1 in just a few hours whereas MECs-vanco failed to eradicate them. inhibition zone assay showed the anti-biofilm activity of the MECs-MDP1 hydrogel too. These findings emphasize that MECs-MDP1 hydrogel would be suggested as a biocompatible wound-dressing candidate with considerable and rapid antibacterial activities to prevent/eradicate VRSA/MRSA bacterial wound infections.

Abstract Decellularization by chemical approaches has harmful effects on extracellular matrix (ECM) proteins, and damages lots of functional peptides and biomolecules present in the ultrastructure. In this study, we employed a combination of chemical and physical decellularization methods to overcome these disadvantages. The induced osmotic pressure by hypertonic/hypotonic solutions dissociated and removed most of cellular membranes significantly without any detergent or chemical agent. In total, 0.025% trypsin solution was found adequate to remove the remaining debrides, and ultimately 1% Triton X-100 was utilized for final cleansing. In addition, conducting all the decellularization processes at 4 °C yielded an ECM with least damages in the ultrastructure which could be inferred by close mechanical strength and swelling ratio to the native vessel, and high quality and quantity of cell attachment, migration and proliferation which were examined by optical microscopy and scanning electron microscopy (SEM) of the histology samples. Moreover, the obtained biological scaffold (BS) had no cytotoxicity according to the MTT assay, and this scaffold is storable at −20 °C. Employing bioreactor for concurrent cyclic tensile and shear stresses improved the cell migration into pores of the BS and made the cells and the scaffold compact in analogous to native tissue. As opening angle test showed by decellularizing of the blood vessel, the residual stress dropped significantly which revealed the role of cells in the amount of induced stress in the structure. However, intact and healthy ECM explicitly recovered upon recellularization and beat the initial residual stress of the native tissue. The tensile test of the blood vessels in longitudinal and radial directions revealed orthotropic behavior which can be explained by collagen fibers direction in the ECM. Furthermore, by the three regions of the stress–strain curve can be elucidated the roles of cells, elastin and collagen fibers in mechanical behavior of the vascular tissues. Graphical Abstract

Objective(s): Bioglass scaffolds, which contain a significant percentage of porosity for tissue engineering purposes, have low strength. For increasing the strength and efficiency of such structures for use in tissue engineering, fabrication of hierarchical meso/macro-porous bioglass scaffolds, developing their mechanical strength by hydrothermal treatment and adjusting pH method, and achieving the appropriate mesopore size for loading large biomolecules, were considered in this study.Materials and Methods: Mesoporous bioglass (MBG) powders were synthesized using cetyltrimethylammonium bromide as a surfactant, with different amounts of calcium sources to obtain the appropriate size of the mesoporous scaffolds. Then MBG scaffolds were fabricated by a polyurethane foam templating method, and for increasing scaffold strength hydrothermal treatment (90 °C, for 5 days) and adjustment pH (pH=9) method was used to obtain hierarchical meso/macro-porous structures. The sample characterization was done by Simultaneous thermal analysis, Fourier transform infrared spectroscopy, Field Emission Scanning electron microscopy, small and wide-angle X-ray powder diffractions, transmission electron microscopy, and analysis of nitrogen adsorption-desorption isotherm. The mechanical strength of scaffolds was also determined.Results: The MBG scaffolds based on 80.28 (wt.) % SiO2- 17.89 (wt.) % CaO- 1.81 (wt.) % P2O5 presented interconnected large pores and pores in the range of 100-150 μm and 6-18 nm, respectively and 0.4 MPa compressive strength. Conclusion: The total pore volume and specific surface area were obtained from the Brunauer-Emmett-Teller theory, 0.709 cm3 g-1 and 213.83 m2 g-1, respectively. These findings could be considered in bone-cartilage tissue engineering.

Objective(s): The high resistance rate of Acinetobacter baumannii and the limited number of available antibiotics have prompted a worldwide effort to develop effective antimicrobial agents. Accordingly, identifying single-chain variable fragment antibodies (scFvs), capable of exerting direct antibacterial activity in an immune system-independent manner, may be making immunocompromised patients more susceptible to A. baumannii infections.Materials and Methods: To isolate bactericidal scFvs targeting A. baumannii, we panned a large human scFv phage display library against whole-cell extensively drug-resistant (XDR) A. baumannii strains grown as biofilm or cultured with human blood or human peripheral blood mononuclear cells plus plasma. The binding of scFv-phages to A. baumannii was assessed by the dot-blot assay. Soluble scFvs, derived from the selected phages, were assessed based on their ability to bind and inhibit the growth of A. baumannii. Results: Five phage clones showed the highest reactivity toward A. baumannii. Among five soluble scFvs, derived from positive phage clones, two scFvs, EB211 and EB279, had high expression yields and displayed strong binding to A. baumannii compared with the controls. Moreover, XDR A. baumannii strains treated with positively-charged scFvs, including EB211, EB279, or a cocktail of EB211 and EB279 (200 µg/ml), displayed lower viability (approximately 50%, 78%, and 40% viability, respectively) compared with PBS-treated bacteria.Conclusion: These results suggest that combining last-resort antibiotics with bactericidal scFvs could provide promising outcomes in immunocompromised individuals with A. baumannii infections.

Abstract Recombinant production of mussel foot proteins among marine-inspired proteinaceous adhesive materials has been attracted high attention for medical applications, due to their exceptional versatility potential of hierarchically arranged nanostructures. Various biochemical and proteinous factors such as amyloid CsgA curli protein have been used as a synergistic factor to enhance the constancy of obtained bio-adhesion but their mechanistic interactions have not yet been deeply investigated widely in different pH conditions. To this end, the present study has first sought to assess molecular simulation and prediction by using RosettaFold to predict the 3-dimensional structure of the fused CsgA subunit and the MFP3 protein followed by in vitro verification. It was developed an ensemble of quantitative structure-activity relationship models relying on simulations according to the surface area and molecular weight values of the fused proteins in acidic to basic situations using PlayMolecule (protein preparation app for MD simulations) online databases followed by molecular dynamic simulation at different pHs. It was found that acidic conditions positively affect adhesive strength throughout the chimeric structure based on comparative structure-based analyses along with those obtained in prevailing literature. Atomic force microscopy analysis was confirmed obtained in silico data which showed enhanced adhesive properties of fused protein after self-assembly in low pH conditions. In conclusion, the augmented model for reactivity predictions not only unravels the performance and explain ability of the adhesive proteins but in turn paves the way for the decision-making process for chimeric subunits modifications needed for future industrial production.

Abstract Chitosan/gelatin‐based nanofibers display excellent biological performance in tissue engineering because of their biocompatible composition and nanofibrous structure with a high surface‐to‐volume ratio mimicking the native extracellular matrix. In this study, to save time and cost of experiments, a response surface methodology based on Box–Behnken design (BBD) is developed to predict the mean diameter of (chitosan:gelatin)/poly(vinyl alcohol) (PVA) nanofibers in three volume ratios of chitosan:gelatin by considering PVA percentage, applied voltage, and flow rate as input variables. The morphology and chemical composition of nanofibers are investigated through scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. The optimum conditions to yield the minimum diameter of nanofibers with chitosan:gelatin ratios of 25:75, 50:50, and 75:25 are found and result in 165, 121, and 92 nm, respectively, which show good accordance with BBD estimated results. The tensile testing indicates that nanofibers containing higher ratio of chitosan:gelatin result in higher tensile stress and lower toughness and tensile strain. The water contact angle analysis (WCA) shows the appropriate hydrophilicity of crosslinked nanofibers. The MTT assay shows excellent cell viability and cell attachment of nanofibers for mouse fibroblast (L929) cells. The results indicate that optimum nanofibers are potent candidates for wound healing applications.

Abstract Methicillin resistance Staphylococcus aureus bacteria (MRSA) are serious hazards of bone implants. The present study was aimed to use the potential synergistic effects of Melittin and tetracycline to prevent MRSA associated bone implant infection. Chitosan/bioactive glass nanoparticles/tetracycline composite coatings were deposited on hydrothermally etched titanium substrate. Melittin was then coated on composite coatings by drop casting method. The surfaces were analyzed by FTIR, XRD, and SEM instruments. Tetracycline in coatings revealed multifunctional behaviors include bone regeneration and antibacterial activity. Releasing ALP enzyme from MC3T3 cells increased by tetracycline, so it is suitable candidate as osteoinductive and antibacterial agent in orthopedic implants coatings. Melittin increased the proliferation of MC3T3 cells. Composite coatings with combination of tetracycline and Melittin eradicate all MRSA bacteria, while coatings with one of them could no t eradicate all of the bacteria. In conclusion, chitosan/bioactive glass/tetracycline/Melittin coating can be suggested as a multifunctional bone implant coating because of its osteogenic and promising antibacterial activity. Graphical abstract

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, which selectively target and kill cancer cells while sparing normal ones. Among them, engineered Herpes simplex virus type 1 (HSV-1) has been proposed as a potential treatment for cancer and was moved to phase III clinical trials. Previous studies showed that design of OV therapy combined with p53 gene therapy increases the anti-cancer activities of OVs. Here, the UL39 gene of the ICP34.5 deleted HSV-1 was manipulated with the insertion of the EGFP-p53 expression cassette utilizing CRISPR/ Cas9 editing approach to enhance oncoselectivity and oncotoxicity capabilities. The ΔUL39/Δγ34.5/HSV1-p53 mutant was isolated using the chorioallantoic membrane (CAM) of fertilized chicken eggs as a complementing membrane to support the growth of the viruses with gene deficiencies. Comparing phenotypic features of ΔUL39/Δγ34.5/HSV1-p53-infected cells with the parent Δγ34.5/HSV-1 in vitro revealed that HSV-1-P53 had cytolytic ability in various cell lines from different origin with different p53 expression rates. Altogether, data presented here illustrate the feasibility of exploiting CAM model as a promising strategy for isolating recombinant viruses such as CRISPR/Cas9 mediated HSV-1-P53 mutant with less virus replication in cell lines due to increased cell mortality induced by exogenous p53.

Despite the fact that the new drugs and targeted therapies have been approved for cancer therapy during the past 30 years, the majority of cancer types are still remain challenging to be treated. Due to the tumor heterogeneity, immune system evasion and the complex interaction between the tumor microenvironment and immune cells, the great majority of malignancies need multimodal therapy. Unfortunately, tumors frequently develop treatment resistance, so it is important to have a variety of therapeutic choices available for the treatment of neoplastic diseases. Immunotherapy has lately shown clinical responses in malignancies with unfavorable outcomes. Oncolytic virus (OV) immunotherapy is a cancer treatment strategy that employs naturally occurring or genetically-modified viruses that multiply preferentially within cancer cells. OVs have the ability to not only induce oncolysis but also activate cells of the immune system, which in turn activates innate and adaptive anticancer responses. Despite the fact that OVs were translated into clinical trials, with T-VECs receiving FDA approval for melanoma, their use in fighting cancer faced some challenges, including off-target side effects, immune system clearance, non-specific uptake, and intratumoral spread of OVs in solid tumors. Although various strategies have been used to overcome the challenges, these strategies have not provided promising outcomes in monotherapy with OVs. In this situation, it is increasingly common to use rational combinations of immunotherapies to improve patient benefit. With the development of other aspects of cancer immunotherapy strategies, combinational therapy has been proposed to improve the anti-tumor activities of OVs. In this regard, OVs were combined with other biotherapeutic platforms, including various forms of antibodies, nanobodies, chimeric antigen receptor (CAR) T cells, and dendritic cells, to reduce the side effects of OVs and enhance their efficacy. This article reviews the promising outcomes of OVs in cancer therapy, the challenges OVs face and solutions, and their combination with other biotherapeutic agents.

Polydopamine (PDA) is a biocompatible cell-adhesive polymer with versatile applications in biomedical devices. Previous studies have shown that PDA coating could improve cell adhesion and differentiation of human mesenchymal stem cells (hMSCs). However, there is still a knowledge gap in the effect of PDA-mediated protein adsorption on the epigenetic status of MSCs. This work used gelatin-coated cell culture surfaces with and without PDA underlayer (Gel and PDA-Gel) to culture and differentiate primary human adipose-derived stem cells (hASCs). The properties of these two substrates were significantly different, which, in combination with a variation in extracellular matrix (ECM) protein bioactivity, regulated cell adhesion and migration. hASCs reduced focal adhesions by downregulating the expression of integrins such as αV, α1, α2, and β1 on the PDA-Gel compared to the Gel substrate. Interestingly, the ratio of H3K27me3 to H3K27me3+H3K4me3 was decreased, but this only occurred for upregulation of AGG and BMP4 genes during chondrogenic differentiation. This result implies that the PDA-Gel surface positively affects the chondrogenic, but not adipogenic and osteogenic, differentiation. In conclusion, for the first time, this study demonstrates the sequential effects of PDA coating on the biophysical property of adsorbed protein and then focal adhesions and differentiation of hMSCs through epigenetic regulation. This study sheds light on PDA-mediated mechanotransduction.

Background and purpose: Some skin injuries are caused by occupational skin exposures that are resulted from production of reactive oxygen species and oxidative stress (ROS). Thus, protecting the skin is a key issue. Recently, several surveys have introduced novel alternatives such as nanofibers for skin protection. In this research, Poly (ε-caprolactone) (PCL)/gelatin (Gt)/ Vitamin E (VE) was investigated as a nanofiber antioxidant protective layer in occupational skin exposures. Materials and methods: Vitamin E was combined with PCL/Gt nanofiber mats by electrospinning method. The diameter of nanofibers and chemical characteristics were determined using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Adhesion characteristics and proliferation of human dermal fibroblast cells (HDFC) on nanofibers were analyzed by Methylthiazol Tetrazolium (MTT) assay. Finally, we studied the application of PCL/Gt/VE nanofibers against oxidation stress induced by tert-butyl hydroperoxide (t-BHP). Results: With increase in Vitamin E levels, the PCL/Gt/VE nanofibers were found to be thiner. The culture of HDF cells on nanofibers showed that adding Vitamin E to PCL/Gt mats caused increased attachment and proliferation of the cells. PCL/Gt/VE nanofibers significantly assisted human skin cells against ROS (P< 0.001). Conclusion: According to current study, PCL/Gt/VE nanofibers could be an appropriate candidate for protecting skin against ROS as a novel method to maintain workers’ health.

Objective Chimeric animal exhibits less viability and more fetal and placental abnormalities than normal animal. This study was aimed to determine the impact of mouse embryonic stem cells (mESCs) injection into the mouse embryos on H3K9me3 and H3K4me3 and cell lineage gene expressions in chimeric blastocysts. Materials And Methods In our experiment, at the first step, incorporation of the GFP positive mESCs (GFP-mESCs) 129/Sv into the inner cell mass (ICM) of pre-compacted and compacted morula stage embryos was compared. At the second and third steps, H3K4me3 and H3K9me3 status as well as the expression of Oct4, Nanog, Tead4, and Cdx2 genes were determined in the following groups: i. In vitro blastocyst derived from in vivo morula subjected to mESCs injection (blast/chimeric), ii. In vivo derived blastocyst (blast/in vivo), iii. In vitro blastocyst derived from culture of morula in vivo (blast/morula), and iv. In vitro blastocyst derived from morula in vivo subjected to sham injection (blast/sham). Results Subzonal injection of GFP-mESCs at the pre-compacted embryos produced more chimeric blastocysts than compacted embryos (P

Although high-dose IL-2 has clear antitumor effects, severe side effects like severe toxicity and activation of Tregs by binding of IL-2 to high-affinity IL-2R, hypotension, and vascular leak syndrome limit its applications as a therapeutic antitumor agent. Here in this study, a rational computational approach was employed to develop and design novel triple-mutant IL-2 variants with the aim of improving IL-2-based immunotherapy. The affinity of the mutants towards IL-2Rα was further computed with the aid of molecular dynamic simulations and umbrella sampling techniques and the obtained results were compared to those of wild-type IL-2. In vitro experiments by flow cytometry showed that the anti-CD25 mAb was able to bind to PBMC cells even after mutant 2 preincubation, however, the binding strength of the mutant to α-subunit was less than of wtIL-2. Additionally, reduction of IL-2Rα subunit affinity did not significantly disturb IL-2/IL2Rβγc subunits interactions.

The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.

Differentiation of stem cells to Schwann is considered efficient way for nerve regeneration since the sources of human Schwann cells are limited for clinical application. It is demonstrated that mimicking micromechanical forces or micro/nanotopographical environments that stem cells are experienced in vivo could control their fate. Here, the potency of substrates with imprinted cell-like topographies for direct differentiation of adipose-derived mesenchymal stem cells (ADSCs) into Schwann cells (SCs) is reported. For the preparation of substrates with imprinted SC-Like topographies, SCs are isolated from the sciatic nerve, grown, fixed, and then SC morphologies are transferred to polydimethylsiloxane (PDMS) substrates by mold casting. Subsequently, mesenchymal stem cells (MSCs) are seeded on the SC-imprinted substrates and their differentiation to SCs is evaluated by immunocytochemistry, real-time PCR, and western blotting. Analysis of morphology and expression of SC-specific markers show that MSCs cultured on the imprinted substrates have the typical SC-like morphology and express SC-specific markers including S100b, p75NTR, and Sox10. It is believed that specific cell-like topographies and related micromechanical cues can be sufficient for direct differentiation of ADSCs into Schwann cells by cell-imprinting method as a physical technique.

Objective Mycoplasmas spp. is among major contaminants of eukaryotic cell cultures. They cause a wide range of problems associated with cell culture in biology research centers or biotechnological companies. Mycoplasmas are also resistant to several antibiotics. Plasmocin™ has been used to treat cell lines but Plasmocin™-resistant strains have been reported. InvivoGen has developed a new anti-Mycoplasma agent called Plasmocure™ in order to eliminate resistant Mycoplasma contamination. The aim of this study was the selection of the best antibiotics for treatment of mycoplasma in cell cultures. Materials And Methods In this experimental study, a total of 100 different mammalian cell lines contaminated with different Mycoplasma species were evaluated by microbiological culture (as the gold standard method), indirect DNA fluorochrome staining, enzymatic (MycoAlert™), and universal or species-specific polymerase chain reaction (PCR) detection methods. In this study, animal and human cell lines available in National Cell Bank of Iran, were treated with Plasmocure™. The treatment efficacy and cytotoxicity of Plasmocure™ were compared with those of commonly used antibiotics such as BM- cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. Results Plasmocure™ is comprised of two antibiotics that act through various mechanisms of action than those in Plasmocin™. Two-week treatment with Plasmocure™ was enough to completely eliminate Mycoplasma spp. A moderate toxicity was observed during Mycoplasma treatment with plasmocure™; But, after elimination of Mycoplasma, cells were fully recovered. Mycoplasma infections were eliminated by Plasmocure™, BM-cyclin, Plasmocin™, MycoRAZOR™, sparfloxacin and enrofloxacin. However, the outcome of the treatment process (i.e. the frequency of complete cure, regrowth or cell death) varied among different antibiotics. Conclusion The highest number of cured cell lines was achieved by using Plasmocure™ which also had the lowest regrowth rate after a period of four months. As a conclusion; Plasmocure™ might be considered an effective antibiotic to treat Mycoplasma infections in mammalian cell cultures especially for precious or vulnerable cells.

The increasing incidences of cancer at the global scale have recently resulted in the invention of various biotechnology approaches among which the oncolytic virotherapy is a new strategy for the treatment of multiple tumors. Herpes simplex virus (HSV) based vectors are one of the most studied oncolytic agents, worldwide. Moreover, syngeneic animal models are the principal parts of the oncolytic virotherapies investigation. The effects of a dual fluorescent γ34.5 deleted vector-HSV-GR- on three mouse tumor cell lines were studied in this work. We previously generated a dual fluorescent labeled oncolytic HSV-HSV-GR- (both copies of γ34.5 were inactivated by insertion of two distinct fluorescent dyes, GFP and mCherry) in our laboratory; subsequently, they were used as oncolytic viruses. The three 4T1, TC-1, and CT26 cell lines were infected with HSV-GR. The infection efficacy and the elimination potency of HSV-GR were analyzed by photomicrography and flow cytometry methods. HSV-GR showed a significant efficiency to infect the cell lines examined. Flow cytometry analyses demonstrated that HSV-GR infected 89.3%, 86.1%, and 92.4% of 4T1, TC-1, and CT26 cells, respectively. Moreover, propidium iodide (PI) staining of infected cells indicated that HSV-GR could kill 27.9%, 21.2%, and 21.3% of 4T1, TC-1, and CT26 cells, respectively. Interestingly, HSV-GR infected cells were capable of expressing both GFP and mCherry at the same time. The promising effects of the oncolytic virus HSV-GR in the mouse syngeneic tumor cell system have shed more light on the therapeutic potential of this anti-cancer approach.

Despite the advent of promising technologies in tissue engineering, finding a biomimetic 3D bio-construct capable of enhancing cell attachment, maintenance, and function is still a challenge in producing tailorable scaffolds for bone regeneration. Here, osteostimulatory effects of the butterfly wings as a naturally porous and non-toxic chitinous scaffold on mesenchymal stromal cells are assessed. The topographical characterization of the butterfly wings implied their ability to mimic bone tissue microenvironment, whereas their regenerative potential was validated after a 14-day cell culture. In vivo analysis showed that the scaffold induced no major inflammatory response in Wistar rats. Topographical features of the bioconstruct upregulated the osteogenic genes, including COL1A1, ALP, BGLAP, SPP1, SP7, and AML3 in differentiated cells compared to the cells cultured in the culture plate. However, butterfly wings were shown to provide a biomimetic microstructure and proper bone regenerative capacity through a unique combination of various structural and material properties. Therefore, this novel platform can be confidently recommended for bone tissue engineering applications.

Background: Basic fibroblast growth factor (FGF2) is one of the most potent proangiogenic proteins involving in tumor angiogenesis. Interaction of heparin with FGF2 and FGF2 receptor (FGFR1) form a ternary complex that is prerequisite for FGF2 proangiogenic activity. Therefore, this interaction can be an important target for inhibition of angiogenesis. Material and Method: In this study, we performed screening studies by computer-aided techniques to find a small molecule interfering with this interaction. Based on ionic interactions, we found seventeen small molecules which had the capability of angiogenic inhibition. According to important negative charge distances, benzene-1, 2, 4-tricarboxylic acids, known as Trimellitic acid (TMLA), was chosen. MTT viability test, real time PCR, tube formation assay, and Flowcytometry technique were used to evaluate TMLA effect. Results: Here, the viability of HUVECs was decreased following exposure to TMLA only at high concentrations. According to real time PCR, gene expression was dramatically decreased in comparison to negative control, indicating that TMLA is an effective agent on reduction of CD31 expression. At half maximal inhibitory concentration (13mM), TMLA inhibited HUVEC tube formation process. Annexin V-FITC / PI flow cytometry technique revealed that TMLA inhibitory effect was via apoptosis. Conclusion: With respect to findings of the present study, it is possible to extend FGF2 inhibitors with much specificity based on ionic interactions strategy.

Objective Most people experience bone damage and bone disorders during their lifetimes. The use of autografts is a suitable way for injury recovery and healing. Mesenchymal stem cells (MSCs) are key players in tissue engineering and regenerative medicine. Their proliferation potential and multipotent differentiation ability enable MSCs to be considered as appropriate cells for therapy and clinical applications. Differentiation of stem cells depends on their microenvironment and biophysical stimulations. The aim of this study is to analyze the effects of an electromagnetic field on osteogenic differentiation of stem cells. Materials and Methods In this experimental animal study, we assessed the effects of the essential parameters of a pulsatile electromagnetic field on osteogenic differentiation. The main purpose was to identify an optimum electromagnetic field for osteogenesis induction. After isolating MSCs from male Wistar rats, passage-3 (P3) cells were exposed to an electromagnetic field that had an intensity of 0.2 millitesla (mT) and frequency of 15 Hz for 10 days. Flow cytometry analysis confirmed the mesenchymal identity of the isolated cells. Pulsatile electromagnetic field-stimulated cells were examined by immunocytochemistry and real-time polymerase chain reaction (PCR). Results Electromagnetic field stimulation alone motivated the expression of osteogenic genes. This stimulation was more effective when combined with osteogenic differentiation medium 6 hours per day for 10 days. For the in vivo study, an incision was made in the cranium of each animal, after which we implanted a collagen scaffold seeded with stimulated cells into the animals. Histological analysis revealed bone formation after 10 weeks of implantation. Conclusion We have shown that the combined use of chemical factors and an electromagnetic field was more effective for inducing osteogenesis. These elements have synergistic effects and are beneficial for bone tissue engineering applications.

Current therapies for acute myeloid leukemia (AML), are associated with high relapse rates. Hence, development of new therapeutic strategies is crucial to circumvent this problem. Bivalent antibody technology has been used to engineer novel antibody fragments with increased avidity, by assembling two scFv in a single molecule. Here, we present accompanying data from construction and characterization experiments of a biscFv antibody targeting CD123, the most important biomarker of leukemic cancer stem cells which play a key role in relapsed AML after chemotherapy. Data in this article are related to the research paper “Development of a novel engineered antibody targeting human CD123” Moradi-Kalbolandi S. et al. (2016) [1].

Background: Pasteurella multocida is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate. Materials and Methods: In this study, the fusion protein (PlpE1 + 2 + 3) and full PlpE genes (PlpE-Total) were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 Escherichia coli. Overlap polymerase chain reaction (PCR) using different primers for 5ˈ and 3ˈ end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning. Results: Cloning of both PlpE1 + 2 + 3 and PlpE-Total into the pET28a vector and their transform into the BL21 (DE3) E. coli host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration. Conclusion: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 E. coli have resulted in sufficient expression, which shows that Top10 E. coli may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

Objective(s): Study of non-coding RNAs is considerable to elucidate principal biological questions or design new therapeutic strategies. miRNAs are a group of non-coding RNAs that their functions in PI3K/AKT signaling and apoptosis pathways after T cell activation is not entirely clear. Herein, miRNAs expression and their putative targets in the mentioned pathways were studied in the activated CD4+T cells. Materials and Methods: Herein, proliferation rate and IL-2 secretion were measured in treated and untreated cells by IL-2. Putative targets of up-regulated miRNAs were predicted by bioinformatics approaches in the apoptotic and PI3K/AKT signaling pathways. Then the expression of two putative targets was evaluated by quantitative RT-PCR. Results: Proliferation rate of treated cells by IL-2 increased in a dose- and time- dependent manner. Naive and activated CD4+T cells induced by different dose of IL-2 secreted abundant amounts of IL-2. Also, in IL-2 un-induced cells (IL-2 depleted cells) after 3 days, decrease of proliferation has been shown. In silico analysis predicted putative targets of up-regulated miRNAs such as AKT1, AKT3 and apoptotic genes in the activated cells induced or un-induced by IL-2. Decrease of AKT3 was shown by Q-RT-PCR as a potential target of miRNAs overexpressed in IL-2 depleted cells. But there was no significant difference in AKT1 expression in two cell groups. Conclusion: Our analysis suggests that decrease of AKT3 was likely controlled via up-regulation of specific miRNAs in IL-2 depleted cells. Also it seems that miRNAs play role in induction of different apoptosis pathways in IL-2 induced and un-induced cells.

Objective(s): To improve water stability of electrospun chitosan/ Polyethylene oxide (PEO) nanofibers, genipin, a biocompatible and nontoxic agent, was used to crosslink chitosan based nanofibers. Materials and Methods: Different amounts of genipin were added to the chitosan/PEO solutions, chitosan/PEO weight ratio 90/10 in 80 % acetic acid, and the solutions were then electrospun to form nanofibers. The spun nanofibers were exposed to water vapor to complete crosslinking. The nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, swelling test, MTT cytotoxicity, and cell attachment. Results: SEM images of electrospun mats showed that genipin-crosslinked nanofibers retained their fibrous structure after immerging in PBS (pH=7.4) for 24 hours, while the uncrosslinked samples lost their fibrous structure, indicating the water stability of genipin-crosslinked nanofibers. The genipin-crosslinked mats also showed no significant change in swelling ratio in comparison with uncrosslinked ones. FTIR-ATR spectrum of uncrosslinked and genipin-crosslinked chitosan nanofibers revealed the reaction between genipin and amino groups of chitosan. Cytotoxicity of genipin-crosslinked nanofibers was examined by MTT assay on human fibroblast cells in the presence of nanofibers extraction media. The genipin-crosslinked nanofibers did not show any toxic effects on fibroblast cells at the lowest and moderate amount of genipin. The fibroblast cells also showed a good adhesion on genipin-crosslinked nanofibers. Conclusion: This electrospun matrix would be used for biomedical applications such as wound dressing and scaffold for tissue engineering without the concern of toxicity.

Nanofibrous structures mimicking the native extracellular matrix have attracted considerable attention for biomedical applications. The present study aims to design and produce drug-eluting core-shell fibrous scaffolds for wound healing and skin tissue engineering. Aloe vera extracts were encapsulated inside polymer fibers containing chitosan, polycaprolactone, and keratin using the co-axial electrospinning technique. Electron microscopic studies show that continuous and uniform fibers with an average diameter of 209 ± 47 nm were successfully fabricated. The fibers have a core-shell structure with a shell thickness of about 90 nm, as confirmed by transmission electron microscopy. By employing Fourier-transform infrared spectroscopy, the characteristic peaks of Aloe vera were detected, which indicate successful incorporation of this natural herb into the polymeric fibers. Tensile testing and hydrophilicity measurements indicated an ultimate strength of 5.3 MPa (elongation of 0.63%) and water contact angle of 89°. In-vitro biological assay revealed increased cellular growth and adhesion with the presence of Aloe vera without any cytotoxic effects. The prepared core-shell fibrous mats containing medical herbs have a great potential for wound healing applications.

Background: Anaphase promoting complex (APC) plays a critical role in cell division and mitotic exit. This protein complex may have a pivotal role in the cell cycle control affecting pathological conditions such as cancer. APC is recommended as the target of many anti-cancer agents due to its importance in cancer pathogenesis. This study aimed to evaluate the inhibitory effects of curcumin and 2-methoxyestradiaul on APC. Methods: Hela cells were treated with various concentrations of curcumin and 2-methoxyestradiaul, and their cytotoxic effects were investigated after 24, 48, 72 and 96 hours by MTT assay. Expression of securin, cyclin B and the APC substrates were investigated using immune-blotting. Finally, cell cycle analysis was performed using DRAQ5 staining and flowcytometry. Findings: Respectively, 20 and 30 percent of cell-death after 24-hour treatment with curcumin and 2-methoxyestradiaul was seen. Also, securin and cyclin levels were raised after 24 hours of treatment. Furthermore, the levels of the substrates (securin and cyclin B) increased 48 hours after treatment. Results of fluorescence-activated cell sorting (FACS) analysis showed that treated cells were arrested in G2 phase of cell cycle which revealed cell arrest in G2 phase of cell cycle. 2-methoxyestradiaul showed a better APC inhibition effect than curcumin (P = 0.02). Conclusions: Our results revealed that APC is a suitable target for cancer treatment and curcumin and 2-methoxyestradiaul have inhibitory effects on the activity of this complex.

Background: Crimean Congo hemorrhagic fever virus (CCHFV) is a member of the Bunyaviridae family and Nairovirus genus. It has a negative-sense, single stranded RNA genome approximately 19.2 kb, containing the Small, Medium, and Large segments. CCHFVs are relatively divergent in their genome sequence and grouped in seven distinct clades based on S-segment sequence analysis and six clades based on M-segment sequences. Our aim was to obtain new insights into the molecular epidemiology of CCHFV in Iran. Methods: We analyzed partial and complete nucleotide sequences of the S and M segments derived from 50 Iranian patients. The extracted RNA was amplified using one-step RT-PCR and then sequenced. The sequences were ana­lyzed using Mega5 software. Results: Phylogenetic analysis of partial S segment sequences demonstrated that clade IV-(Asia 1), clade IV-(Asia 2) and clade V-(Europe) accounted for 80 %, 4 % and 14 % of the circulating genomic variants of CCHFV in Iran respectively. However, one of the Iranian strains (Iran-Kerman/22) was associated with none of other sequences and formed a new clade (VII). The phylogenetic analysis of complete S-segment nucleotide sequences from selected Ira­nian CCHFV strains complemented with representative strains from GenBank revealed similar topology as partial sequences with eight major clusters. A partial M segment phylogeny positioned the Iranian strains in either associa­tion with clade III (Asia-Africa) or clade V (Europe). Conclusion: The phylogenetic analysis revealed subtle links between distant geographic locations, which we pro­pose might originate either from international livestock trade or from long-distance carriage of CCHFV by infected ticks via bird migration.

Background: Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect.Methods: Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit.Results: The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. Conclusion: The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties.

Tissue-engineered gingival graft was used for regenerating facial gingiva around an implant at lower left first premolar area with insufficient attached gingiva. Engineered gingival graft was produced by mixing 250 ml bovine skin collagen with 250 ml nutritional medium containing human gingival fibroblasts (2×105). 3 months post-surgery, there were gains in the attached gingiva compared to pre-surgery. The histological examination revealed a keratinized tissue on the treated site. Based on the result of this investigation, this graft was safe and capable of generating keratinized gingiva.

Objective: Mesenchymal stem cells (MSCs) can be expanded and differentiated intomany mature cell types including smooth muscle cells (SMCs). In addition to growth factor,cyclic stretch contributes to differentiation of stem cells. Mechanical stimuli are criticalto morphological changes, development, regeneration, differentiation and pathology ofmesenchymal tissues. The aim of this study is to investigate effects of cyclic stretch withdiffering amplitudes on morphology and differentiation of mesenchymal stem cells.Materials and Methods: Mesenchymal stem cells are extracted from human bone marrow.Cells are cultured on silicone membrane and exposed to cyclic stretch by a custommade device. Cellular images are captured before and after tests. Effects of 5% and 15%uniaxial strain with 1Hz frequency and 1-8 hour durations on morphology of human mesenchymalstem cells are investigated. It is assumed that environmental factors such asmechanical loading regulate MSCs differentiation to SMCs. Fractal analysis is used toquantify alterations in cellular morphology. An image processing method with a designedcode is used for evaluation of fractal dimension parameter.Results: Results demonstrate statistically significant change in cell morphology due tomechanical stretch. By elevation of strain amplitude and number of load cycles, fractaldimensions of cell images decrease. Such decrease is equivalent to alignment of cells bymechanical stimulus. Cells are differentiated to SMCs purely by cyclic stretch. The initiationand rate of differentiation depend on mechanical conditions.Conclusion: To produce functional SMCs for engineered tissues, MSCs can be exposed to uniaxialcyclic stretch. The functionality of differentiated SMCs depends on loading conditions.

Objective: Helicobacter pylori (H. pylori) cytotoxin and its heterogeneity amongst strains hasbeen closely linked to the varying infection-associated clinical outcomes. In order to determinethe decisive role of the vacuolating cytotoxin (vacA) gene mosaicism in its corresponding geneexpression and phenotype, we aimed to characterize vacA alleles of different H. pylori strainsin addition to the resulting protein and its vacuolating activity in epithelial cell culture.Materials and Methods: vacA gene polymorphism was determined for 80 H. pylori strainsisolated from dyspeptic patients, using multiplex gene-specific polymerase chain reaction(PCR). VacA protein was detected by immuno-blotting assay using a polyclonal anti-VacAantibody. In vitro cytotoxicity assay was conducted on HeLa cells in order to evaluate thevacuolating cytotoxin activity.Results: Genotyping revealed the following strain distribution: 26 (32.5%) s1m1, 35(43.8%) s1m2, and 19 (23.8%) s2m2 subtypes. Infection with s1m1 type strain was significantlyassociated with gastric cancer as compared to non-ulcer dyspepsia (p=0.005)and peptic ulcer disease (p=0.008). A 95-kDa immuno-reactive band that represented thevacuolating toxin was demonstrated in SDS-PAGE analysis of concentrated culture filtrate(CCF) of H. pylori strains. H. pylori CCFs induced HeLa cell vacuolation which correlatedwith the strain genotype; s1m1 strains demonstrated higher levels of vacuolation as comparedto s1m2 strains, whereas s2m2 strains showed no detectable cytotoxic activity.Conclusion: The current study confirmed the relatively high cytotoxic activity of s1m1type H. pylori strains which infect the majority of patients suffering from gastric cancer andmay be partly responsible for the pathogenesis of this mortal disease.

Objective: This study was performed to determine whether melatonin atphysiological concentrations (0.01-10nM) could affect the proliferation andosteogenic differentiation of Rat ADSCs in vitro.Materials and Methods: ADSCs were isolated from the fat of adult rats. Aftercell expansion in culture media and through three passages, osteogenesiswas induced on a monolayer culture with osteogenic medium with or withoutmelatonin at physiological concentrations (0.01-10nM). After 4 weeks cultureswere examined for mineralization by Alizarin Red S and von Kossa staining andfor alkaline phosphatase (ALP) activity by ALP kit. Cell viability and apoptosiswere also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-ulfophenyl)-2H-tetrazolium assay and flowcytometry, respectively. Allassays were performed in triplicate.Results: The results indicated that at physiological concentrations, melatoninsuppressed proliferation and differentiation of ADSCs. These data indicate thatADSCs exposed to melatonin, had a lower ALP activity in contrast to the cellsexposed to the osteogenic medium alone. Similarly, the mineral deposition(calcium level) also decreased. The flow cytometry proved that the cell growthdecreased and the apoptotic cells increased.Conclusion: These results suggest that physiological concentration ofmelatonin has a negative effect on ADSCs osteogenesis.

Background: Osteogenesis driven by adipose-derived stem cells (ADSCs) is regulated by physiological and pathological factors. Accumulating evidence from in vitro and in vivo experiments suggests that melatonin may have an influence on bone formation. However, little is known about the effects of melatonin on osteogenesis, which thus remains to be elucidated. This study was performed to determine whether melatonin at physiological concentrations (0.01-10 nM) could affect the in vitro proliferation and osteogenic differentiation of rat ADSCs.Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced in a monolayer culture using osteogenic medium with or without melatonin at physiological concentrations (0.01-10 nM). After four weeks, the cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity using an ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTT) assay and flow cytometry, respectively.Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to osteogenic medium alone. Similarly, mineral deposition (calcium level) also decreased in the presence of melatonin. Flow cytometry confirmed that cell growth had decreased and that the numbers of apoptotic cells had increased.Conclusion: These results suggest that the physiological concentration of melatonin has a negative effect on ADSC osteogenesis.

According to significant improvements in the tissue engineering field over the past several years, lung tissue cells have recently attracted more attention due to the high prevalence and diversity in related diseases. However, selection of an appropriate cell type, screening of suitable conditions for growth and proliferation, as well as subsequent implantation into the body to repair and regenerate damaged tissues are considered as important issues in this context. It should also be noted that most studies have been described in animal models, but not in humans. Because of the high regenerative capacity, predominant immunomodulatory feature, and inhibition of T-lymphocyte proliferation, mesenchymal stem cells (MSCs) may play an important role in the reconstruction of damaged tissues including bronchioles in pulmonary diseases. Interestingly, clinical trial studies demonstrated that MSCs have the significant potential to treat a wide variety of diseases including acute myocardial infarction (AMI), liver cirrhosis, crohn’s disease, and graft-versus-host disease (GVHD).

Background: Anaphase promoting complex (APC) is the biggest Cullin-RING E3 ligase and is very important in cell cycle control; many anti-cancer agents target this. APC controls the onset of chromosome separation and mitotic exit through securin and cyclin B degradation, respectively. Its APC3 subunit identifies the APC activators-Cdh1 and Cdc20. Materials and Methods: The structural model of the APC3 subunit of APC was developed by means of computational techniques; the binding of a natural inhibitory compound to APC3 was also investigated. Results: It was found that APC3 structure consists of numerous helices organized in anti-parallel and the overall model is superhelical of tetratrico-peptide repeat (TPR) domains. Furthermore, binding pocket of the natural inhibitory compound as APC3 inhibitor was shown. Conclusion: The findings are beneficial to understand the mechanism of the APC activation and design inhibitory compounds.

As a contribution to the functionality of nerve guide conduits (NGCs) in nerve tissue engineering, here we report a conduit processing technique through introduction and evaluation of topographical, physical and chemical cues. Porous structure of NGCs based on freeze-dried silk/single walled carbon nanotubes (SF/SWNTs) has shown a uniform chemical and physical structure with suitable electrical conductivity. Moreover, fibronectin (FN) containing nanofibers within the structure of SF/SWNT conduits produced through electrospinning process have shown aligned fashion with appropriate porosity and diameter. Moreover, fibronectin remained its bioactivity and influenced the adhesion and growth of U373 cell lines. The conduits were then implanted to 10 mm left sciatic nerve defects in rats. The histological assessment has shown that nerve regeneration has taken places in proximal region of implanted nerve after 5 weeks following surgery. Furthermore, nerve conduction velocities (NCV) and more myelinated axons were observed in SF/SWNT and SF/SWNT/FN groups after 5 weeks post implantation, indicating a functional recovery for the injured nerves. With immunohistochemistry, the higher S-100 expression of Schwann cells in SF/SWNT/FN conduits in comparison to other groups was confirmed. In conclusion, an oriented conduit of biocompatible SF/SWNT/FN has been fabricated with acceptable structure that is particularly applicable in nerve grafts.

Different research groups have extensively studied the associations of cytokine gene polymorphisms in different diseases. The role of cytokines gene polymorphisms in multiple sclerosis (MS), as a chronic Immune-mediated neurodegenerative disease, has been previously reported in the various populations. For determining pro-inflammatory cytokine gene polymorphisms, 100 relapsing remitting multiple sclerosis (RRMS) Iranian patients and 140 normal individuals as control enrolled in this study. DNA of each sample was extracted by a modified salting out method. Cytokine single gene nucleotide polymorphisms including IL-1α -889, IL-1β (-511 and +3962), IL-1R pst1 1970, IL-1RA mspal 11100, and TNF-α (-308 and -238) were determined by using the PCR-SSP method. The results of our data indicate the decrease in frequency of IL-1α TC-889 genotype (p=0.002), IL-1β TC +3962 genotype (p=0.004), IL-1R T pst1 1970 allele (p= 0.0001), IL-1 RA TC Mspa1 11100 genotype (p=0.009), TNF-α A-308 allele (p=0.0002) and AG genotype (p=0.00001) in the patients group versus normal subjects. On the other hand the frequency of IL-1α TT -889 genotype (p=0.028), IL-1R C pst1 1970 allele (p=0.0001) and CC genotype (p=0.00006), TNFα G -308 allele (p=0.0002) and GG genotype (p=0.000001) decreased significantly in the patients versus normal subjects. These results suggest that polymorphic variations of these pro-inflammatory cytokines may play an important role in susceptibility of Iranian multiple sclerosis patients.

Background: There is a novel hypothesis in that antibodies may have specificity for two distinct antigens that have been named "dual specificity." This hypothesis was evaluated for some defined therapeutic monoclonal antibodies (mAbs) such as Trastuzumab, Pertuzumab, Bevacizumab, and Cetuximab. In silico design and construction of expression vectors for trastuzumab monoclonal antibody also in this work were performed. Materials and Methods: First, in bioinformatics studies the 3D structures of concerned mAbs were obtained from the Protein Data Bank (PDB). Three-dimensional structural alignments were performed with SIM and MUSTANG softwares. AutoDock4.2 software also was used for the docking analysis. Second, the suitable genes for trastuzumab heavy and light chains were designed, synthesized, and cloned in the prokaryotic vector. These fragments individually were PCR amplified and cloned into pcDNA™ 3.3-TOPO® and pOptiVEC™ TOPO® shuttle vectors, using standard methods. Results: First, many bioinformatics tools and softwares were applied but we did not meet any new dual specificity in the selected antibodies. In the following step, the suitable expression cascade for the heavy and light chains of Trastuzumab therapeutic mAb were designed and constructed. Gene cloning was successfully performed and created constructs were confirmed using gene mapping and sequencing. Conclusions: This study was based on a recently developed technology for mAb expression in mammalian cells. The obtained constructs could be successfully used for biosimilar recombinant mAb production in CHO DG44 dihydrofolate reductase (DHFR) gene deficient cell line in the suspension culture medium.