Publications by authors named "Morteza Oladnabi"

21 Publications

  • Page 1 of 1

In silico drug repurposing for the treatment of heart diseases using gene expression data and molecular docking techniques.

Biochem Biophys Res Commun 2021 10 5;572:138-144. Epub 2021 Aug 5.

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Golestan University of Medical Sciences, Gorgan, Iran; Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran. Electronic address:

Heart diseases are known as the most primary causes of mortality worldwide. Although many therapeutic approaches and medications are proposed for these diseases, the identification of novel therapeutics in fatal heart conditions is promptly demanded. Besides, the interplay between gene expression data and molecular docking provides several novel insights to discover more effective and specific drugs for the treatment of the diseases. This study aimed to discover potent therapeutic drugs in the heart diseases based on the expression profile of heart-specific genes exclusively. Initially, the heart-specific and highly expressed genes were identified by comparing the gene expression profile of different body tissues. Subsequently, the druggable-genes were identified using in silico techniques. The interaction between these druggable genes with more than 1600 FDA approved drugs was then investigated using the molecular docking simulation. By comprehensively analyzing RNA-sequencing data obtained from 949 normal tissue samples, 48 heart-specific genes were identified in both the heart development and function. Notably, of these, 24 heart-specific genes were capable to be considered as druggable genes, among which only MYBPC3, MYLK3, and SCN5A genes entered the molecular docking process due to their functions. Afterward, the pharmacokinetics properties of top 10 ligands with the highest binding affinity for these proteins were studied. Accordingly, methylergonovine, fosaprepitant, pralatrexate, daunorubicin, glecaprevir, digoxin, and venetoclax drugs were competent, in order to interact with the target proteins perfectly. It was shown that these medications can be used as specific drugs for the treatment of heart diseases after fulfilling further experiments in this regard.
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http://dx.doi.org/10.1016/j.bbrc.2021.07.076DOI Listing
October 2021

Correlation between ELF-PEMF exposure and Human RPE Cell Proliferation, Apoptosis and Gene Expression.

J Ophthalmic Vis Res 2021 Apr-Jun;16(2):202-211. Epub 2021 Apr 29.

Department of Clinical Biochemistry and Medical Genetics, Molecular and Cell Biology Research Center, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Purpose: Emerging evidence implies that electromagnetic fields (EMFs) can negatively affect angiogenesis. In this regard, the effects of extremely low frequency pulsed electromagnetic field (ELF-PEMF) exposure on the relative expression level of angiogenic factors involved in the pathogenesis of ocular disorders were evaluated in human retinal pigment epithelial (hRPE) cells in order to investigate a noninvasive therapeutic method for patients with several ocular diseases associated with neovascularization.

Methods: After separating hRPE cells from globes, hRPE cells were exposed to 15 mT of ELF-PEMF (120 Hz) at 5, 10, and 15 min for seven days. Cell proliferation and apoptosis of treated cells were evaluated via ELISA assay. Moreover, relative expression changes of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 were performed using real-time RT-PCR.

Results: ELF-PEMF exposure had no significant effects on the apoptosis and proliferation rate of hRPE cells. Expression level of HIF-1α, CTGF, VEGFA, MMP-2, cathepsin D, and E2F3 was downregulated following 5 min of ELF-PEMF exposure.

Conclusion: As ELF-PEMF showed inhibitory effects on the expression of angiogenic genes in hRPE cells with no cytotoxic or proliferative side effects, it can be introduced as a useful procedure for managing angiogenesis induced by retinal pathogenesis, although more studies with adequate follow-up in animal models are needed.
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http://dx.doi.org/10.18502/jovr.v16i2.9084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126745PMC
April 2021

WDR81 Gene Silencing Can Reduce Exosome Levels in Human U87-MG Glioblastoma Cells.

J Mol Neurosci 2021 Aug 6;71(8):1696-1702. Epub 2021 May 6.

Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran.

Glioblastoma is a very invasive and prevalent brain tumor that affects 15 in 100,000 persons over the age of 70 years. Studies have shown that the expression of the WD repeat domain 81 (WDR81) gene, which is effective in vesicular transport and inhibition of autophagy, is increased in glioblastoma. The decreased autophagy was found to be related to the increased production of exosomes, which is a major factor in the pathogenesis of glioblastoma. The PI-3kinase complex is a pre-autophagic complex that is highly active in the absence of WDR81. The WDR81 gene, as a negative regulator of PI3K activity, prevents autophagy and increases exosome secretion by preventing the formation of the class III PI3K complex. Therefore, targeted reduction of exosomes can be considered an effective strategy for reducing the pathogenesis of glioblastoma. This study aimed to assess the effect of WDR81 gene silencing with siRNA on exosome levels in a U87-MG cell line. Culturing of U87-MG cells was carried out in Dulbecco's modified Eagle medium (DMEM) containing 5% FBS and 1% penicillin/streptomycin. Thereafter, silencing of WDR81 was performed using WDR81 siRNA, whose gene expression level was determined via real-time qRT-PCR. Cell viability was evaluated using the MTT assay. The exosomes were extracted from a cell culture using the Exocib kit. The size accuracy of the exosomes was confirmed by dynamic light scattering (DLS). Finally, the protein content and RNA of the exosomes were assessed. WDR81 gene expression of siRNA-transfected cells was decreased to 82% after 24 h compared to the non-transfected control cells. The analysis of the exosomes showed that the concentration of exosomes and their RNA and protein content in the siRNA-transfected cells decreased significantly compared to the non-transfected control cells. No considerable difference was observed in cell viability after transfection with either WDR81-specific siRNAs or scrambled control siRNAs. Our findings showed that silencing the WDR81 gene could reduce the level of exosomes in human U87-MG glioblastoma cells. Therefore, the reduced exosome content may be suggested as a new gene therapy strategy for targeted therapy of glioblastoma by increasing autophagy via activation of PI3KIII. However, more studies are needed in this regard.
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http://dx.doi.org/10.1007/s12031-021-01849-zDOI Listing
August 2021

Toll-Like Receptor (TLR)-9 rs352140 Polymorphism is an Immunopathology Protective Factor in Parkinson's Disease in the Northern Iranian Population.

Iran J Immunol 2020 12;17(4):313-323

Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran.

Background: Neuroinflammation and immunopathology in Parkinson's disease (PD) are believed to be associated with genetic and environmental factors.

Objective: We conducted the current study to evaluate the Toll-like receptors (TLR4 and TLR9) genes polymorphism in patients with Parkinson's disease in northern Iran.

Methods: We extracted DNA from peripheral blood samples of 100 sporadic cases of Parkinson's disease and 100 healthy-matched controls with the mean age of 69.98 and 71.94 years, respectively. Subsequently, single-nucleotide polymorphisms (SNPs) of TLR4 and TLR9 were genotyped using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR). Results were confirmed employing Sanger sequencing. For the analysis of our data, we used SNPStats and SPSS 22 software.

Results: Our findings indicated that the allele distribution for rs352140 of TLR9 gene was significantly different in the PD group compared with the healthy controls (p=0.02). Moreover, rs352140 T allele was observed to be correlated with PD reduced risk (TT + TC vs. CC). The dominant rs352140 model was approved as the most acceptable inheritance model for fitting the data (OR 0.41, 95% CI 0.23-0.75, p=0.0031). Additionally, haplotype analysis revealed a significant correlation between TLR9 polymorphisms and Parkinson's disease.

Conclusion: The results of this study indicated that rs352140T of TLR9 gene was a protective factor in Parkinson's disease. Furthermore, this SNP could be regarded as a prognostic factor. However, this conclusion should be confirmed by further investigations.
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http://dx.doi.org/10.22034/iji.2020.87454.1803DOI Listing
December 2020

Variants in Intron 4 of PD-1 Gene are Associated with the Susceptibility to SLE in an Iranian Population.

Iran J Immunol 2020 Sep;17(3):204-214

Department of Microbiology, School of Medicine, Golestan University of Medical Sciences, Gorgan, Iran.

Background: Programmed cell death protein 1 (PD-1) is a negative co-stimulatory molecule with immunomodulatory properties. Recently, PD-1 gene defects have attracted attention in the pathogenesis of SLE.

Objective: Here, we assessed the association of PD-1 gene polymorphisms in intron 4 and haplotypes with the susceptibility to SLE.

Methods: Seventy-six SLE patients and 159 healthy controls were included. We screened the polymorphisms by amplifying the intron 4 of the PD-1 gene with the specific primers followed by sequencing.

Results: Two distinct SNPs were identified (rs6705653 and rs41386439) within the intron 4 of the PD-1 gene. The AA genotype of +7499 (G/A) SNP was associated with the higher risk of SLE [OR=3.31, 95% CI (1.25-8.76), p-value=0.045], while A allele was identified as a risk allele [OR=1.75, 95% CI (1.10-2.76), p-value=0.015]. However, no significant association was observed between the allele and the genotype frequencies of +7209 (C/T) polymorphic region of the PD-1 gene and susceptibility to SLE. Haplotype analysis showed the significantly higher presence of H2 haplotype (AC; +7499/+7209) [OR=1.70, 95% CI (1.24-2.33), p-value=0.0012] in SLE patients.

Conclusion: To the best of our knowledge, this is the first report of the significant association of PD-1 +7499 (G/A) SNP with the SLE susceptibility and the first detection of both polymorphic loci in a population from Iran. However, more investigations are necessary to confirm these findings.
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http://dx.doi.org/10.22034/iji.2020.83046.1610DOI Listing
September 2020

Molecular and biochemical mechanisms of human iris color: A comprehensive review.

J Cell Physiol 2020 12 2;235(12):8972-8982. Epub 2020 Jun 2.

Gorgan Congenital Malformations Research Center, Golestan University of Medical Sciences, Gorgan, Iran.

Eye color is determined as a polymorphism and polygenic trait. Brown is the most common eye color in the world, accounting for about 79%, blue eye color for about 8-10%, hazel for 5%, and green for 2%. Rare-colored eyes include gray and red/violet. Different factors are involved in determining eye color. The two most important factors are the iris pigment and the way light is scattered from the iris. Gene expression determines the iris pigmentation and how much melanin is present in the eye, which is the number of melanin subunits that identify eye color. The genes involved in the pigmentation of single-nucleotide polymorphism (SNP) have a significant role; and even some genes are included only in the eye color through SNP. MicroRNAs also affect melanocyte synthesis, which is usually affected by the downregulation of essential genes involved in pigmentation. In this study, we assess the biochemical pathways of melanin synthesis, and the role of each gene in this pathway also has been examined in the signaling pathway that stimulates melanin synthesis.
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http://dx.doi.org/10.1002/jcp.29824DOI Listing
December 2020

Distinct genetic variation and heterogeneity of the Iranian population.

PLoS Genet 2019 09 24;15(9):e1008385. Epub 2019 Sep 24.

Cologne Center for Genomics, University of Cologne, Cologne, Germany.

Iran, despite its size, geographic location and past cultural influence, has largely been a blind spot for human population genetic studies. With only sparse genetic information on the Iranian population available, we pursued its genome-wide and geographic characterization based on 1021 samples from eleven ethnic groups. We show that Iranians, while close to neighboring populations, present distinct genetic variation consistent with long-standing genetic continuity, harbor high heterogeneity and different levels of consanguinity, fall apart into a cluster of similar groups and several admixed ones and have experienced numerous language adoption events in the past. Our findings render Iran an important source for human genetic variation in Western and Central Asia, will guide adequate study sampling and assist the interpretation of putative disease-implicated genetic variation. Given Iran's internal genetic heterogeneity, future studies will have to consider ethnic affiliations and possible admixture.
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http://dx.doi.org/10.1371/journal.pgen.1008385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759149PMC
September 2019

Cannabinoid CB2 Receptor Functional Variation (Q63R) Is Associated with Multiple Sclerosis in Iranian Subjects.

J Mol Neurosci 2020 Jan 13;70(1):26-31. Epub 2019 Aug 13.

Department of Neurology, Sayyad Hospital, Golestan University of Medical Sciences, Gorgan, Iran.

The cannabinoid system has been identified as a critical endogenous regulator of immune homeostasis through immunomodulatory actions. This system is one of the main regulatory systems of the central nervous system (CNS). Variations in the cannabinoid CB2 receptor gene (CNR2) could affect intracellular signaling and reduce system function, which has been associated with an unbalanced immune response and increased risk of a variety of autoimmune inflammatory disorders. The present study investigated the relationship between CNR2 rs35761398 (Q63R) functional variation and multiple sclerosis (MS). A total of 100 Iranian MS patients and 100 healthy controls were enrolled in the study and genotyped through TaqMan assay. The co-dominant, dominant, recessive, over-dominant, and additive inheritance models were analyzed using SNPStats software. A significant genetic association was observed between Q63R polymorphism and MS. The dominant model was accepted as the best inheritance model to fit the data (OR 2.70, 95% CI 1.47-4.97, p = 0.001). The data implied the involvement of the CNR2 gene in susceptibility to MS in Iranian patients.
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http://dx.doi.org/10.1007/s12031-019-01395-9DOI Listing
January 2020

Iranome: A catalog of genomic variations in the Iranian population.

Hum Mutat 2019 11 17;40(11):1968-1984. Epub 2019 Aug 17.

Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.

Considering the application of human genome variation databases in precision medicine, population-specific genome projects are continuously being developed. However, the Middle Eastern population is underrepresented in current databases. Accordingly, we established Iranome database (www.iranome.com) by performing whole exome sequencing on 800 individuals from eight major Iranian ethnic groups representing the second largest population of Middle East. We identified 1,575,702 variants of which 308,311 were novel (19.6%). Also, by presenting higher frequency for 37,384 novel or known rare variants, Iranome database can improve the power of molecular diagnosis. Moreover, attainable clinical information makes this database a good resource for classifying pathogenicity of rare variants. Principal components analysis indicated that, apart from Iranian-Baluchs, Iranian-Turkmen, and Iranian-Persian Gulf Islanders, who form their own clusters, rest of the population were genetically linked, forming a super-population. Furthermore, only 0.6% of novel variants showed counterparts in "Greater Middle East Variome Project", emphasizing the value of Iranome at national level by releasing a comprehensive catalog of Iranian genomic variations and also filling another gap in the catalog of human genome variations at international level. We introduce Iranome as a resource which may also be applicable in other countries located in neighboring regions historically called Greater Iran (Persia).
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http://dx.doi.org/10.1002/humu.23880DOI Listing
November 2019

Evaluation of cytokeratin 19 as a prognostic tumoral and metastatic marker with focus on improved detection methods.

J Cell Physiol 2019 12 1;234(12):21425-21435. Epub 2019 May 1.

Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran.

In the last few years, there has been a growing interest in Cytokeratin 19 (CK19) studies in the cancer research field. CK19 belongs to the Type I CKs, serves as a useful research tool in prognosis, diagnosis, and management of the tumors. In this paper, we dissect the metastatic potential of CK19, its relation with cancer stem cells and retinal epithelial cells behavior, its application as a tumor marker and its role among 30 cancers such as thyroid, thoracic, lung, pancreatic, cervical, colorectal, and so forth. CK19 expressed in several cancer types because of its metastatic potential. This paper also presents modified detection methods of CK19 in disseminated tumor cells.
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http://dx.doi.org/10.1002/jcp.28768DOI Listing
December 2019

Extremely low frequency-pulsed electromagnetic fields affect proangiogenic-related gene expression in retinal pigment epithelial cells.

Iran J Basic Med Sci 2019 Feb;22(2):128-133

Medical Cellular and Molecular Research Center, Golestan University of Medical Sciences, Gorgan, Iran.

Objectives: It is known that extremely low frequency-pulsed electromagnetic fields (ELF-PEMF) influence multiple cellular and molecular processes. Retinal pigment epithelial (RPE) cells have a significant part in the emergence and pathophysiology of several ocular disorders, such as neovascularization. This study assessed the impact of ELF-PEMF on the proangiogenic features of RPE cells.

Materials And Methods: Primary cultured RPE cells were treated with ELF-PEMF (50 Hz) for three days. Using ELISA assay, we evaluated the effects of treatment on RPE cell proliferation and apoptosis. Also, RT-PCR was used to determine the gene expression of proangiogenic factors, such as matrix metalloproteinase-2 (MMP-2), MMP-9, vascular endothelial growth factors receptor 2 (VEGFR-2), hypoxia-inducible factor 1 (HIF-1α), VEGFA, cathepsin D, connective tissue growth factor (CTGF), E2F3, tissue inhibitors of metalloproteinases 1 (TIMP-1), and TIMP-2.

Results: No noticeable changes were observed in cell proliferation and cell death of ELF-PEMF-exposed RPE cells, while transcript levels of proangiogenic genes (HIF-1α, VEGFA, VEGFR-2, CTGF, cathepsin D, TIMP-1, E2F3, MMP-2, and MMP-9) increased significantly.

Conclusion: RPE cells are important for homeostasis of the retina. ELF-PEMF increased the gene expression of proangiogenic factors in RPE cells, which highlights concerns about the impact of this treatment on human health.
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http://dx.doi.org/10.22038/ijbms.2018.25023.6214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396987PMC
February 2019

CNKSR1 gene defect can cause syndromic autosomal recessive intellectual disability.

Am J Med Genet B Neuropsychiatr Genet 2018 12 18;177(8):691-699. Epub 2018 Nov 18.

Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.

The advent of high-throughput sequencing technologies has led to an exponential increase in the identification of novel disease-causing genes in highly heterogeneous diseases. A novel frameshift mutation in CNKSR1 gene was detected by Next-Generation Sequencing (NGS) in an Iranian family with syndromic autosomal recessive intellectual disability (ARID). CNKSR1 encodes a connector enhancer of kinase suppressor of Ras 1, which acts as a scaffold component for receptor tyrosine kinase in mitogen-activated protein kinase (MAPK) cascades. CNKSR1 interacts with proteins which have already been shown to be associated with intellectual disability (ID) in the MAPK signaling pathway and promotes cell migration through RhoA-mediated c-Jun N-terminal kinase (JNK) activation. Lack of CNKSR1 transcripts and protein was observed in lymphoblastoid cells derived from affected patients using qRT-PCR and western blot analysis, respectively. Furthermore, RNAi-mediated knockdown of cnk, the CNKSR1 orthologue in Drosophila melanogaster brain, led to defects in eye and mushroom body (MB) structures. In conclusion, our findings support the possible role of CNKSR1 in brain development which can lead to cognitive impairment.
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http://dx.doi.org/10.1002/ajmg.b.32648DOI Listing
December 2018

Gene Silencing of TGFβRII Can Inhibit Glioblastoma Cell Growth

Asian Pac J Cancer Prev 2018 Sep 26;19(9):2681-2686. Epub 2018 Sep 26.

Cellular and Molecular Biology Research Center, Health Research Institute, Babol University of Medical Sciences, Babol, Iran. Email:

Objective: Glioblastoma (GBM) is the most malignant and aggressive type of glioma, associated with a high rate of mortality. The transforming growth factor-β receptor II (TGFβ RII) is involved in glioma initiation and progression. On the other hand, TGFβ RII silencing is critical to the inhibition of GBM. Therefore, we aimed to determine the effects of specific TGFβ RII siRNA on the survival of U-373MG cells. Methods: TGFβ RII siRNA was transfected, and qRT-PCR was performed to examine TGFβ RII mRNA expression. Cell survival was determined using colorimetric MTT assay, and platelet-derived growth factor-BB (PDGF-BB) level was measured in the culture supernatant using ELISA assay. Result: Our findings indicated that specific siRNAs could dose-dependently suppress TGFβ RII mRNA expression after 48 hours. In addition, treatment with TGFβ RII siRNA significantly reduced tumor cell survival and decreased the amount of PDGF-BB protein in the cell culture supernatant. Conclusion: Our results suggest that TGFβ RII silencing can be a promising complementary treatment for glioma.
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http://dx.doi.org/10.22034/APJCP.2018.19.9.2681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249455PMC
September 2018

Enzymatic characterization of a NADH-dependent diaphorase from Lysinibacillus sp. strain PAD-91.

Protein Expr Purif 2018 06;146:1-7

Department of Biology, Bandar Jask Branch, Islamic Azad University, Bandar Jask, Iran. Electronic address:

Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this paper, we present recombinant expression, characterization and medium optimization of a lipoamide dehydrogenase (DLD) with NADH-dependent diaphorase activity from a Lysinibacillus sp. strain. DLD encoding sequence showed an open reading frame of 1413-bp encoding a 470 amino acid chain. Lysinibacillus sp. DLD catalyzed the NADH-dependent reduction of electron acceptors and exhibited diaphorase activity. The molecular mass of the isolated enzyme was found to be about 50 kDa, and determined to be a monomeric protein. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 7.5 and 30 °C. The K and V values were estimated to be 0.025 mM and 1.33 μmol/min, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO. By Scaling up fermentation from flask to bioreactor, enzyme activity was increased to 487.5 U/ml. This study provides data on the identification, characterization and medium optimization of a NADH-dependent diaphorase from a newly isolated Lysinibacillus sp. PAD-91.
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http://dx.doi.org/10.1016/j.pep.2018.01.005DOI Listing
June 2018

Genetics of intellectual disability in consanguineous families.

Mol Psychiatry 2019 07 4;24(7):1027-1039. Epub 2018 Jan 4.

Meybod Genetics Research Center, Welfare Organization, Yazd, 89651, Iran.

Autosomal recessive (AR) gene defects are the leading genetic cause of intellectual disability (ID) in countries with frequent parental consanguinity, which account for about 1/7th of the world population. Yet, compared to autosomal dominant de novo mutations, which are the predominant cause of ID in Western countries, the identification of AR-ID genes has lagged behind. Here, we report on whole exome and whole genome sequencing in 404 consanguineous predominantly Iranian families with two or more affected offspring. In 219 of these, we found likely causative variants, involving 77 known and 77 novel AR-ID (candidate) genes, 21 X-linked genes, as well as 9 genes previously implicated in diseases other than ID. This study, the largest of its kind published to date, illustrates that high-throughput DNA sequencing in consanguineous families is a superior strategy for elucidating the thousands of hitherto unknown gene defects underlying AR-ID, and it sheds light on their prevalence.
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http://dx.doi.org/10.1038/s41380-017-0012-2DOI Listing
July 2019

Recombinant expression, characterization and application of a dihydrolipoamide dehydrogenase with diaphorase activity from Bacillus sphaericus.

3 Biotech 2017 Jun 8;7(2):153. Epub 2017 Jun 8.

Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran.

Diaphorases are flavin-containing enzymes with potential applications in biotransfomation reactions, biosensor design and in vitro diagnostic tests. In this communication, we describe recombinant expression, characterization and application of a lipoamide dehydrogenase (DLD) with diaphorase activity from a strain of Bacillus sphaericus. The DLD gene consisting of 1413 bp encoding a protein of 470 amino acids was expressed in Escherichia coli BL21 (DE3) and the recombinant enzyme was characterized. B. sphaericus DLD catalyzed the reduction of NAD by dihydrolipoamide and exhibited NADH-dependent diaphorase activity. The molecular weight of purified enzyme was about 50 kDa, and determined to be a monomeric protein. Diaphorase was active and stable from pH 7.0 to 9.0 with an optimal activity at pH 8.5. It showed its maximal activity at temperature of 30 °C and was almost stable at temperatures between 25 and 30 °C. Different metal ions and inhibitors showed no influence on the activity of target enzyme. The K and V values for NADH were estimated to be 0.33 mM and 200.0 U/ml, respectively. Moreover, recombinant B. sphaericus diaphorase exhibited considerable potential to be used as a component of diagnostic tests for the quantification of metabolites. In conclusion, considering the properties of diaphorase from B. sphaericus PAD-91, it can have potential application as a diagnostic enzyme.
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http://dx.doi.org/10.1007/s13205-017-0763-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465047PMC
June 2017

Purification and Characterization of Recombinant Darbepoetin Alfa from Leishmania tarentolae.

Mol Biotechnol 2016 Sep;58(8-9):566-72

Enzyme Technology Laboratory, Biochemistry Department, Metabolic and Genetic Research Group, Pasteur Institute of Iran, Tehran, Iran.

Darbepoetin alfa is a biopharmaceutical glycoprotein that stimulates erythropoiesis and is used to treat anemia, which associated with renal failure and cancer chemotherapy. We herein describe the structural characterization of recombinant darbepoetin alfa produced by Leishmania tarentolae T7-TR host. The DNA expression cassette was integrated into the L. tarentolae genome through homologous recombination. Transformed clones were selected by antibiotic resistance, diagnostic PCRs, and protein expression analysis. The structure of recombinant darbepoetin alfa was analyzed by isoelectric focusing, ultraviolet-visible spectrum, and circular dichroism (CD) spectroscopy. Expression analysis showed the presence of a protein band at 40 kDa, and its expression level was 51.2 mg/ml of culture medium. Darbepoetin alfa have 5 isoforms with varying degree of sialylation. The UV absorption and CD spectra were analogous to original drug (Aranesp), which confirmed that the produced protein was darbepoetin alfa. Potency test results revealed that the purified protein was biologically active. In brief, the structural and biological characteristics of expressed darbepoetin alfa were very similar to Aranesp which has been normally expressed in CHO. Our data also suggest that produced protein has potential to be developed for clinical use.
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http://dx.doi.org/10.1007/s12033-016-9954-xDOI Listing
September 2016

The Rapid and Sensitive Quantitative Determination of Galactose by Combined Enzymatic and Colorimetric Method: Application in Neonatal Screening.

Appl Biochem Biotechnol 2016 May 28;179(2):283-93. Epub 2016 Jan 28.

Department of Biology, Bandar Jask Branch, Islamic Azad University, Bandar Jask, Iran.

The quantitative measurement of galactose in blood is essential for the early diagnosis, treatment, and dietary monitoring of galactosemia patients. In this communication, we aimed to develop a rapid, sensitive, and cost-effective combined method for galactose determination in dry blood spots. This procedure was based on the combination of enzymatic reactions of galactose dehydrogenase (GalDH), dihydrolipoyl dehydrogenase (DLD), and alkaline phosphates with a colorimetric system. The incubation time and the concentration of enzymes used in new method were also optimized. The analytical performance was studied by the precision, recovery, linearity, and sensitivity parameters. Statistical analysis was applied to method comparison experiment. The regression equation and correlation coefficient (R (2)) were Y = 0.0085x + 0.032 and R (2) = 0.998, respectively. This assay exhibited a recovery in the range of 91.7-114.3 % and had the limit detection of 0.5 mg/dl for galactose. The between-run coefficient of variation (CV) was between 2.6 and 11.1 %. The within-run CV was between 4.9 and 9.2 %. Our results indicated that the new and reference methods were in agreement because no significant biases exist between them. Briefly, a quick and reliable combined enzymatic and colorimetric assay was presented for application in newborn mass screening and monitoring of galactosemia patients.
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http://dx.doi.org/10.1007/s12010-016-1993-zDOI Listing
May 2016

Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

Protein Expr Purif 2016 Feb 3;118:120-5. Epub 2015 Nov 3.

Department of Biochemistry, Genetics and Metabolism Research Group, Pasteur Institute of Iran, Tehran 13164, Iran. Electronic address:

Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa.
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http://dx.doi.org/10.1016/j.pep.2015.10.013DOI Listing
February 2016

New evidence for the role of calpain 10 in autosomal recessive intellectual disability: identification of two novel nonsense variants by exome sequencing in Iranian families.

Arch Iran Med 2015 Mar;18(3):179-84

Genetics Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran.

Background: Knowledge of the genes responsible for intellectual disability, particularly autosomal recessive forms, is rapidly expanding. Increasing numbers of the gene show great heterogeneity and supports the hypothesis that human genome may contain over 2000 causative genes with a critical role in brain development.

Methods: Since 2004, we have applied genome-wide SNP genotyping and next-generation sequencing in large consanguineous Iranian families with intellectual disability, to identify the genes harboring disease-causing mutations. The current study paved the way for identification of responsible genes in two unrelated Iranian families.

Results: We found two novel nonsense mutations, p.C77* and p.Q115*, in the calpain catalytic domain of CAPN10, which is a cysteine protease known to be involved in pathogenesis of noninsulin-dependent diabetes mellitus. Another different mutation in this gene (p.S138_R139ins5) has previously been reported in an Iranian family. All of these patients have common clinical features in spite of specific brain structural abnormalities on MRI.

Conclusions: Different mutations in CAPN10 have already been found in three independent Iranian families. These results have strongly supported the possible role of CAPN10 in human brain development. Altogether, we proposed CAPN10 as a promising candidate gene for intellectual disability, which should be considered in diagnostic gene panels.
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http://dx.doi.org/0151803/AIM.008DOI Listing
March 2015
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