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Nazari, M. ,
Emamzadeh, R. ,
Masoudi-khorram, N. ,
Nazari, M. ,
Abdolmaleki, F. ,
Kangarani-farahani, M. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (1873376X) 1263
The authors regret that the following series of corrections are needed in this article. Please add the following authors: 1) Fereshteh Abdolmaleki, Endocrine Research Center, Institute of Endocrinology and Metabolism, Iran University of Medical Sciences, Tehran, Iran; 2) Mohammad Kangarani-Farahani: Mahboubbioesearch co, Tehran, Iran. These authors were responsible for the following tasks for this article: Fereshteh Abdolmaleki, Writing – review; Mohammad Kangarani-Farahani, Writing – review. The authors would like to apologise for any inconvenience caused. © 2025 Elsevier Inc.
Biochimie (03009084) 235pp. 59-67
Engineered affiprobes that detect a specific target by generating a target-dependent signal, play an important role in biomedical sciences such as breast cancer monitoring. In this study, a new bioluminescence affiprobe consisting of an HER3-binding affibody and a bioluminescent protein, C-SRLc8, a thermostable variant of Renilla luciferase has been developed. The bioluminescent affibody, called C-SRLuc8‒Z08699 was expressed in the Escherichia coli and purified by affinity chromatography. The suitability of the new affiprobe to detected HER3 positive cells was evaluated. It was found that the new affiprobe allowed the detection of HER3 expressing cells with a detection limit of 14578 cells per 200 μl assay volume and a working range of 14000–50000 cells. The new affiprobe can be used for detecting, studying and monitoring the HER3-expressing cells using a luminometer in fast and inexpensive assays. © 2025 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
Enzyme and Microbial Technology (18790909) 185
The utilization of polyhistidine tags (His-tag) for the purification and analysis of recombinant proteins is a widely adopted technique in biotechnology. Considering the high costs associated with antibody-based methods, the development of cost-effective techniques for protein identification following purification could significantly lower research expenses. This study developed a novel His-tag aptasensor, combining an anti-His tag aptamer with a G-quadruplex-based DNAzyme, which demonstrates limits of detection (LODs) of 0.29 μM and 0.73 μM for a His-tagged protein in calorimetric and point-of-care assays, respectively. These LODs are significantly lower than typical protein concentrations obtained through Ni-NTA affinity chromatography, indicating that the His-tag aptasensor provides an efficient solution for in vitro analysis and post-purification monitoring of His-tagged proteins. © 2025 Elsevier Inc.
Biocatalysis and Biotransformation (10292446) 43(1)pp. 61-70
Renilla luciferase, a luminescent enzyme, is utilized in gene expression analysis and biosensor technology, and extensive research has been conducted on its structure and function. Glycine is an osmolyte that plays a key role in protein stabilization against denaturation. However, its impact on enzyme properties is unpredictable. This study aimed to investigate the effect of glycine on the kinetics and stability of Renilla luciferase. The data revealed that glycine at a concentration range of 0.1 to 0.6 M improved enzyme kinetics and thermal stability. The highest catalytic efficiency was observed at a concentration of 0.5 M. Molecular dynamics simulations demonstrated that in the presence of 0.5 M glycine, substrate access to the enzyme’s active site was enhanced while the root-mean-square fluctuation (RMSF) of the protein backbone was reduced. Additionally, the analysis of protein-water hydrogen bonding interactions showed an increase in the hydrogen bonding between water molecules and Renilla luciferase. The present study may be used for the formulation of Renilla luciferase for commercial purposes. © 2024 University of Isfahan. Published by Informa UK Limited, trading as Taylor & Francis Group.
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences (1873376X) 1261
Human serum albumin (HSA) is an important protein in plasma with various biological functions in the human body. Due to its unique features in the binding and transfer of ligands and pharmaceutical molecules, HSA is extensively used in therapeutics and pharmaceutical approaches. Commercial albumin is produced by a multi-step process of plasma fractionation. However, this traditional method has some limitations such as risk of contamination, low quality, and quantity of the purified final protein. In this study, we developed two affinity chromatography platforms for the purification of human serum albumin. The recombinant albumin-binding domain (ABD) was expressed and purified using molecular biology techniques. Two types of commercial beads—Cyanogen bromide-activated Sepharose 4B and amine-functionalized magnetic particles—were then functionalized with the recombinant ABD. Protein purification using chromatography columns demonstrated that HSA can be purified to 95 % purity in a single step. Circular dichroism (CD) spectroscopy revealed structural similarities in HSA purified through affinity chromatography and fractionation using the Cohen method. Furthermore, the study of aspirin binding to HSA demonstrated that proteins purified via affinity chromatography and those fractionated by the Cohen method exhibited identical drug-binding affinities. The results of this study may have important implications for the clinical purification of human serum albumin. © 2025 Elsevier B.V.
Enzyme and Microbial Technology (18790909) 180
Prostate cancer is the most prevalent cancer in men. At present, the diagnosis and screening of prostate cancer rely on the essential biomarker known as prostate-specific antigen (PSA). The main purpose of this study was to develop a novel immunoassay for the detection of PSA based on a tri-part split-nanoluciferase system and a nanobody targeting PSA. In our approach, two small components of the split-nanoluciferase, referred to as β9 and β10, were individually fused to two anti-PSA nanobodies, N7 and N23. When these proteins bind to PSA and in the presence of the third nanoluciferase component, called Δ11S, the split-nanoluciferase components are brought into close proximity, facilitating the reassembly of the active nanoluciferase and activation of luminescence. These proteins were expressed in a bacterial expression system, purified, and employed for the intended immunoassay. The developed immunoassay demonstrated the capability to sensitively detect PSA within a linear range from 1.0 to 20.0 ng/mL with LOD of 0.4 ng/mL, and the results obtained through this immunoassay agreed with those derived from the ELISA. Our study indicates that the homogeneous immunoassay developed with nanobodies exhibits remarkable specificity for PSA and can serve as a reliable, fast, and user-friendly test for detecting PSA. © 2024 Elsevier Inc.
Akbari, A. ,
Emamzadeh, R. ,
Nazari, M. ,
Brandstetter, T. ,
Rühe, J. International Journal of Biological Macromolecules (01418130) 259
The growth factor Anterior Gradient 2 (AGR2) has been shown to have an effective role in tissue regeneration, but remained largely unexplored in localized tissue engineering applications. Alginate beads have been proven as safe carriers for protein encapsulation, but they suffer from fragility and uncontrolled protein release. For such alginate systems, little is known about how changes in concentrations and ion-crosslinking affect protein release and accumulation in 3-D matrices. To address these questions, an engineered interpenetrating polymer network (IPN) has been used to synthesize a novel hybrid system consisting of AGR2 loaded beads composed of calcium-crosslinked sodium alginate (SA) and carboxymethyl cellulose (CMC). These beads are embedded in films consisting of SA and polyvinyl alcohol (PVA), using a simple ion gelation technique. We assess protein release kinetics and accumulation within the hybrid system by varying polymer concentrations and cross-linking parameters. The IPN hybrid system maintains controlled release over two weeks, without an initial burst period. Through this approach efficicnt delivery of AGR2 is achieved which in turn effectively mediates cell migration and proliferation, resulting in excellent cell viability and complete wound closure. The described release system opens new perspectives in tissue engineering. © 2023
Protein Journal (18758355) 43(4)pp. 739-750
Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide and results in the emission of a photon of light. Although Renilla luciferase has various applications in biotechnology, its low thermal stability limits the development of its applications. Arginine is a well-known stabilizing amino acid that plays a key role in protein stabilization against inactivation. However, its impact on enzyme properties is unpredictable. This study investigates the impact of arginine on the kinetics and thermal stability of Renilla luciferase. The enzyme's performance was significantly enhanced in the presence of arginine, with catalytic efficiency increasing by 3.31-fold and 3.08-fold when exposed to 0.2 M and 0.3 M arginine, respectively. Additionally, arginine improved the thermal stability of Renilla luciferase. Molecular dynamics simulation showed that the addition of 0.2 M arginine reduced the binding of coelenteramide, the reaction product and an enzyme inhibitor, to the active site of the Renilla luciferase. Therefore, the release of the product was accelerated, and the affinity of Renilla luciferase for coelenterazine increased. Furthermore, Molecular dynamics studies indicated an increased network of water molecules surrounding Renilla luciferase in the presence of 0.2 M arginine. This network potentially enhances the hydrophobic effect on the protein structure, ultimately improving enzyme stability. The findings of this study hold promise for the development of commercial kits incorporating Renilla luciferase. © The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2024.
Biochemical Engineering Journal (1873295X) 209
Liver cancer is one of the most common cancers and the third leading cause of cancer deaths worldwide. Diagnosis and screening for liver cancer rely on the alpha-fetoprotein (AFP) biomarker. This study aimed to pioneer a novel assay for AFP detection utilizing a tri-part split-luciferase system in conjunction with nanobodies targeting AFP. The strategy involved fusing anti-AFP nanobodies P5 and P15 to the split-nanoluciferase components β9 and β10, respectively. Upon binding to AFP and in the presence of the third nanoluciferase component Δ11S, the proximity-induced reassembly of split-nanoluciferase components triggers luciferase activation and luminescence emission. Following expression in a bacterial system, purification, and assay implementation, the developed assay exhibited high sensitivity in detecting AFP within a linear range of 1–20 ng/ml, with a Limit of Detection (LOD) of 0.5 ng/ml. The assay results were in line with those obtained from ELISA, indicating its efficiency. This study highlights the specificity of the homogenous assay developed with nanobodies for AFP, offering a reliable and user-friendly test for AFP detection. © 2024 Elsevier B.V.
Protein Expression and Purification (10960279) 214
A new variant of Renilla luciferase, named Met C-SRLuc 8, was obtained from a random mutagenesis library and expressed in Escherichia coli BL21 (DE3) plys and purified. The results of the enzyme's binding affinity, kinetic stability, and bioinformatic studies demonstrated that leucine 59, located within the hot-spot foldon in the N-terminal domain of the protein, plays a significant role in the stability and activity of Renilla luciferase. These findings may facilitate the engineering of different variants of this enzyme to achieve thermally stable versions for various biotechnological applications. © 2023
Khoshnevisan, G. ,
Emamzadeh, R. ,
Nazari, M. ,
Oliayi, M. ,
Sariri, R. Biochemistry and Biophysics Reports (24055808) 37
Renilla luciferase catalyzes the oxidation of coelenterazine to coelenteramide, resulting in the emission of a photon of light. This study investigated the impact of sorbitol on the structural and kinetic properties of Renilla luciferase using circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations. Our investigation, carried out using circular dichroism and fluorescence analyses, as well as a thermal stability assay, has revealed that sorbitol induces conformational changes in the enzyme but does not improve its thermal stability. Moreover, through kinetic studies, it has been demonstrated that at a concentration of 0.4 M, sorbitol enhances the catalytic efficiency of Renilla luciferase. However, at higher concentrations, sorbitol results in a decrease in catalytic efficiency. Additionally, molecular dynamics simulations have shown that sorbitol increases the presence of hydrophobic pockets on the enzyme's surface. These simulations have also provided evidence that at a concentration of 0.4 M, sorbitol facilitates substrate access to the active site of the enzyme. Nevertheless, at higher concentrations, sorbitol obstructs substrate trafficking, most likely due to its impact on the gateway to the active site. This study may provide insights into the kinetic changes observed in enzymes with buried active sites, such as those with α/β hydrolase fold. © 2023 The Authors
Medical Microbiology and Immunology (14321831) 212(4)pp. 263-270
Adult T-cell leukemia/lymphoma (ATLL) is pathogen-caused cancer that is progressed after the infection by human T-cell leukemia virus type 1. Four significant subtypes comprising acute, lymphoma, chronic, and smoldering have been identified for this cancer. However, there are no trustworthy prognostic biomarkers for these subtypes. We utilized a combination of two powerful network-based and machine-learning algorithms including differential co-expressed genes (DiffCoEx) and support vector machine-recursive feature elimination with cross-validation (SVM-RFECV) methods to categorize disparate ATLL subtypes from asymptomatic carriers (ACs). The results disclosed the significant involvement of CBX6, CNKSR1, and MAX in chronic, MYH10 and P2RY1 in acute, C22orf46 and HNRNPA0 in smoldering subtypes. These genes also can classify each ATLL subtype from AC carriers. The integration of the results of two powerful algorithms led to the identification of reliable gene classifiers and biomarkers for diverse ATLL subtypes. © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Bmc Medical Genomics (17558794) 16(1)
Background: Adult T-cell Leukemia/Lymphoma (ATLL) is a rapidly progressing type of T-cell non-Hodgkin lymphoma that is developed after the infection by human T-cell leukemia virus type 1 (HTLV-1). It could be categorized into four major subtypes, acute, lymphoma, chronic, and smoldering. These different subtypes have some shared clinical manifestations, and there are no trustworthy biomarkers for diagnosis of them. Methods: We applied weighted-gene co-expression network analysis to find the potential gene and miRNA biomarkers for various ATLL subtypes. Afterward, we found reliable miRNA-gene interactions by identifying the experimentally validated-target genes of miRNAs. Results: The outcomes disclosed the interactions of miR-29b-2-5p and miR-342-3p with LSAMP in ATLL_acute, miR-575 with UBN2, miR-342-3p with ZNF280B, and miR-342-5p with FOXRED2 in ATLL_chronic, miR-940 and miR-423-3p with C6orf141, miR-940 and miR-1225-3p with CDCP1, and miR-324-3p with COL14A1 in ATLL_smoldering. These miRNA-gene interactions determine the molecular factors involved in the pathogenesis of each ATLL subtype and the unique ones could be considered biomarkers. Conclusion: The above-mentioned miRNAs-genes interactions are suggested as diagnostic biomarkers for different ATLL subtypes. © 2023, The Author(s).
Process Biochemistry (18733298) 130pp. 245-255
Human Follistatin-like 1, is a protein with multiple disulfide bonds involved in important physiological processes. The current research aimed to evaluate the relationship between the reduction of disulfide bonds and the function of human FSTL1. Human FSTL1 was expressed in a bacterial expression system, purified, and reduced using 4–16 mM dithiothreitol (DTT). The results showed that the proliferation in the presence of the reduced human FSTL1 with 16 mM was better than that of the non-reduced protein. While circular dichroism spectroscopy showed that the content of the secondary structure of human FSTL1 did not change significantly, fluorescence spectroscopy studies show that the surface hydrophobicity of human FSTL1 decreased after reduction of disulfide bonds. Molecular dynamics studies showed that the radius of gyration decreased in the presence of 16 mM DTT. Our results showed that the reduction of disulfide bonds in human FSTL1 increased the hydrophilicity of the surface and might have a compensatory effect on the decrease in the hydrophilicity caused by the lack of post-translational modifications in the bacterial expression system. The present study may be useful for future pathological studies. © 2023 Elsevier Ltd
Nori, Z.Z. ,
Bahadori, M. ,
Moghadam, M. ,
Tangestaninejad, S. ,
Mirkhani, V. ,
Mohammadpoor baltork, I. ,
Jafari, S.S. ,
Emamzadeh, R. ,
Alem, H. Journal of Drug Delivery Science and Technology (17732247) 81
A unique multi-functional magnetic nanocarrier containing thiol groups was designed and applied in drug delivery, hyperthermia and as a contrast agent in MRI. The polymeric nanoparticles were prepared by modification of Fe3-δO4@Au magnetic nanoparticles with a thiol-ended dendrimer. This platform introduces suitable carriers through the grafting of thiol-ended dendrimer for the further binding of an anticancer drug as 6-mercaptopurine (6-MP). The drug is released when exposed to the reductive environmental conditions of cancer cells. Cytotoxicity of the MNP-CC2-6-MP with human breast cancer cell line MCF-7 was examined. Also, the MNP, MNP@Au, and MNP-CC2 nanomaterials were used as magnetic hyperthermia tools and as a contrast agent in MRI and the results were compared with each other. The effect of RF hyperthermia and the released drug in cancer cells were simultaneously investigated as well and confirmed the therapeutic efficiency of those new composite nanomaterials. This magneto dendrimer with multimodal cancer therapy can be selected as an ideal source for biomedicine study. © 2023
Analytical Methods (17599679) 15(32)pp. 3924-3931
For several decades, researchers have been using protein-fragment complementation assay (PCA) approaches for biosensing to study protein-protein interaction for a variety of aims, including viral infection, cellular apoptosis, G protein coupled receptor (GPCR) signaling, drug and substrate screening, and protein aggregation and protein editing by CRISPR/Cas9. As a reporter, NanoLuc (NLuc), a smaller and the brightest engineered luciferase derived from deep-sea shrimp Oplophorus gracilirostris, has been found to have many benefits over other luminescent enzymes in PCA. Inspired by the split green fluorescent protein (GFP) and its β-barrel structure, two split NLuc consisting of peptide fragments have been reported including the binary and ternary NLuc systems. NanoBiT® (large fragment + peptide) has been used extensively. In contrast, tripart split NLuc (large fragment + 2 peptides) has been applied and hardly used, while it has some advantages over NanoBiT in some studies. Nevertheless, tripart NLuc has some drawbacks and challenges to overcome but has several potential characteristics to become a multifunctional and powerful tool. In this review, several aspects of tripart NLuc are studied and a brief comparison with NanoBiT® is given. © 2023 The Royal Society of Chemistry.
Nazari, M. ,
Emamzadeh, R. ,
Jahanpanah, M. ,
Yazdani, E. ,
Radmanesh, R. International Journal of Biological Macromolecules (01418130) 219pp. 1122-1134
High expression of receptor tyrosine-protein kinase erbB-3 (HER3) has been found in several malignancies such as breast cancer. In this study, we designed, produced and evaluated a new affitoxin consisting of a truncated form of diphtheria toxin and a HER3-binding affibody domains. The new affitoxin was expressed in Escherichia coli and purified by affinity chromatography. We evaluated the suitability of affitoxin to kill HER3 positive breast cancer cells with MTT and apoptosis assays. The protein synthesis inhibition was also evaluated. The IC50 value in HER3 negative cells is about 10 times more than HER3 positive cells in new design of affitoxin. The specificity of affitoxin for binding to HER3 positive cells was also investigated with binding assay with flow cytometry. The results show that, the new affitoxin is an anti-cancer molecule with specific binding to HER3 positive cells and may open a new window for the treatment of HER3-positive cancers. © 2022
Research In Pharmaceutical Sciences (17355362) 17(3)pp. 284-293
Background and purpose: Growth hormone (GH) has been known as a crucial metabolic hormone expressed at the pituitary and the other number of cells and tissues and responsible for body growth. Because of the short half-life of GH, daily subcutaneous injections were shown to be more effective for GH therapy. This represents a burden for patients. So, there is a strong effort from the industry to create a long-acting form of GH and lots of technologies like GH fusion proteins are used to increase GH half-life. Experimental approach: In this study, the Fc domain of human IgG1 with serine-glycine linkers was attached to the C-terminal of a GH superagonist via molecular cloning. The presence of recombinant vector in E. coli host was confirmed by PCR. SDS-PAGE and western blot analysis showed the expression of recombinant proteins in the bacterial lysate. The binding ability to growth hormone receptors is determined by ELISA. Findings / Results: Our results showed that the novel SupGH-Fc has a good binding affinity to its receptor in ELISA in comparison to standard GH, although it has a big size. Conclusion and implications: Our data in this study clearly demonstrated the expression of the SupGH-Fc in a recombinant protein expression system. It is an introduction to the production of the new recombinant GH, which can bind to its receptor more effectively than commercial growth hormones and also might have a longer half-life. © 2022 Wolters Kluwer Medknow Publications. All rights reserved.
Frontiers in Immunology (16643224) 13
Severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2) is the causative virus of the pandemic coronavirus disease 2019 (COVID-19). Evaluating the immunological factors and other implicated processes underlying the progression of COVID-19 is essential for the recognition and then the design of efficacious therapies. Therefore, we analyzed RNAseq data obtained from PBMCs of the COVID-19 patients to explore coding and non-coding RNA diagnostic immunological panels. For this purpose, we integrated multiple RNAseq data and analyzed them overall as well as by considering the state of disease including severe and non-severe conditions. Afterward, we utilized a co-expressed-based machine learning procedure comprising weighted-gene co-expression analysis and differential expression gene as filter phase and recursive feature elimination-support vector machine as wrapper phase. This procedure led to the identification of two modules containing 5 and 84 genes which are mostly involved in cell dysregulation and innate immune suppression, respectively. Moreover, the role of vitamin D in regulating some classifiers was highlighted. Further analysis disclosed the role of discriminant miRNAs including miR-197-3p, miR-150-5p, miR-340-5p, miR-122-5p, miR-1307-3p, miR-34a-5p, miR-98-5p and their target genes comprising GAN, VWC2, TNFRSF6B, and CHST3 in the metabolic pathways. These classifiers differentiate the final fate of infection toward severe or non-severe COVID-19. The identified classifier genes and miRNAs may help in the proper design of therapeutic procedures considering their involvement in the immune and metabolic pathways. Copyright © 2022 Zarei Ghobadi, Emamzadeh, Teymoori-Rad and Afsaneh.
Emamzadeh, R. ,
Ghobadi, M.Z. ,
Afsaneh, E. ,
Ghobadi, M.Z. ,
Emamzadeh, R. ,
Afsaneh, E. Bmc Cancer (14712407) 22(1)
Background: Adult T-cell Leukemia/Lymphoma (ATLL) is a cancer disease that is developed due to the infection by human T-cell leukemia virus type 1. It can be classified into four main subtypes including, acute, chronic, smoldering, and lymphoma. Despite the clinical manifestations, there are no reliable diagnostic biomarkers for the classification of these subtypes. Methods: Herein, we employed a machine learning approach, namely, Support Vector Machine-Recursive Feature Elimination with Cross-Validation (SVM-RFECV) to classify the different ATLL subtypes from Asymptomatic Carriers (ACs). The expression values of multiple mRNAs and miRNAs were used as the features. Afterward, the reliable miRNA-mRNA interactions for each subtype were identified through exploring the experimentally validated-target genes of miRNAs. Results: The results revealed that miR-21 and its interactions with DAAM1 and E2F2 in acute, SMAD7 in chronic, MYEF2 and PARP1 in smoldering subtypes could significantly classify the diverse subtypes. Conclusions: Considering the high accuracy of the constructed model, the identified mRNAs and miRNA are proposed as the potential therapeutic targets and the prognostic biomarkers for various ATLL subtypes. © 2022, The Author(s).
PLoS ONE (19326203) 17(1 January)
Human T-cell Leukemia Virus type-1 (HTLV-1) is an oncovirus that may cause two main life-threatening diseases including a cancer type named Adult T-cell Leukemia/Lymphoma (ATLL) and a neurological and immune disturbance known as HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). However, a large number of the infected subjects remain as asymptomatic carriers (ACs). There is no comprehensive study that determines which dysregulated genes differentiate the pathogenesis routes toward ATLL or HAM/TSP. Therefore, two main algorithms including weighted gene co-expression analysis (WGCNA) and multi-class support vector machines (SVM) were utilized to find major gene players in each condition. WGCNA was used to find the highly co-regulated genes and multi-class SVM was employed to identify the most important classifier genes. The identified modules from WGCNA were validated in the external datasets. Furthermore, to find specific modules for ATLL and HAM/TSP, the non-preserved modules in another condition were found. In the next step, a model was constructed by multi-class SVM. The results revealed 467, 3249, and 716 classifiers for ACs, ATLL, and HAM/TSP, respectively. Eventually, the common genes between the WGCNA results and classifier genes resulted from multi-class SVM that also determined as differentially expressed genes, were identified. Through these step-wise analyses, PAIP1, BCAS2, COPS2, CTNNB1, FASLG, GTPBP1, HNRNPA1, RBBP6, TOP1, SLC9A1, JMY, PABPC3, and PBX1 were found as the possible critical genes involved in the progression of ATLL. Moreover, FBXO9, ZNF526, ERCC8, WDR5, and XRCC3 were identified as the conceivable major involved genes in the development of HAM/TSP. These genes can be proposed as specific biomarker candidates and therapeutic targets for each disease. © 2022 Zarei Ghobadi, Emamzadeh. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PLoS ONE (19326203) 16(2 February)
Adult T-cell leukemia/lymphoma (ATLL) is virus-caused cancer that originates from the infection by human T-cell leukemia virus type 1. ATLL dysregulates various biological pathways related to the viral infection and cancer progression through the dysexpression of miRNAs and mRNAs. In this study, the potential regulatory subnetworks were constructed aiming to shed light on the pathogenesis mechanism of ATLL. For this purpose, two mRNA and one miRNA expression datasets were firstly downloaded from the GEO database. Next, the differentially expressed genes and miRNAs (DEGs and DE-miRNAs, respectively), as well as differentially co-expressed gene pairs (DCGs), were determined. Afterward, common DEGs and DCGs targeted by experimentally validated DE-miRNAs were explored. The oncogenic and anti-oncogenic miRNA-mRNA regulatory subnetworks were then generated. The expression levels of four genes and two miRNAs were examined in the blood samples by qRT-PCR. The members of three oncogenic/anti-oncogenic subnetworks were generally enriched in immune, virus, and cancer-related pathways. Among them, FZD6, THBS4, SIRT1, CPNE3, miR-142-3p, and miR-451a were further validated by realtime PCR. The significant up-regulation of FZD6, THBS4, and miR-451a as well as downregulation of CPNE3, SIRT1, and miR-142-3p were found in ATLL samples than normal samples. The identified oncogenic/anti-oncogenic subnetworks are pieces of the pathogenesis puzzle of ATLL. The ultimate winner is probably an oncogenic network that determines the final fate of the disease. The identified genes and miRNAs are proposed as novel prognostic biomarkers for ATLL. Copyright: © 2021 Ghobadi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Virology Journal (1743422X) 18(1)
Background: Human T-cell Leukemia Virus type-1 (HTLV-1) is a retrovirus that causes two diseases including Adult T-cell Leukemia/Lymphoma (ATLL cancer) and HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP, a neurodegenerative disease) after a long latency period as an asymptomatic carrier (AC). There are no obvious explanations about how each of the mentioned diseases develops in the AC carriers. Finding the discriminative molecular factors and pathways may clarify the destiny of the infection. Methods: To shed light on the involved molecular players and activated pathways in each state, differentially co-expressed modules (DiffCoEx) algorithm was employed to identify the highly correlated genes which were co-expressed differently between normal and ACs, ACs and ATLL, as well as ACs and HAM/TSP samples. Through differential pathway analysis, the dysregulated pathways and the specific disease-genes-pathways were figured out. Moreover, the common genes between the member of DiffCoEx and differentially expressed genes were found and the specific genes in ATLL and HAM/TSP were introduced as possible biomarkers. Results: The dysregulated genes in the ATLL were mostly enriched in immune and cancer-related pathways while the ones in the HAM/TSP were enriched in immune, inflammation, and neurological pathways. The differential pathway analysis clarified the differences between the gene players in the common activated pathways. Eventually, the final analysis revealed the involvement of specific dysregulated genes including KIRREL2, RAB36, and KANK1 in HAM/TSP as well as LTB4R2, HCN4, FZD9, GRIK5, CREB3L4, TACR2, FRMD1, LHB, FGF3, TEAD3, GRIN2D, GNRH2, PRLH, GPR156, and CRHR2 in ATLL. Conclusion: The identified potential prognostic biomarkers and therapeutic targets are proposed as the most important platers in developing ATLL or HAM/TSP. Moreover, the proposed signaling network clarifies the differences between the functional players in the activated pathways in ACs, ATLL, and HAM/TSP. © 2021, The Author(s).
Nazari, M. ,
Minai-tehrani, A. ,
Mousavi, S. ,
Zamani koukhaloo, S. ,
Emamzadeh, R. International Journal of Biological Macromolecules (01418130) 189pp. 948-955
Human epidermal growth factor receptor 3 (HER3) has rapidly gained much attention as a promising target for cancer treatment. The increasing recognition of HER3 roles in a number of HER family-driven cancers has led to studies aimed at targeting this receptor and developing HER3-targeted platforms with the ability to deliver therapeutic genes. We have previously indicated that the flexible linker and one unit of RALA in affibody-based platform could target HER3 and deliver its cargo. Based on the previous finding, in a new class of affibody-based platforms, we used two different linkers and RALA units and then compared their effectiveness on targeting and delivering specified genes to HER3 positive cells. Our results clearly showed that our biopolymeric platforms can successfully condense DNA into nanoparticles and object the overexpressed HER3 receptors and then transfer specific genes. Our affibody-based platform containing a rigid linker and one RALA unit presents an adequate transfection efficacy and low toxicity (based on MTT and apoptosis assays), however, the platform containing two RALA units and a flexible linker demonstrated high transfection efficacy while having modest toxicity in HER3 positive breast cancer cells. This may pave the way for further innovative applications of recombinant biopolymer when stable and economical productions need to be definitely considered. © 2021 Elsevier B.V.
Mortazavi, M. ,
Hosseinkhani, S. ,
Torkzadeh-mahani, M. ,
Lotfi, S. ,
Emamzadeh, R. ,
Ghasemi, Y. Current Proteomics (18756247) 18(3)pp. 424-434
Aim: The purpose of this study was to conduct in silico analysis of the Lampyroidea maculata luciferase enzyme. Background: Bioluminescence is the production and emission of light by the luciferase enzymes in a living organism. The luciferase enzyme has been widely used in biotechnology due to its excel-lent properties. Objective: Recently, the new gene of the luciferase from the Lampyroidea maculata, has been cloned and characterized. Methods: In the following, in silico analysis of this enzyme were conducted by structural modeling in the I-TASSER web server. Finally, the binding site properties were studied using the AutoDock Vina. In the following, the codon usage bias parameters as the CAI, CBI, ENC, and the base composition of this sequence were studied. After analysis of the base composition, it was found that the coding DNA sequence is rich in AT. Moreover, the indices GC1, GC2, and GC3 were computed to establish the relationship among three codon positions. On the other hand, the GC2 has a much low-er frequency. Results: By molecular modeling, some rare codons were identified that may have a critical role in the structure and function of this luciferase. The GC3% of the CDs was 17/304 and GC3 Skewness was 0.115. The GC content at the first codon position (GC1) and second codon position (GC2) was compared with that of the third codon position (GC3). AutoDock Vina was used in the molecular docking that recognizes some residues that yield closely related to the ADSL binding site. This bioinformatics analyzes play an important role in the design of new drugs. Conclusion: Previous studies show that mutation pressure and natural selection reasons for codon usage variation among different genes. If the mutational pressure was the only effective reason for the codon usage bias, then the frequency of nucleotides C and G should be equal to A and T at the synonymous third codon position. By these analyses, a new understanding of the sequence and structure of this enzyme was created, and our findings can be used in some fields of clinical and in-dustrial biotechnology. © 2021 Bentham Science Publishers.
Rezaei, M. ,
Rabbani, M. ,
Zarkesh-esfahani, H. ,
Emamzadeh, R. ,
Abtahi, H. Archives of Microbiology (1432072X) 203(5)pp. 2591-2596
This study was designed to introduce the recombinant Lactococcus lactis MG1363 as a cell factory candidate for production of recombinant Brucella melitensis Omp16-Human IL2 (r-Omp16-IL2) and to suggest it as a promising safe, non-pathogenic mucosal live vaccine against brucellosis. Three groups of BALB/c mice (10 mice per group) were intragastrically administrated with phosphate-buffered saline (PBS), L. lactis harboring the empty pAMJ2008 plasmid and with L. lactis expressing rOmp-IL2. The first two groups were classified as control groups and the third one is indicated as treatment group. Another group was injected by the intraperitoneal (i.p.) route with purified rOmp16-IL2 protein. The total serum IgG of each group was assessed with indirect ELISAs at two days before immunization and also two weeks after the last immunization. Results showed that BALB/c mice intragastrically administrated with L. lactis expressing rOmp-IL2 had dominant IgG response compared to the control (PBS administrated) group (P < 0.05). The level of IgG was significantly increased by intraperitoneally injection of recombinant Omp-IL2 in adjuvant compared to the intragastrically administration of PBS and L. lactis/pAMJ2008 as control groups, and also compared to L. lactis/pAMJ2008-rOmp-IL2 (P < 0.05). Our findings provide the use of L. lactis rOmp16-IL2 as a new promising alternative safe strategy than presently live attenuated vaccines toward developing an oral vaccine or subunit-based vaccine against brucellosis. © 2021, The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.
Journal of Applied Microbiology (13652672) 130(2)pp. 493-503
Aims: Diagnosis of Staphylococcus aureus is important in various diseases from hospital-acquired infections to foodborne diseases. This work reports two new luminescent affiprobes for specific detection of S. aureus. Methods and Results: To develop advanced luminescent affiprobes, enhanced green fluorescent protein (EGFP) was flanked by single and double repeats of ZpA963 affibody using molecular biology studies. The recombinant proteins including fluorescent monomeric affibody (fA1) and fluorescent dimeric affibody (fA2) were expressed in the bacterial expression system, purified and used to identify the S. aureus. Fluorescence microscope and flow cytometry results demonstrated that the treated samples with fA1 and fA2 had relatively high fluorescent mean intensities in comparison to the untreated S. aureus cells. Moreover, it was revealed that ‘fA2’ affiprobe had lower dissociation constant value (about 25-fold) and was more effective for detection of S. aureus than the ‘fA1’ affiprobe. In addition, the binding of the affiprobes for some other pathogenic bacteria i.e. Escherichia coli, Bacillus cereus, Enterococcus faecalis and Staphylococcus saprophyticus was examined. Expectedly, no cross-reaction was observed for binding the constructed affiprobes to these bacteria, eliminating possibilities for false positive results. Conclusions: The results show that ‘fA1’ affiprobe and ‘fA2’ affiprobe are two new efficient luminescent affiprobes for detecting S. aureus. Significance and Impact of the Study: We developed a new approach for detection of Staphylococcus aureus in a simple one-step process and in low concentrations of probes. In the best of our knowledge, this is the first study to direct detection of bacterial cells by affiprobes and may be used to develop new diagnostic kits. © 2020 The Society for Applied Microbiology
Aliramaei, M.R. ,
Rabbani, M. ,
Rahmani, M.R. ,
Zarkesh-esfahani, H. ,
Emamzadeh, R. Microbial Pathogenesis (10961208) 142
Helicobacter pylori is a gram negative pathogen which commonly colonizes in the human gastric mucosa from early childhood and persists throughout life. CagL is a 27-kDa protein that is located at the tip of T4SS pili and highly conserved among pathogenic H. pylori strains. Lactic acid bacteria especially Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. In this study H. pylori CagL gene was cloned in pAMJ2008 vector and transferred to Lactococcus lactis MG1363 as the host for CagL antigen production. This recombinant bacterium was orally subjected to mice, and the immune response to CagL was evaluated by ELISA. Intracellular expression of CagL protein was confirmed by Western blot analysis. Mucosal immunization of mice with the recombinant L. lactis significantly stimulated CagL-Specific antibodies: IgA, IgG, cytokine IL-17 and IN-γ. Moreover, the specific anti-CagL IgA response was detected in the feces of immunized mice. These results indicate that CagL of H. pylori was successfully expressed in L. lactis and the recombinant bacteria can be potentially used as an edible vaccine against H. pylori infection. © 2019
Rezaei, M. ,
Rabbani, M. ,
Esfahani, S.H.Z. ,
Emamzadeh, R. ,
Abtahi, H. Canadian Journal of Microbiology (00084166) 66(1)pp. 39-45
The use of the food-grade bacterium Lactococcus lactis as a new cell factory is a promising alternative expression system for producing a desired protein. The Omp16-IL2 fusion protein antigen was cloned, expressed, and purified in this study. The Omp16-IL2 fusion gene was designed and cloned in pGH plasmid with appropriate restriction sites and subcloned in pAMJ2008 expression vector digested with the same enzymes. The purified recombinant constructed pAMJ-rOmp-IL2 was introduced into L. lactis subsp. cremoris MG1363 by electrotransfor-mation. Finally, the expression and purification of Omp16-IL2 fusion protein was investigated. This study reports the construction of a recombinant L. lactis expressing the Omp16-IL2 fusion protein as an oral Lactococcus-based vaccine, as compared with commonly used live attenuated vaccines, for future studies against brucellosis. © 2020, Canadian Science Publishing. All rights reserved.
Nazari, M. ,
Emamzadeh, R. ,
Minai-tehrani, A. ,
Nasir shirazi, M. ,
Naimi, A. Analytical Methods (17599679) 11(33)pp. 4233-4241
A novel receptor tyrosine-protein kinase erbB-2 (HER2)-binding affiprobe-named ZHER2:342-RLuc-was designed, produced and assessed in terms of function to detect HER2 positive cells and for preliminary clinical diagnosis. The ZHER2:342 affibody with affinity for HER2 was fused to Renilla luciferase, and the purified affiprobe was used for detection of HER2 positive cells as well as evaluation of surgical specimens from breast cancer patients. The results showed that the affiprobe can specifically bind to HER2 positive cells, and therefore, it may be used for molecular detection. © 2019 The Royal Society of Chemistry.
International Journal of Biological Macromolecules (01418130) 138pp. 755-763
Molecular probes have become an important tool to decipher cell and molecular events, as well as contributing to clinical diagnosis. In this study, we report on a novel bioluminescence affiprobe and evaluate its functionality by monitoring HER2 expression in the prostate cancer cells. The proposed bioluminescence affiprobe consists of a binding domain, an HER2 specific affibody molecule and a bioluminescent domain, recombinant Renilla luciferase. The experiments indicate that bioluminescence affiprobe can serve as a reliable and user-friendly probe for the detection of human HER2 positive prostate cells as well as ex vivo based detection of HER2-positive human prostate cancer specimens using luminometeric-based assays. © 2019 Elsevier B.V.
Nazari, M. ,
Zamani koukhaloo, S. ,
Mousavi, S. ,
Minai-tehrani, A. ,
Emamzadeh, R. ,
Cheraghi, R. Macromolecular Bioscience (16165195) 19(11)
Despite the initial successes of gene delivery applications, they faced on several intrinsic drawbacks including toxicity and immunogenicity. Therefore, alternative gene-delivery systems derived from recombinant peptides have emerged and is rapidly developing. Human epidermal growth factor receptor-3 (HER3) shows high activity in tumor resistance to anti-human epidermal growth factor receptor 2 (HER2) therapies. In this study, an affibody molecule against HER3 is conjugated to a biomimetic peptide RALA (an amphipathic and cationic peptide enriched with arginine) and the ability of the fusion vector for targeting HER3 and afterward delivering specific genes in breast cancer cells is evaluated. The results demonstrate that the biopolymeric platform, which contains an affibody-conjugated RALA peptide, can effectively condense DNA into nanoparticles and target the overexpressed HER3 receptors in breast cancer cells and transfer specific genes. The use of such a recombinant biopolymer may pave the way for the development of sensitive and effective diagnostic and treatment tool for breast cancer. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Rezaei, M. ,
Rabbani, M. ,
Zarkesh-esfahani, H. ,
Emamzadeh, R. ,
Abtahi, H. Infectious Disorders - Drug Targets (22123989) 19(1)pp. 36-45
Background: Brucellosis is an infectious disease caused by Brucella bacteria that cause disease in animals and humans. Brucellosis is one of the most common zoonotic diseases transmitted from animals-to-human through direct contact with infected animals and also consumption of unpasteurized dairy products. Due to the wide incidence of brucellosis in Iran and economical costs in industrial animal husbandry, Vaccination is the best way to prevent this disease. All of the available commercial vaccines against brucellosis are derived from live attenuated strains of Brucella but because of the disadvantage of live attenuated vaccines, protective subunit vaccine against Brucella may be a good candidate for the production of new recombinant vaccines based on Brucella Outer Membrane Protein (OMP) antigens. In the present study, comprehensive bioinformatics analysis has been conducted on prediction software to predict T and B cell epitopes, the secondary and tertiary structures and antigenicity of Omp16 antigen and the validation of used software confirmed by experimental results. Conclusion: The final epitope prediction results have proposed that the three epitopes were predicted for the Omp16 protein with antigenicity ability. We hypothesized that these epitopes likely have the protective capacity to stimulate both the B-cell and T-cell mediated immune responses and so may be effective as an immunogenic candidate for the development of an epitope-based vaccine against brucellosis. © 2019 Bentham Science Publishers.
Khoshnevisan, G. ,
Emamzadeh, R. ,
Nazari, M. ,
Rasa, S.M.M. ,
Sariri, R. ,
Hassani, L. International Journal of Biological Macromolecules (01418130) 117pp. 617-624
Renilla Luciferase is a bioluminescent enzyme which is broadly implemented as protein reporter in biology-related researches. In this study, new evidences on the kinetics, structure, and dynamics of Renilla luciferase solvated in binary mixtures of glycerol and water using MD simulation along with experimental procedures including fluorescence and CD spectroscopy were obtained. The results indicated that the Renilla luciferase activity decreased at 0.8 and 1.2 M of glycerol through the obstruction of enzyme emitter site. The present study may describe a new molecular mechanism of decreasing enzyme activity in the presents of glycerol. © 2018 Elsevier B.V.
Journal of Cellular Biochemistry (07302312) 119(2)pp. 1780-1790
Renilla Luciferase (RLuc) is a blue light emitter protein which can be applied as a valuable tool in medical diagnosis. But due to lack of the crystal structure of RLuc-ligand complex, the functional motions and catalytic mechanism of this enzyme remain largely unknown. In the present study, the active site properties and the ligand-receptor interactions of the native RLuc and its red-shifted light emitting variant (Super RLuc 8) were investigated using molecular docking approach, molecular dynamics (MD) analysis, and MM-PBSA method. The detailed analysis of the main clusters led to identifying a lid-like structure and its functional motions. Furthermore, an induced-fit mechanism is proposed where ligand-binding induces conformational changes of the active site. Our findings give an insight into the deeper understanding of RLuc conformational changes during binding steps and ligand-receptor pattern. Moreover, our work broaden our understanding of how active site geometry is adjusted to support the catalytic activity and red-shifted light emission in Super RLuc 8. © 2017 Wiley Periodicals, Inc.
Gene (18790038) 610pp. 112-117
Hepcidin25 is a small cysteine-rich peptide hormone known as a new class of antimicrobial peptides. The purpose of the present study was to express, purify and investigate the antibacterial properties of recombinant human hepcidin25 protein production in Escherichia coli. Human hepcidin25 gene was optimized and fused to a small ubiquitin-related modifier (SUMO) gene for higher expression. Then SUMO-hepcidin25 was cloned into the pET-32a (+) vector and expressed in E. coli Origami. The fusion protein with a molecular weight of approximately 35 kDa was analyzed on SDS-PAGE gel. The highest expression was observed after 6 h induction and the fusion protein consisted approximately 47% of the total cellular protein. The purified SUMO-hepcidin25 purity was determined to be higher than 95%, with a final yield of 3.9 mg l−1 of media. The recombinant hepcidin25 showed antibacterial activity against both Gram negative (Klebsiella pneumonia) and Gram positive (Staphylococcus aureus and Bacillus cereus) bacteria with minimum inhibitory concentrations (MICs) of 150 μg ml− 1, 18.7 μg/ml− 1 and 37.5 μg/ml− 1, respectively. These results indicated that thioredoxin and SUMO dual fusion system is an efficient production system for synthesis functional human hepcidin25. © 2017 Elsevier B.V.
Biochimica et Biophysica Acta - Proteins and Proteomics (15709639) 1865(2)pp. 252-259
Renilla luciferase (RLuc), also known as Renilla-luciferin 2-monooxygenase, is a light producing enzyme used in many biotechnological applications such as bioreporters. However, its kinetics stability -especially at higher temperatures- is a limiting factor for developing thermostable bioreporters. The aim of this study was to improve the stability of super Renilla luciferase 8 (SRLuc 8) which is a red-emitter variety of RLuc at higher temperatures, by introduction of a disulfide bridge into its structure. In this study, the choice of the proper disulfide bond formation was based on computational methods and enzyme functionality (active site position) which is called geometric-functional method. N45 and A71 at the N-terminal of the enzyme were selected for directed evolution. The engineered luciferase was called C-SRLuc 8 and its activity and stability were assayed. The results indicated that the kinetic stability of C-SRLuc 8 increased significantly at 60 °C to 70 °C as compared to SRLuc 8; the residual activity of C-SRLuc 8 was approximately 20% after incubation at 65 °C for 5 min. Moreover, the enzyme activity decreased compared with SRLuc 8. The molecular basis of the structural changes was considered using molecular dynamics simulations and the results indicated that the N45C/A71C crosslink was involved in a hotspot foldon which seemed to be the rate-limiting step of conformational collapse at higher temperatures. The present study may provide an opportunity for the development of the next-generation of thermostable RLuc-based biosensors. © 2016
Nazari, M. ,
Zarnani, A. ,
Ghods, R. ,
Emamzadeh, R. ,
Najafzadeh, S. ,
Minai-tehrani, A. ,
Mahmoudian, J. ,
Yousefi, M. ,
Vafaei, S. ,
Massahi, S. Protein Expression and Purification (10960279) 133pp. 139-151
Placenta specific -1 (PLAC1) has been recently introduced as a small membrane-associated protein mainly involved in placental development. Expression of PLAC1 transcript has been documented in almost one hundred cancer cell lines standing for fourteen distinct cancer types. The presence of two disulfide bridges makes difficult to produce functional recombinant PLAC1 in soluble form with high yield. This limitation also complicates the structural studies of PLAC1, which is important for prediction of its physiological roles. To address this issue, we employed an expression matrix consisting of two expression vectors, five different E. coli hosts and five solubilization conditions to optimize production of full and truncated forms of human PLAC1. The recombinant proteins were then characterized using an anti-PLAC1-specific antibody in Western blotting (WB) and enzyme linked immunosorbent assay (ELISA). Structure of full length protein was also investigated using circular dichroism (CD). We demonstrated the combination of Origami™ and pCold expression vector to yield substantial amount of soluble truncated PLAC1 without further need for solubilization step. Full length PLAC1, however, expressed mostly as inclusion bodies with higher yield in Origami™ and Rosetta2. Among solubilization buffers examined, buffer containing Urea 2 M, pH 12 was found to be more effective. Recombinant proteins exhibited excellent reactivity as detected by ELISA and WB. The secondary structure of full length PLAC1 was considered by CD spectroscopy. Taken together, we introduced here a simple, affordable and efficient expression system for soluble PLAC1 production. © 2017 Elsevier Inc.
Safavi, A. ,
Emamzadeh, R. ,
Nazari, M. ,
Ehsani m., ,
Zarkesh-esfahani, H. ,
Rahgozar, S. Molecular BioSystems (1742206X) 13(3)pp. 470-475
The human immunodeficiency virus (HIV) destroys CD4+ lymphocytes and monitoring these cells is one of the best techniques for studying HIV infection. In the present study a novel bioluminescent probe, super RLuc8-sFv, is developed in order to detect human CD4+ cells by fusion of an anti-human CD4 sFv to the C-terminus of super RLuc8. The results indicate that the probe can bind to CD4+ cells via its sFv domain; also it emits visible light through its signalling domain. Super RLuc8-sFv provides a new gateway for detection of human CD4+ cells using luminometric-based assays and may reduce the difficulties involved in, and the cost of, HIV-related diagnostic tests. © The Royal Society of Chemistry.
Rahnama, S. ,
Saffar, B. ,
Kahrani, Z.F. ,
Nazari, M. ,
Emamzadeh, R. Enzyme and Microbial Technology (18790909) 96pp. 60-66
Renilla luciferase is a bioluminescent enzyme which is broadly used as a reporter protein in molecular biosensors. In this study, a novel luciferase with desired light emission wavelength and thermostability is reported. The results indicated that the new luciferase, namely super RLuc8, had a red-shifted spectrum and showed stable light emission. Super RLuc8 showed a 10-fold (p-value = 0.0084) increase in the thermostability at 37 °C after 20 min incubation, in comparison to the native enzyme. The optimum temperature of the mutant increased from 30 to 37 °C. Molecular dynamics simulation analysis indicated that the increased thermostability was most probably caused by a better structural compactness and more local rigidity in the regions out of the emitter site. © 2016 Elsevier Inc.
International Journal of Biological Macromolecules (01418130) 105pp. 66-73
Renilla luciferase (Rluc) from Renilla reniformis is an appropriate protein reporter for the detection of specific molecular targets due to its bioluminescent feature, although its relatively low stability limits the application. To investigate the effects of trehalose and sucrose as chemical chaperones on the kinetic stability of Rluc, we assayed the activity of the enzyme in the presence of these additives at high temperatures and to comprehend the mechanism of stability, molecular dynamic (MD) simulation was carried out. In the presence of trehalose a thermostabilizing effect which was considerable in comparison with other systems was observed. It is proposed that a wide radial like network of trehalose molecules supports α-helix structures that are located in the N-terminus and C-terminus of the protein. However, in the water simulation box, these helices alter to instable structures at high temperatures. Reduction of the fluctuation of these helices in the presence of trehalose molecules, may prevent the protein from unfolding and increase its shelf life. © 2017 Elsevier B.V.
Aminjafari, A. ,
Miroliaei, M. ,
Angelova, V.T. ,
Emamzadeh, R. ,
Djukic, M.M. ,
Djuric, A. ,
Saso, L. Electronic Journal Of Biotechnology (07173458) 24pp. 43-48
Background: Synthesized aminocoumarins are heterocyclic compounds possessing potential for the treatment of insulin-dependent diabetes mellitus with unexplored anti-glycative action. Results: In this study 4-aminocoumarin derivatives (4-ACDs) were evaluated in vitro for antiglycation (AG) activities by using the human serum albumin (HSA)/glucose system, for 8 weeks of incubation. The glycation and conformational alteration of HSA in the presence of the tested compounds were evaluated by Congo red assay, fluorescence and circular dichroism spectroscopy. The antioxidant (AO) capacity were also tested by four different assays including: DPPH (2,2′-diphenyl-1-picrylhydrazyl radical), ABTS (2,2-azinobis (3-ethylbenzothiazoline-6-sulphonate) diammonium salt), FRAP (ferric reducing antioxidant power) and β-carotene-linoleic acid assay. The tested compounds showed AG and AO effects. The intensity of the accomplished AO potential is related to the type of the used assay. Significant alterations in the secondary (monitored by CD spectropolarimetry) and tertiary structure (assessed by spectrofluorimetry) of HSA upon glycation were mitigated by the 4-ACDs, suggesting their suppressive role in the late stage (post-Amadori) of the HSA glycation. Conclusions: By the analogues, in vitro ascertained AO and AG properties of 4-ACDmay be recognized as rationale for their protective role against oxidative changes of proteins, thereby precluding diabetic complications in humans. © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved.
Mirbagheri m., M. ,
Nahvi i., I. ,
Emamzadeh, R. ,
Emtiazi, G. ,
Shirani, E. International Journal of Environmental Science and Technology (17351472) 13(1)pp. 31-38
The neutraceutical and pharmaceutical application of essential fatty acids is much cleared. Alpha-linolenic acid (ALA) is omega-3 fatty acid and generally known to have beneficial effects in CVS, CNS and other diseases. The purpose of the present investigation is to produce essential fatty acid, especially ALA by Mucorcircinelloides from oil wastes. Five oil wastes collected from food industries were used as carbon sources, and the contents of total lipids, biomass and fatty acids were examined during 168 h. The ability of oil waste degradation was determined by measuring of biochemical oxygen demand (BOD) and chemical oxygen demand (COD). Interestingly, 76 % reduction in BOD and 68 % reduction in COD by this strain were achieved, and M. circinelloides could be a good candidate for oil waste treatment. In order to enhance ALA production, fermentation variables were chosen in accordance with the fractional design and further optimized by the response surface method. The statistical model was constructed via central composite design. Following the optimization step, ALA production increased by approximately 44.3 %, when compared to the screening step. The results indicate that carrying out the fermentation under the conditions of oil waste 4.37 %, yeast extract at 0.65 g/l, (NH4)2SO4 at 0.38 g/l, an agitation rate of 180 rpm and fermentation time of 3 days will increase the ALA production up to 108.57 mg/l. In this study, a new renewable source of ALA was employed and optimized successfully for the production of valuable fatty acids. © 2015, Islamic Azad University (IAU).
International Journal of Biological Macromolecules (01418130) 93pp. 1253-1260
Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6–66 mM) and isopropanol (19.3–76 mM) the Km changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the “non-binding” model was proposed. © 2016 Elsevier B.V.
Ghaedizadeh, S. ,
Emamzadeh, R. ,
Nazari, M. ,
Rasa, S.M.M. ,
Zarkesh-esfahani, H. ,
Yousefi, M. Biochemical Engineering Journal (1873295X) 105pp. 505-513
Renilla luciferase is a bioluminescent enzyme which is broadly implemented as cellular marker in many biology-related researches. In order to understand how the of enzyme is influenced by ionic liquids, we performed a combined experimental and molecular dynamics simulation study of Renilla luciferase in ionic liquids. Our results revealed a main tunnel in the structure of Renilla luciferase, and demonstrated that the conformational changes of the main tunnel are directly related to enzyme activity and the enzyme activity is decreased severely in the presence of [BMIM][BF4] and [BMIM][PF6]. While the main tunnel shows a screw in shape in the region near to active site in the presence of [BMIM][BF4], the entrance to the main tunnel is modulated by alanine 150 and isoleucine 265 in the presence of [BMIM][PF6]. The protein-ionic liquid interactions also have impact on the structure of enzyme, especially in [BMIM][PF6], where interactions of Renilla luciferase (with α/β Fold) with fluorine anions causes a conformational collapse in the exposed α-helices. Based on the results, the structural distortions in Renilla luciferase in the presence of ionic liquids is started from the outer layer of the enzyme, a model which is called the "α-shield collapse" model. © 2015 Elsevier B.V.
RSC Advances (20462069) 5(70)pp. 56578-56582
A new and advanced CD4-binding reporter has been designed, produced and functionally assayed for the detection of T-helper cells. The sFv fragment fused N-terminally to the EGFP is a fluorescence probe successfully employed for counting T CD4+ cells using flow cytometry. © The Royal Society of Chemistry 2015.
Farzannia a., ,
Roghanian, R. ,
Zarkesh-esfahani, H. ,
Nazari, M. ,
Emamzadeh, R. Analyst (00032654) 140(5)pp. 1438-1441
A novel and advanced Fc-binding probe-FcUni-RLuc namely-has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence-HWRGWV-was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells. This journal is © The Royal Society of Chemistry.
Iranian Journal Of Biotechnology (23222921) 13(2)pp. 25-30
Background: The production of waste pollutants has become a major problem for many food and oil industries. However, oil wastes can provide alternative substrates for industry, which could help to solve environmental pollution problems. Furthermore, oil wastes can be used as substrates to produce unsaturated fatty acids, which are important for health. Objectives: The production of fatty acids in fungi using oil wastes and renewable substrates were investigated. Material and Methods: Oil waste sources were obtained from food factories and restaurants (F1, F2, F3, R1, and R2). Cunninghamella echinulata DSM1905 and Rhizopus stolonifer DSM2194 were used to treat the wastes. Changes in lipid and fatty acid contents, biomass, and pH were monitored. Results: C. echinulata produced about 18.4 and 20.1% gamma linolenic acid (GLA) from the R1 and R2 oil wastes, respectively. It also produced 9.3% and 12.4% linolenate from the F2 and F3 wastes. R. stolonifer produced 21% GLA from R1 and 9.3% linolenate from F3. C. echinulata reduced biological oxygen demand (BOD) and chemical oxygen demand (COD) by 67%-74% and 50%-98%, respectively. R. stolonifer reduced BOD by 36%-74% and COD by 10%- 78%. Conclusions: This study emphasized the abilities of oleaginous fungi to utilize oil wastes as carbon sources to reduce BOD and COD of the wastes, producing essential fatty acids. © 2015, Kowsar Medical Publishing Company. All rights Reserved.
Analytical Methods (17599679) 6(12)pp. 4199-4204
Apoptosis is a tightly controlled biochemical process for cell death. Although the induction of apoptosis is an important mechanism for the screening of many valuable products such as drugs, due to the false signals that usually occur during the isolation of cells from the surface of the culture plate, techniques have limitations in measuring apoptosis in adherent cells. In this study, we investigated the use of RLuc/Annexin V, a probe obtained by the fusion of Renilla luciferase (RLuc) with Annexin V and bound to phosphatidylserine (PS) on the surface of suspended apoptotic cells, as a potentially luminescent probe to assay apoptosis in adherent cells such as Chinese Hamster Ovary (CHO) cells. The probe was overexpressed in Escherichia coli BL21 (DE3) and purified by immobilized metal ion chromatography. The probe assayed for detection of apoptosis in CHO cells. The results show that RLuc/Annexin V binds to the CHO cells with no additional treatment for cell suspension, and the signal of RLuc can be detected by a luminometer. The new assay based on RLuc/Annexin V was named as adherent state apoptosis assay (ASA). This may be a new method for studying apoptosis in adherent cells in a rapid, reliable, and non-invasive way. © 2014 the Partner Organisations.
Applied Biochemistry and Biotechnology (02732289) 173(1)pp. 103-115
S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium-S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N-H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits. © Springer Science+Business Media 2014.
RSC Advances (20462069) 4(85)pp. 45128-45135
Apoptosis is a well-organized mechanism developed by eukaryotic organisms during maturation. The importance of apoptosis as a major form of cellular suicide with a causative or contributing role in a variety of diseases has become progressively obvious. A large body of evidence implicates the association of apoptosis with an arranged series of physical and biochemical alterations comprising the nucleus, cytoplasm and cell membrane. Understanding the process of apoptosis is not only important for maintaining physiological conditions, but also is critical for therapy development. In this review, we briefly outline the various ways to detect apoptosis with distinct assays and molecules, and provide an outlook on the latest imaging techniques for the visualization of phosphatidylserine (PS) externalization, with specific focus on annexin V based targeting of PS and its conjugates. Furthermore, the distinct advantages and disadvantages of each technique are reviewed. Recognition of the major benefits and drawbacks of each assay based on annexin V conjugates would allow us to use the appropriate method to detect apoptosis for instance, in a variety of diseases, such as cancer and will open novel prospects for treatment approaches. © 2014 the Partner Organisations.
Current Microbiology (03438651) 69(2)pp. 149-157
The purpose of the present investigation was to produce erythritol by Yarrowia lipolytica mutant without any by-products. Mutants of Y. lipolytica were generated by ultra-violet for enhancing erythrose reductase (ER) activity and erythritol production. The mutants showing the highest ER activity were screened by triphenyl tetrazolium chloride agar plate assay. Productivity of samples was analyzed by thin-layer chromatography and high-performance liquid chromatography equipped with the refractive index detector. One of the mutants named as mutant 49 gave maximum erythritol production without any other by-products (particularly glycerol). Erythritol production and specific ER activity in mutant 49 increased to 1.65 and 1.47 times, respectively, in comparison with wild-type strain. The ER gene of wild and mutant strains was sequenced and analyzed. A general comparison of wild and mutant gene sequences showed the replacement of Asp270 with Glu270 in ER protein. In order to enhance erythritol production, we used a three component-three level-one response Box-Behnken of response surface methodology model. The optimum medium composition for erythritol production was found to be (g/l) glucose 279.49, ammonium sulfate 9.28, and pH 5.41 with 39.76 erythritol production. © 2014 Springer Science+Business Media.
Hashempoor, T. ,
Bamdad, T. ,
Arasteh, J. ,
Emamzadeh, R. ,
Haj-sheikhol-eslami, A. ,
Asadi, R. ,
Merat, S. Govaresh (15607186) 18(1)pp. 7-15
Background : The mechanism behind the apparent lack of effective antiviral immune response in patients with chronic hepatitis C virus (HCV) infection is poorly understood. Although multiple levels of abnormalities have been identified in innate and adaptive immunity, it is postulated that production of specific cytokines such as IL-10 may contribute to the induction and maintenance of HCV persistence. Production of IL-10 by CD4+,CD25 +,IL-10+ regulatory T cells with regulatory capacity (Tregs) appears to be one of the viral mechanisms that alter the antiviral immune response. As the first report, that attempts to mimic physiological forces that can occur during HCV infection, in this study we evaluate the ability of HCV-core antigens in increasing the frequency of CD4 +,CD25+,IL-10+ regulatory T cells. Materials and Methods: We analyzed peripheral blood mononuclear cells (PBMCs) from chronic HCV-infected patients (n=19) and normal controls (n=6) to determine the effect of the HCV-core antigen in the frequency of HCV-specific IL-10 production. PBMCs of different groups were isolated, cultured and stimulated with core antigen. Then, an in-house triple-stain flow cytometric method was used to investigate the frequency of CD4+,CD25+,IL-10 producing cells. Results: Following incubation of PBMCs with HCV-core antigen, a population of CD4+,CD25+,IL-10+ cells (regulatory T cells) increased. However we observed no increase in Tregs in the negative controls. Conclusion: The study supports the view that specific CD4+,CD25 +,IL-10+ T cells may be implicated in host immune tolerance during an HCV infection. It is likely that HCV vaccine candidates avoid epitopes that lead to strong IL-10 production.
Enzyme and Microbial Technology (18790909) 51(4)pp. 186-192
In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction and is an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly loses its activity at temperatures above 30 °C and this leads to reduced sensitivity and precision in analytical applications. Residue Thr346 in a connecting loop (341-348) of firefly luciferase is located in a disallowed region of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles (ψ, φ) with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were analyzed using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability. © 2012 Elsevier Inc.
Nazari, M. ,
Emamzadeh, R. ,
Hosseinkhani, S. ,
Cevenini, L. ,
Michelini, E. ,
Roda, A. Analyst (00032654) 137(21)pp. 5062-5070
The Ca2+-dependent binding of Annexin V to phosphatidylserine on cell surfaces is a reliable marker for apoptosis that is widely used in flow cytometry based apoptosis assays. In this paper, we report a new class of Annexin V-based probes for apoptosis. Luciferase from Renilla reniformis (RLuc) was linked to Annexin V and expressed successfully in a soluble form in Escherichia coli BL21 (DE3). The new probe, Rluc/Annexin V, was purified and functionally assayed for detection of apoptosis in actinomycin D-induced apoptotic Jurkat cells. Moreover, the spontaneous apoptosis in neutrophils was shown using the new probe. The results indicate that Rluc/Annexin V can bind to the apoptotic cells, and the signal of Renilla luciferase can be detected by luminometric measurements. The availability of Rluc/Annexin V may be of potential commercial interest for improving current apoptosis assays. © 2012 The Royal Society of Chemistry.
Enzyme and Microbial Technology (18790909) 47(4)pp. 159-165
In the firefly light organ, luciferin-regenerating enzyme (LRE) plays an important role in the recycling of oxyluciferin into luciferin. In this study, the LRE cDNA was cloned from total RNA extracted from lantern of Lampyris turkestanicus (an Iranian firefly) using PCR-based techniques including reverse transcription-polymerase chain reaction, 5'-RACE (5'-rapid amplification of cDNA ends) and 3'-RACE. The full-length cDNA encoding LRE of L. turkestanicus (T-LRE) was 1047 with 924. bp open-reading frame encoding the protein of 307 amino acid residues with a calculated molecular mass of 33.5. kDa. The deduced amino acid sequence of T-LRE showed 46 and 45% identity to A-LRE (LRE from the American firefly - Photinus pyralis) and G-LRE (LRE from a Japanese firefly - Luciola cruciata), respectively. The cDNA encoding LRE of L. turkestanicus was expressed in recombinant Escherichia coli and it is shown that T-LRE improves the luminescent signal of firefly luciferase. © 2010 Elsevier Inc.
Farashahi yazd e., ,
Sadeghizadeh, M. ,
Hosseinkhani, S. ,
Khalaj-kondori m., ,
Emamzadeh, R. Journal Of The Iranian Chemical Society (1735207X) 6(4)pp. 831-837
Thermostable DNA polymerases are widely used in DNA amplification reactions such as the Polymerase Chain Reaction (PCR), requiring the activity of the enzymes at high temperatures. The aim of the present study was to assess the potential biotechnological capabilities of Iranian thermostable DNA polymerases. To this end, we cloned the gene encoding a DNA polymerase from a novel thermophilic eubacterium, Bacillus sp. G (2006). Phylogentic analysis of this gene revealed that the new isolate belongs to the genera Bacillus. Sequence analysis of the fragment produced by degenerate primers also showed that it consists of 2,631 bp encoding an 876 amino acid protein, and subsequent amino acid sequence analysis of this DNA polymerase showed that it belongs to family A-type DNA polymerases. The expression vector pET28a (+) was chosen for expression of the gene fragment in the mesophilic host bacterium E. coli BL21. This expression vector has some advantages such as attachment of a Poly-His tag to the N-terminus of the protein for the ease of purification and a powerful promoter of lac-Z induced by IPTG. The band corresponding to the protein product was observed in the molecular weight range of about 100KDa on the SDS-PAGE gel after heat and Ni+2-NTA column chromatography. Using the dot blot technique, the polymerase activity of the enzyme was qualitatively confirmed at 70 °C. Therefore, it is suggested that optimizations of this activity could make this enzyme appropriate for PCR processes in future.
Tafreshi, N.K. ,
Sadeghizadeh, M. ,
Emamzadeh, R. ,
Ranjbar, B. ,
Naderi-manesh, H. ,
Hosseinkhani, S. Biochemical Journal (14708728) 412(1)pp. 27-33
The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as Km and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging. © The Authors.
