javad ranjbari

 Treatment of critical-sized bone defects is challenging. Tissue engineering as a state-of-the-art method has been concerned with treating these non-self-healing bone defects. Here, we studied the potentials of new three-dimensional nanofibrous scaffolds (3DNS) with and without human adipose mesenchymal stem cells (ADSCs) for reconstructing rat critical-sized calvarial defects (CSCD).

 Scaffolds were made from 1- polytetrafluoroethylene (PTFE), and polyvinyl alcohol (PVA) (PTFE/ PVA group), and 2- PTFE, PVA, and graphene oxide (GO) nanoparticle (PTFE/ PVA/GO group) and seeded by ADSCs and incubated in osteogenic media (OM). The expression of key osteogenic proteins including Runt-related transcription factor 2 (Runx2), collagen type Iα (COL Iα), osteocalcin (OCN), and osteonectin (ON) at days 14 and 21 of culture were evaluated by western blot and immunocytochemistry methods. Next, 40 selected rats were assigned to five groups (n=8) to create CSCD which will be filled by scaffolds or cell-containing scaffolds. The groups were denominated as the following order: Control (empty defects), PTFE/PVA (PTFE/PVA scaffolds implant), PTFE/PVA/GO (PTFE/PVA/GO scaffolds implant), PTFE/PVA/Cell group (PTFE/PVA scaffolds containing ADSCs implant), and PTFE/PVA/GO/Cell group (PTFE/PVA/GO scaffolds containing ADSCs implant). Six and 12 weeks after implantation, the animals were sacrificed and bone regeneration was evaluated using computerized tomography (CT), and hematoxylin-eosin (H&E) staining.

 Based on the in-vitro study, expression of bone-related proteins in ADSCs seeded on PTFE/PVA/GO scaffolds were significantly higher than PTFE/PVA scaffolds and TCPS (P<0.05). Based on the in-vivo study, bone regeneration in CSCD were filled with PTFE/PVA/GO scaffolds containing ADSCs were significantly higher than PTFE/PVA scaffolds containing ADSCs (P<0.05). CSCD filled with cell-seeded scaffolds showed higher bone regeneration in comparison with CSCD filled with scaffolds only (P<0.05).

 The data provided evidence showing new freeze-dried nanofibrous scaffolds formed from hydrophobic (PTFE) and hydrophilic (PVA) polymers with and without GO provide a suitable environment for ADSCs due to the expression of bone-related proteins. ADSCs and GO in the implanted scaffolds had a distinct effect on the bone regeneration process in this in-vivo study.

Nowadays, mesenchymal stem cells (MSCs) are the most widely used cell sources for bone regenerative medicine. Electrospun polyacrylonitrile (PAN)-based scaffolds play an important role in bone tissue engineering due to their good mechanical properties, which could be enhanced by the presence of nanoparticles such as nanoclay. This study evaluated the in-vitro effect of different concentrations of nanoclay in surface characteristic properties of PAN-based electrospun nanofiber scaffolds and the osteogenic differentiation ability of adipose-derived mesenchymal stem cells (AD-MSCs). After electrospinning nanofibers, their structure were assessed through some characterization tests. Then AD-MSCs isolation and characterization were done, and the cell attachment and the biocompatibility were determined. Finally, osteogenic differentiation-related markers, genes, and proteins were studied. Clay-PAN25% electrospun nanofiber scaffold could support attachment, proliferation, and osteogenic differentiation of AD-MSCs better than other groups. Also, nanoclay could enhance the properties of PAN-based scaffolds, such as fiber diameter, topography, surface charge, hydrophilicity, roughness, and degradation, as well as osteogenic differentiation of cells. As a result, Clay-PAN25% with the highest concentration of nanoclay was found as a promising biodegradable and cost-effective scaffold for osteogenic differentiation of AD-MSCs.

Pseudomonas aeruginosa infection is one of the major challenges in burn patients. This organism is resistant to a wide range of antibiotics. The aim of this study was to investigate the antibiotic susceptibility and genetic relatedness in Pseudomonas aeruginosa isolated from patients admitted to Motahari Burn Center, Tehran, Iran.

This study was conducted on 186 burn patients with Pseudomonas aeruginosa colonization following admission in Motahari Burn Center during one year. Antibiotic resistance test was performed by disc diffusion method and genetic relatedness was evaluated by PCR-RAPD analysis.

The highest and lowest percentage of resistance was observed against ceftizoxime and ciprofloxacin. The genotyping study by RAPD PCR technique revealed 57 different genotypes, among which RAPD 5, RAPD 8, and RAPD 9 were the most prevalent patterns and produced by 14%, 9%, and 7% of the isolates, respectively.

In total, no association was found between RAPD genotypes and antibiotic resistance patterns, and death rate.

Acetate accumulation in the culture medium is known as an inhibitor in recombinant protein production in Escherichia coli. Various approaches have been proposed and evaluated to overcome this challenge and reduce the concentration of acetate. In this study, we examined the effect of acetate kinase A antisense on acetate production rate in E. coli We also used PAMAM dendrimers as a suitable delivery agent for antisense transformation into E. coli host cell. This study aimed to decrease acetate production as a by-product using an antisense-dendrimer complex to increase mass cell and subsequently recombinant Albumin production in E. coli. Here, to study the effect of this treatment on recombinant protein production, we used pET22b/ HAS construct. The ackA gene expression was inhibited by designed antisense to reduce acetate concentration in culture medium. AckA antisense was transferred to E. coli by PAMAM dendrimer. Finally, ackA expression and recombinant Albumin production were evaluated Real-Time PCR and densitometry, respectively. Our data showed, designed antisense lead to reduction of acetate kinase gene expression and subsequently acetate concentration in the culture medium. Finally, acetate concentration reduction and cell mass increase result in enhanced recombinant Alb production in the treated group (1.25 mg.mL-1) compare to the control group ( 0.59 mg.mL-1). Reduction of acetate in E. coli fermentation process decreased the recombinant Alb production following cell growth and cell mass increase. In the current study, we showed that an antisense can be a useful tool for ackA gene expression reduction. Also, we noted that PAMAM dendrimer could be a proper delivery agent for oligonucleotide antisense transformation into bacterial cells.

Simulating hydrophobic-hydrophilic composite face with hierarchical porous and fibrous architectures of bone extracellular matrix (ECM) is a key aspect in bone tissue engineering. This study focused on the fabrication of new three-dimensional (3D) scaffolds containing polytetrafluoroethylene (PTFE), and polyvinyl alcohol (PVA), with and without graphene oxide (GO) nanoparticles using the chemical cross-linking and freeze-drying methods for bone tissue application. The effects of GO on physicochemical features and osteoinduction properties of the scaffolds were evaluated through an in vitro study.

After synthesizing the GO nanoparticles, two types of 3D scaffolds, PTFE/PVA (PP) and PTFE/PVA/GO (PPG), were developed by cross-linking and freeze-drying methods. The physicochemical features of scaffolds were assessed and the interaction of the 3D scaffold types with human adipose mesenchymal stem cells (hADSCs) including attachment, proliferation, and differentiation to osteogenic like cells were investigated.

GO nanoparticles were successfully synthesized with no agglomeration. The blending of PTFE as a hydrophobic polymer with PVA polymer and GO nanoparticles (hydrophilic compartments) were successful. Two types of 3D scaffolds had nano topographical structures, good porosities, hydrophilic surfaces, thermal stabilities, good stiffness, as well as supporting the cell attachments, proliferation, and osteogenic differentiation. Notably, GO incorporating scaffolds provided a better milieu for cell behaviors.

Novel multiscale porous nanofibrous 3D scaffolds made from PTFE/ PVA polymers with and without GO nanoparticles could be an ideal candidate for bone tissue engineering as a 3D template.

Insulin-Like Growth Factor-1 (IGF-1) is a small peptide with 70 amino acids and 7.6 kDa molecular weight that acts as the major mediator of growth hormone. According to the previous studies, recombinant production of human IGF-1 (rhIGF-1) in E. coli has resulted in an inactive form of protein (inclusion body). There are several strategies to transform inclusion body to a soluble form. Production in the form of fusion proteins as a suitable strategy, helps researcher in recombinant production of proteins in the soluble and active form. In current study, NusA fusion protein was used to produce IGF-1 soluble form, instead of insoluble protein. In previous study, rhIGF-1 was optimally expressed in inclusion body with 1.2 g/L concentration. rhIGF1 -NusA construct was cloned and expressed in E. coli, then, cell lysate was analyze by SDS-PAGE and densitometry techniques, to assay soluble and insoluble form of rhIGF-1. Results showed that rhIGF-1 concentration in soluble phase was 0.14 g/L, indicating that about 12% of total expression of rhIGF-1 was in the soluble form through NusA-fusion protein strategy. These results confirmed that some fusion proteins like NusA could improve the solubility of recombinant proteins expressed in heterogeneous bacterial hosts.HIGHLIGHTSFusion proteins is a suitable strategy for recombinant production of proteins in e soluble form.NusA fusion tag improves the solubility of recombinant proteins expressed in bacterial hosts. NusA fusion protein convert IGF-1 insoluble form to soluble form in E. coli.

Researchers add serum to a classical medium at concentrations of 5 to 10% (v/v) to grow cellsin-vitro culture media. Unfortunately, serum is a poorly defined culture medium componentas its composition can vary considerably while serum-free cell culture media are an excellentalternative to standard serum-containing media and offer several major advantages. Advantagesof using serum-free media include a lower risk of infectious agents, lower risk of interferingcomponents, less contaminant, avoids ethical issues. According to previous studies insulin,selenium, transferrin and glucose are important component of serum that affect cell growth. Inthe present study, we optimized amount of these factors in order to serum free culture mediumfabrication. Response surface methodology (RSM) was employed for optimization of key factorsin serum free medium to enhance recombinant human GM-CSF (rhGM-CSF) production in CHOcell line. Four important process parameters including insulin concentration (0-2 g/L), transferrinconcentration (0-1 g/L), selenium concentration (0-0.001 g/L) and glucose concentration (0-5g/L) were optimized to obtain the best response of rhGM-CSF production using the statisticalBox–Behnken design. The experimental data obtained were analyzed by analysis of variance(ANOVA) and fitted to a second-order polynomial equation using multiple regression analysis.Numerical optimization applying desirability function was used to identify the optimumconditions for maximum production of rhGM-CSF. The optimum conditions were found to beinsulin concentration = 1.1 g/L, transferrin concentration = 0.545 g/L, selenium concentration =0.000724 g/L and glucose = 1. 4 g/L. Maximum rhGM-CSF production was found to be 3.5 g/L.

Selection of a suitable host strain is an important step in recombinant production of heterologous proteins. As the prokaryotic systems and especially Escherichia coli (E. coli) cells are the most attractive host in therapeutic protein production, various derivatives of this organism have been developed to overcome the limitations exist in recombinant protein production, such as inefficient expression and folding of target proteins with eukaryotic origin. This review summarized key strategies for E. coli engineering and introduced some new and applicable engineered E. coli strains that produce more complex proteins for therapeutic and research use in the future.

Protein-coated nanoparticles have diverse applications in biomedical science. The protein hydrophobic domains or surface electrostatic charge conducts adsorption of proteins to different surfaces. This property can be customized to immobilize specific molecules on solid supports for experimental screenings or purification processes. To develop highly selective affinity ligands—such as aptamers—against specific protein targets, protein coated magnetic particles have been successfully applied. This approach could be highly efficient in affinity ligand development against coagulation factor VIII.In this study, magnetic nanoparticles were prepared by co-precipitation method and, then, a gold coating was run on the MNPs’ surface. The gold coating could add some attractive specifications to the protein immobilized nanoparticles during the aptamer selection process, such as simultaneous affinity determination of aptameric oligonucleotides by fluorescence-based methods. The gold surface has been indicated as a specific feature for covalent binding to the sulphur functional groups of various molecules. In proteins, sulphur units of cysteine or methionine might be bound covalently to the gold surface. In addition, nonspecific and non-covalent attachment of proteins to the gold particles may be performed. Therefore, a series of samples containing different mass ratios of protein to gold magnetic nanoparticles (GMNPs) were evaluated to find the best conditions for coagulation factor VIII immobilization. The results showed that the best condition for high coating efficiency was 48 h incubation at 4 ºC of protein and GMNPs with a mass ratio of 0.5% in PBS 25mM, with pH=7 as binding buffer.

Human insulin-like growth factor I (hIGF-I) is a kind of growth factor with clinical significance in medicine. The major objective of this study is over- production of recombinant human insulin-like growth factor I(rhIGF-I) through a developed process by recruiting effective factors in order to achieve the most recombinant protein. Up to now E. coli expression system has been widely used as a host to produce rhIGF-1 with high yields. Batch cultures as non-continuous fermentation were carried out to overproduce rhIGF-I in E. coli. The effects of culture medium type, induction temperature and amount of inducer on cell growth and IGF-I production were investigated in shaking flask. Taguchi design of experiments (DOE) method was used as the statistical method. Analysis of experimental data showed that maximum production of rhIGF-I was occurred in 32y culture medium at 28°C and 0.05 Mm IPTG. Under this condition, 0.7 g/L of rhIGF-I was produced as the inclusion bodies. Following optimization of these three factors, we have also optimized the amount of glucose and induction time in 5 liter top bench bioreactor. Full factorial design of experiment method was used for these two factors as the statistical method. 10 g/l and OD600=5 were selected as the optimum point of Glucose amount and induction time, respectively. Finally we have had 1.26 g/l rhIGF-1 production as the final product that is one of the best reported amounts.

The purpose of this study is to evaluate the effect of Curcuma longa extract on the telomerase gene expression in QU-DB lung cancer and T47D breast cancer cell lines. The present study is an experimental research. Using 3 different phases n-hexane, dichloromethane and methanol, total extract of Curcuma longa in a serial dilution was prepared and three phases was analyzed for determining which phase has more curcuminoids. Then the extract cytotoxicity effect was tested on breast cancer cell line (T47D), and lung cancer cell line (QU-DB) by 24, 48 and 72 h MTT (Dimethyl thiazolyl diphenyl tetrazolium) assay. Then, the cells were treated with serial concentrations of the extract. Finally, total protein was extracted from the control and test groups, its quantity was determined and telomeric repeat amplification protocol (TRAP) assay was performed for measurement of possible inhibition of the telomerase activity. Cell viability and MTT-based cytotoxicity assay show that the total extract of Curcuma longa has cytotoxic effect with different IC50s in breast and lung cancer cell lines. Analysis of TRAP assay also shows a significant reduction in telomerase activity on both cancer cells with different levels. Curcuma longa extract has anti-proliferation and telomerase inhibitory effects on QU-DB lung cancer and T47D breast cancer cells with differences in levels of telomerase inhibition.