Expression of functional recombinant human tissue transglutaminase (TG2) using the bac-to-bac baculovirus expression system

Purpose: Tissue transglutaminase (TG2) is a unique multifunctional enzyme. The enzyme possesses enzymatic activities such as transamidation/crosslinking and non-enzymatic functions such as cell migration and signal transduction. TG2 has been shown to be involved in molecular mechanisms of cancers and several neurodegenerative diseases such as Alzheimer's disease. The present study aimed at cloning and expression of full length human TG2 in Bac-to-Bac baculovirus expression system and evaluation of its activity. Methods: pFastBac HTA donor vector containing coding sequence of human TG2 was constructed. The construct was transformed to DH10Bac for generating recombinant bacmid. The verified bacmid was transfected to insect cell line (Sf9). Expression of recombinant TG2 was examined by RT-PCR, SDS-PAGE and western blot analysis. Functional analysis was evaluated by fluorometric assay and gel electrophoresis. Results: Recombinant bacmid was verified by amplification of a band near to 4500 bp. Expression analysis showed that the enzyme was expressed as a protein with a molecular weight near 80 kDa. Western blot confirmed the presence of TG2 and the activity assays including flurometric assay indicated that the recombinant TG2 was functional. The electrophoresis assay conformed that the expressed TG2 was the indeed capable of crosslinking in the presence of physiological concentration calcium ions. Conclusion: Human TG2 was expressed efficiently in the active biological form in the Bacto- Bac baculovirus expression system. The expressed enzyme could be used for medical diagnostic, or studies which aim at finding novel inhibitors of the enzymes . To best of our knowledge, this is probably the first report of expression of full length human tissue transglutaminase (TG2) using the Bac-to-Bac expression system. © 2016 The Authors

A survey on the possibility of utilizing γH2AX as a biodosimeter in radiation workers

Introduction DNA damage is among the main consequences of radiation. Of many different classes of DNA damage, double-strand breaks are the most deleterious. Development of a sensitive biodosimetry method, which utilizes a detection material with a similar construction to the body, seems essential for monitoring radiation workers. In this study, histone H2AX protein was examined as a potential biodosimeter in radiation workers. Moreover, the presence of this protein after in vitro irradiation of blood samples was assessed simultaneously. Materials and Methods Blood samples from 46 radiation workers were analyzed in Golestan province, Iran. Meanwhile, two groups of blood samples (five blood samples in each group) were irradiated in vitro by doses of 1 to 0.2 Gy and 0.09 to 0.01 Gy from a 60Co source, respectively. γH2AX level in lymphocytes was measured, using Western blot technique. ANOVA and Tukey's tests were performed, using SPSS version 16. The significance level was considered to be 0.05. Results The results of Western blotting for the identification of γH2AX protein in radiation workers were negative. However, γH2AX level in lymphocytes of two in vitro irradiated groups showed a significant correlation with the radiation dose (P<0.0001). Conclusion The results showed that γH2AX was a good indicator for acute or local exposure to ionizing radiation, while in chronically exposed individuals, including radiation workers, this protein was useless at least in autoradiography detection method. Regarding the presence of γH2AX protein in blood samples, which were irradiated in vitro at low doses, it can be concluded that this protein has powerful repair mechanisms

The prognostic effect of PTEN expression status in colorectal cancer development and evaluation of factors affecting it: MiR-21 and promoter methylation

Background: PTEN is a tumor suppressor gene which is involved in cellular proliferation, differentiation, and apoptosis. Loss or down-regulation of PTEN plays an important role in human cancers development. In this study, we investigated the effect of miR-21 and promoter methylation on the PTEN expression status in CRC tissues and analyzed association of the PTEN expression status with clinicopathological features in patients with CRC. Results: The PTEN expression was positively detected in 67.2 CRC tissues and all adjacent non-cancerous samples. PTEN mRNA level was negatively correlated with miR-21 level (r = -0.595, P < 0.001). PTEN expression was also correlated directly with the PTEN mRNA level (r = 0.583, P < 0.001) and conversely with miR-21 level (r = -0.632, P < 0.001). PTEN Promoter methylation was significantly associated with PTEN expression status (p = 0.013). PTEN expression was negatively associated with tumor size (p = 0.007) and advanced tumor stage (P = 0.011). Multivariate analysis indicated that tumor stage, tumor differentiation and PTEN expression status were independent prognostic factors for overall carcinoma in CRC patients (P < 0.05). The Kaplan-Meier curve indicated a negative correlation between PTEN expression levels and survival of CRC patients (P = 0.013). Conclusions: This study suggests a high frequency of miR-21 overexpression and aberrant promoter methylation in down-regulation of PTEN expression in colorectal carcinoma. Loss of PTEN may be a prognostic factor for patients with CRC. © 2016 Yazdani et al

Preliminary assessment of various additives on the specific reactivity of anti- rHBsAg monoclonal antibodies

Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage. © 2015, Avicenna Journal of Medical Biotechnology. All rights reserved

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background: Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples. Results: Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA. Conclusions: The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme. © 2014 Mansour Samaei et al.; licensee BioMed Central

Studying the effect of perceptual errors on the decisions made by the investors by effectiveness of information in Tehran Stock Exchange Company

There are many latent factors that are effective on the decisions made by the investors. The factors that the investors are not aware of their effectiveness and make investment decisions. The main purpose of the present research is to study the perceptual factors affecting on the decision making process of the investors and the effect of information on these factors. For this aim, 385 investors of Tehran Stock Exchange Company were selected as a sample through random sampling method and the required data were gathered via the questionnaire. The accuracy of the hypothesizes was tested via a structural equation model. The results obtained from the present study show that the decision making process of the investors is affected by the representative error, overconfidence error and mood state error by 19%. Moreover, overconfidence error is affected by the degree of information by 95% and by the anonymity of the information by 10%. The mood state error is effective on information processing time by 13% and the information processing time is effective on decision making process by 24%.Keywords: Behavioral Finance, Perceptual Error, Decision making, Investmen

Mechanical performance of composite bonded joints in the presence of localised process-induced zero-thickness defects

Processing parameters and environmental conditions can introduce variation into the performance of adhesively bonded joints. The effect of such variation on the mechanical performance of the joints is not well understood. Moreover, there is no validated nondestructive inspection (NDI) available to ensure bond integrity post-process and in-service so as to guarantee initial and continued airworthiness in aerospace sector. This research studies polymer bond defects produced in the laboratory scale single-lap composite-to-composite joints that may represent the process-induced defects occurring in actual processing scenarios such as composite joining and repair in composite aircrafts. The effect of such defects on the degradation of a joint's mechanical performance is then investigated via quasi-static testing in conjunction with NDI ultrasonic C-scanning and pulsed thermography. This research is divided into three main sections: 1- manufacturing carbon fibre-reinforced composite joints containing representative nearly zero-thickness bond defects, 2- mechanical testing of the composite joints, and 3- assessment of the NDI capability for detection of the bond defects in such joints

Expression of an innate immune element (mouse hepcidin-1) in baculovirus expression system and the comparison of its function with synthetic human hepcidin-25

Hepcidin is an innate immune element which decreases the iron absorption from diet and iron releasing from macrophage cell. In contrast to the chemical iron chelators, there has been limited effort applied to the specific use of hepcidin as a new drug for decreasing the iron overload. Hepcidin is produced in different biological systems. For instance, E-coli is used for human hepcidin expression, however, post-translational modification is impaired. We have used a simple baculovirus expression system (BES) to improve the hepcidin folding and activity. Hepcidin Messenger Ribonucleic acid (mRNA) was isolated from mouse liver cells and its complementary Deoxyribonucleic acid (cDNA) was produced and amplified. PFastBac HTB vector was used for recombinant bacmid production. Recombinant baculovirus was produced using SF-9 cell line. The mouse hepcidin-1 protein was expressed in a large quantity and functional tests were performed for this recombinant peptide. The yield of hepcidin in BES was 20 μg/mL and anti-histidine (anti-His) tag antibody was used for the confirmation of hepcidin on western blot nitrocellulose paper. Functional tests showed that mouse hepcidin accumulates iron in the macrophage cell line J774A.1 up to 63%. In addition, our data showed that the mouse hepcidin-1 has less toxicity compared to the synthetic human hepcidin-25 (p = 0.000). © 2011 by School of Pharmacy

A novel diblock copolymer of (monomethoxy poly [ethylene glycol]-oleate) with a small hydrophobic fraction to make stable micelles/polymersomes for curcumin delivery to cancer cells

Curcumin is a potent natural anticancer agent, but its effectiveness is limited by properties such as very low solubility, high rate of degradation, and low rate of absorption of its hydrophobic molecules in vivo. To date, various nanocarriers have been used to improve the bioavailability of this hydrophobic biomaterial. This study investigates the encapsulation of curcumin in a novel nanostructure of monomethoxy poly(ethylene glycol)-oleate (mPEG-OA) and its anticancer effect. Tests were done to determine the critical micelle concentration (CMC), encapsulation efficiency, drug-loading efficiency, and cytotoxicity (against U87MG brain carcinoma cells and HFSF-PI3 cells as normal human fibroblasts) of some nanodevice preparations. The results of fluorescence microscopy and cell-cycle analyses indicated that the in vitro bioavailability of the encapsulated curcumin was significantly greater than that of free curcumin. Cytotoxicity evaluations showed that half maximal inhibitory concentrations of free curcumin and curcumin-loaded mPEG-OA for the U87MG cancer cell line were 48 μM and 24 μM, respectively. The Annexin-V-FLUOS assay was used to quantify the apoptotic effect of the prepared nanostructures. Apoptosis induction was observed in a dose-dependent manner after curcumin-loaded mPEG-OA treatments. Two common self-assembling structures, micelles and polymersomes, were observed by atomic force microscopy and dynamic light scat­tering, and the abundance of each structure was dependent on the concentration of the diblock copolymer. The mPEG-OA micelles had a very low CMC (13.24 μM or 0.03 g/L). Moreover, atomic force microscopy and dynamic light scattering showed that the curcumin-loaded mPEG-OA polymersomes had very stable structures, and at concentrations 1,000 times less than the CMC, at which the micelles disappear, polymersomes were the dominant structures in the dispersion with a reduced size distribution below 150 nm. Overall, the results from these tests revealed that this nanocarrier can be considered as an appropriate drug delivery system for delivering curcumin to cancer cells. © 2014 Erfani-Moghadam et al

Molecular cloning and expression of novel fibroblast growth factor-2 conjugated with immunodominant domains of pseudomonas exotoxin

Angiogenesis is very important in cancer growth and metastasis. Basic fibroblast growth factor (bFGF) as one of the most important angiogenesis factors is an attractive target for cancer vaccine. Due to low immunogenicity, it cannot stimulate an effective immune response. Theoretically, pseudomonas exotoxin (PE) as a potent immunogenic carrier protein when fused to low immunogenic antigens such as bFGF significantly increased immunogenicity of it. In this study, we tried to molecular cloning and expression of bFGF conjugated with immunodominant domains of pseudomonas exotoxin. The coding sequence of fusion protein composed of bFGF linked to PE domains 1b and 2 using EAAAK poly linker. The KDEL sequence was also used in C-terminal coding sequence. It was synthesized and expressed using recombinant DNA technology in the bacterial expression system. Expression of recombinant protein verified using SDS-PAGE and western blot analyses. Finally, it purified using Ni-affinity chromatography. The band close to 37 kDa in SDS-PAGE and western blot analyses was aligned completely to designed sequence. Purified recombinant protein also showed as a clear single band near to 37 kDa