Production and purification of anti-VEGFR2 single chain fragment variable antibody

Introduction: The antibody display technology (ADT) such as phage display (PD) has substantially improved the production of monoclonal antibodies (mAbs) and Ab fragments through bypassing several limitations associated with the traditional approach of hybridoma technology. In the current study, we capitalized on the PD technology to produce high affinity single chain variable fragment (scFv) against Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). VEGFR and its receptor (VEGFR2) play an important role in angiogenesis associated with tumor growth and metastasis. Methods and Results: To pursue production of scFv antibody fragments against human VEGFR2, we performed four rounds of biopanning using stepwise decreased amount of VEGFR2 peptide (1 to 0.1 μg), a semi-synthetic phage antibody library (Tomlinson I + J) and TG1 cells. Antibody clones were isolated and selected through enzyme-linked immunosorbent assay (ELISA) screening. The selected scFv antibody fragments were further characterized by means of ELISA, PCR and Western blot analyses as well as fluorescence microscopy and Wound healing assay. Based upon binding affinity to VEGFR2, 5 clones were selected out of 30 positive clones enriched from PD in vitro selection. The selected scFvs displayed high specificity and binding affinity with Kd values at nm range to human VEGFR2. The immunofluorescence analysis revealed significant binding of the selected scFv antibody fragments to the HUVEC cell line. The effectiveness of the selected scFv fragments was further validated by Western blot analyses and Wound healing assay. Conclusions: Based on these findings, we propose the selected fully human anti- VEGFR2 scFv antibody fragments as potential immunotherapy agents that may be translated into preclinical/clinical applications

The effect of 12 weeks of aerobic exercise intervention on bone mineral density, expression of lymphocyte alkaline phosphatase gene and bone turnover markers in overweight postmenopausal women: a randomized controlled trial

The aim of our study was to examine the effect of 12 weeks of moderate-intensity aerobic exercise on bone mineral density (BMD), lymphocyte alkaline phosphatase (ALP) mRNA expression, and biochemical markers of bone turnover in postmenopausal women (PMWs). Twenty-four healthy sedentary PMWs aged 50-60 years were randomly assigned to exercise (EX, n=12) and control (C, n=12) groups. The EX group performed walking/jogging (50-60min/day, 3days/week at 65%-70% HRmax reserve) for 12-week while the C group participated in no intervention and continued their normal lifestyle. The BMD and lymphocyte ALP mRNA were determined by DXA and qRT-PCR, respectively. After 12 weeks, the increase in the lymphocyte ALP mRNA expression and its serum (P=0.008 and P=0.001), PTH (P=0.001), Vit-D (P=0.002), and VO2max (P=0.001) were significantly higher in the EX group compared to the C group, whereas body fat was significantly decreased (P=0.028). Our study indicates that 12 weeks of moderate-intensity aerobic exercise intervention improves bone turnover by increasing the ALP mRNA expression, serum levels of PTH, ALP, and Vit-D which can lead to the prevention of aging-induced osteopenia among PMWs

Effects of Lactobacillus acidophilus and Bifidobacterium bifidum probiotics on the serum biochemical parameters, and the vitamin D and leptin receptor genes on mice colon cancer

Objective(s): The preclinical reports have shown that specific probiotics like Bifidobacterium bifidum (B. bifidum) and Lactobacillus acidophilus (L. acidophilus) can be applied as the biotherapeutic agents in the inhibition or therapy of colorectal cancer via the modification of gut bacteria. In the previous studies, we have assessed the impact of L. acidophilus and B. bifidum probiotics on gut bacteria concentration and also their chemo-protective impact on mice colon cancer. In the following, we assessed the effects of these probiotics on the  gene expression of vitamin D receptor (VDR) and the leptin receptor (LPR) and the serum biochemical parameters on mice colon cancer. Materials and Methods: Thirty-six male BALB/c mice were equally shared into 4 groups; (i) health with routine dietary foods without any treatment, (ii) azoxymethane (AOM)-induced mice colon cancer with common dietary foods, (iii) and (iv) AOM-induced mice colon cancer with oral consumption of L. acidophilus and B. bifidum (1×109 cfu/g) for 5 months, respectively. Then, the serum total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), alanine transaminase, alkaline phosphatase, and albumin and also VDR and LPR genes expression were evaluated. Results: Oral consumption of L. acidophilus and B. bifidum probiotics significantly decreased the triglycerides, alkaline phosphatase, LDL, and also the VDR and LPR gene expression in mice colon cancer (

Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies

Investigating Quantitative analysis of the gene expression of calcium/calmodulin-dependent protein kinase IV by the effect of Olibanum alcoholic extract in PC12 cell line

Background & Objective: Long-term memory depends on protein synthesis. The product of Camkiv gene promotes memory via activating its proteins. The treatment of laboratory animals by Olibanum leads to memory improvement and the recovery of Alzheimer. Therefore, the aim of this study is the evaluation of Olibanum ethanolic extract on the Camkiv expression in PC12 cells. Materials & Methods: Olibanum toxicity on the cell viability was investigated by MTT test. Cells were treated with concentrations 10,25,40,55,70 and 85 μg/ml of extract in time intervals 12,24,48 and 72 hours and their absorption rate was measured. Then, cells were treated by concentrations 2 and 20 μg/ml of extract in mentioned times. Extracted RNA was converted into cDNA and real-time PCR performed. Results: Cell death was raised by increasing time and concentration of extract treatment. IC50 values were obtained as 71.01, 52.95, 21.05 and 13.85 μg/ml in 12, 24, 48 and 72 hours of treatment, respectively. Besides, concentrations 2 and 20 μg/ml significantly increased Camkiv expression following 24 hour treatment. The maximum expression of Camkiv was observed in 48 hour treatment. The effect of Olibanum on gene upregulation was stable until 72 hours. Conclusion: The Olibanum ethanolic extract can remarkably upregulate Camkiv expression for a long time. These results are consistent with the previous studies indicating the effect of Olibanum on upregulation of Bdnf, Camkiv‌-downstream gene. However, regarding the existence of the two-direction pathway in the expression regulation of Bdnf and Camkiv‌, comprehensive studies are required to determine exact mechanism of Olibanum function in the brain

Effect of moderate-intensity aerobic training on alkaline phosphatase gene expression and serum markers of bone turnover in sedentary postmenopausal women

Background: Studies show that aerobic exercise prevents osteoporosis in menopause by stimulating osteoblastic cells. Therefore, the purpose of this study was to investigate the effect of 12 weeks of moderate-intensity aerobic exercise on alkaline phosphatase gene expression, serum levels of alkaline phosphatase, parathyroid hormone, and calcium in sedentary women. Methods: This investigation is a semi-experimental study that was performed in September 2015 at Urmia University, Iran. The statistical population was all healthy and sedentary postmenopausal women 50 to 65 years old in Urmia city. Twenty sedentary postmenopausal women with an average age 60.12±2.12 yr, weight 72.35±10.50 kg, and body mass index 29.46±3.24 kg/m2 voluntarily and bona fide participated in this study, and then subjects were randomly divided to the Exercise/E (10 women) and Control/C (10 women) groups by random sampling method. E group performed of 12 weeks walking and jogging moderate-intensity aerobic exercise at 65-70% maximal heart rate of training, three sessions per week and per session 50-60 (min), but the C group participated in no intervention. Twenty-four hours before and after the 12-week training program were taken blood samples in order to measure of alkaline phosphatase gene expression and serum markers of bone in the E and C Groups. Evaluation of gene expression and serum markers of bone were measured by real-time reverse transcription PCR (RT-PCR) and Auto-analyzer (Biotechnica, Italy)/ ELISA reader (Awareness Inc., USA) machines, respectively. Data analysis included descriptive and inferential (ANCOVA test) statistics using SPSS version 23 (Chicago, IL, USA) and a significance level of P≥0.05 was considered. Results: The results showed that alkaline phosphatase gene expression and parathyroid hormone after 12 weeks of moderate-intensity aerobic exercise in between-groups were significantly increased (P=0.027 and P=0.006, respectively), while serum levels of calcium and alkaline phosphatase were not significantly different (P=0.941 and P=0.990, respectively). Conclusion: The results suggest that 12 weeks of aerobic exercise of walking and jogging at 65-70% maximal heart rate of training increases alkaline phosphatase gene expression and parathyroid hormone in sedentary postmenopausal women

Variations of Serum Leptin and Resistin levels in Healthy non- Diabetic women with different degrees of Obesity

ABSTRACT The role of leptin and resistin serum concentrations in pathogenesis of obesity is considered an emerging topic issue in clinical biochemistry researches. This study was carried out to evaluate the variation of leptin and resistin serum levels in women with different grades of obesity. A total of 149 non-diabetic women were included in this study Adipose tissue is an endocrine organ that secretes a wide variety of proteins which contribute to regulation of body weigh

Human amniotic fluid stem cells (hAFSCs) expressing p21 and cyclin D1 genes retain excellent viability after freezing with (dimethyl sulfoxide) DMSO

Human amniotic fluid stem cells (hAFSCs) have features intermediate between embryonic and adult SCs, can differentiate into lineages of all three germ layers, and do not develop into tumors in vivo. Moreover, hAFSCs can be easily obtained in routine procedures and there is no ethical or legal limitations regarding their use for clinical and experimental applications. The aim of this study was to assess the effect of slow freezing/thawing and two different concentrations of DMSO (10% DMSO + 90% fetal bovine serum [FBS] and 5% DMSO + 95% FBS) on the survival of hAFSCs. hAFSCs were obtained from 5 pregnant women during amniocentesis at 16–22 weeks of gestation. The expression of pluripotency markers (Octamer-binding transcription factor 4 [Oct4] and NANOG) by reverse transcription polymerase chain reaction and cell surface markers (cluster of differentiation [CD31], CD44, CD45, and CD90) by flow cytometry was analyzed before and after the slow-freezing. Cell viability was assessed by trypan blue exclusion or MTT assay. Quantitative mRNA expression of Oct4, NANOG, cyclin D1 and p21 was determined by real-time PCR before and after the slow-freezing. Pluripotency of hAFSCs was confirmed by NANOG and POU5F1 (Oct4) gene expression before and after slow-freezing. All hAFSC cultures were positive for CD44 and CD90. A higher viability of hAFSCs was observed after freezing with 90% FBS + 10% DMSO. There was increased expression of NANOG and decreased expression of POU5F1 gene after freezing, compared to control cells (before freezing). DMSO and the process of freezing did not significantly change the expression of p21 and cyclin D1 genes in hAFSCs. Overall, our results indicate the applicability of slow-freezing and DMSO in cryopreservation of SCs

Clofarabine Has Apoptotic Effect on T47D Breast Cancer Cell Line via P53R2 Gene Expression

Purpose: Clofarabine, a purine nucleoside analogue and inhibitor of Ribonucleotide Reductase (RR), is used for treatment of leukemia. Clofarabine-induced defect in DNA replication, induces p53 and subsequently P53R2 genes as subunit of RR. clofarabine deregulated P53R2 gene expression leading to the elevated levels of P53R2 which impose resistance to DNA damaging drugs. In this study the apoptotic and cytotoxic effects of clofarabine has been investigated on breast cancer cell line. Methods: Cofarabine cytotoxicity on T47D cells has been studied by MTT assay. T47D cells were exposed to the different concentrations of clofarabine for 24, 48 and 72 hours intervals. Relative expression of P53R2 gene has been studied using real-time PCR. Moreover, after treating with clofarabine the apoptotic and necrotic cells were detected using Annexin V and propodium iodide (PI) reagents by flowcytometry technique. Results: MTT assay results showed that the clofarabine IC50 on T47D cell line were 3 and 2.5µM after 48 and 72 h exposure, respectively. Clofarabine did not show any significant cytotoxic effect after 24 h exposure. The analysis of qRT-PCR showed a significant increase in P53R2 gene expression in treated cells with both 2.5 and 3 μM doses and also, the results of flowcytometry revealed 26.91 and 74.46 percent apoptosis induction in 48 and 72h treatments respectively in comparison to the control groups. Conclusion: Our results showed that apoptotic and cytotoxic effects of clofarabine on T47D cell line were in time and dose dependent manner; therefore it could be considered a new candidate in breast cancer therapy