Publications by authors named "Zohreh Hojati"

39 Publications

The Effects of Self-cleavage Intein-ELK16 Tag in the Transcript Steric Hindrance of IFN.

Indian J Clin Biochem 2021 Apr 30;36(2):159-166. Epub 2020 Jan 30.

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, 81746-73441 Iran.

Intervening proteins (Inteins) are identified as protein domains in a precursor protein structure. Inteins can excise itself from precursor protein and join the remaining portions which result in forming an active protein. In this study, the transcript expression level of recombinant human Interferon beta (rhIFNβ) connected to the self-cleavage Intein-ELK16 (LELELKLKLELELKLK) tag was measured by real-time PCR in HEK293T cell line. First, the sequence of RecA (Mtu recA) was obtained from the InBase database to do appropriate changes including adding the restriction sites, kozak sequence, signal peptide and ELK16 sequence by SnapGene software. The RNA secondary structure were also examined using the online RNA Fold 2.2 web server. Next, the construct was inserted into pUC19 plasmid. The sequence of rhIFNβ was also cloned into pBudCE4.1 vector. In the next step, the rhIFNβ was ligated into the construct (self-cleavage tag of ELK16) using T4 DNA ligase and the recombinant construct was transfected into HEK293T cell line. Finally, expression of the cassette was evaluated by real-time PCR. The analysis of secondary RNA structure indicates a minimum free energy of MEF - 261.10 kcal/mol. Our results indicate that IFNβ was upregulated (37.8-fold, < 0.0001) in cells which transfected by rhIFNβ-ELK16 compared to the mock and un-transfected conditions. Altogether, our results show that the presence of mini self-cleavage Intein-ELK16 tag along with the rhIFNβ had no interference in transcription of rhIFNβ in the HEK293T cell line.
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http://dx.doi.org/10.1007/s12291-020-00872-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994496PMC
April 2021

Gene expression profiles of YAP1, TAZ, CRB3, and VDR in familial and sporadic multiple sclerosis among an Iranian population.

Sci Rep 2021 Apr 8;11(1):7713. Epub 2021 Apr 8.

Isfahan Neuroscience Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.

Alterations in the regulatory mechanisms that control the process of myelination in the nervous system, may lead to the impaired myelination in the Multiple sclerosis. The Hippo pathway is an important mediator of myelination in the nervous system and might contribute to the pathophysiology of MS. This study examined via qPCR the RNA expression of YAP1, TAZ, and CRB3 as the key effectors of the Hippo pathway and also, VDR in the peripheral blood of 35 sporadic, 37 familial MS patients; and also 34 healthy first-degree relatives of the familial MS patients (HFR) and 40 healthy individuals without a family history of the disease (control). The results showed the increased expression of VDR in the sporadic group, as compared to other groups. There was also an increased expression of TAZ in the familial and HFR groups, as compared to the control group. The familial and sporadic patients displayed a significantly lower level of expression of YAP1 in comparison to the HFR group. The increased expression level in the sporadic patients and control group, as compared to the HFR group, was seen in CRB3. We also assessed different clinical parameters and MRI characteristics of the patients. Overall, these findings suggest that Hippo pathway effectors and also VDR gene may play a potential role in the pathophysiology of the sporadic and familial forms of MS. Confirmation of different gene expression patterns in sporadic and familial MS groups may have obvious implications for the personalization of therapies in the disease.
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http://dx.doi.org/10.1038/s41598-021-87131-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032816PMC
April 2021

Computational study for suppression of CD25/IL-2 interaction.

Biol Chem 2021 01 19;402(2):167-178. Epub 2020 Nov 19.

Department of Clinical Oncology, Queen Elizabeth Hospital, HKSAR, China.

Cancer recurrence presents a huge challenge in cancer patient management. Immune escape is a key mechanism of cancer progression and metastatic dissemination. CD25 is expressed in regulatory T (Treg) cells including tumor-infiltrating Treg cells (TI-Tregs). These cells specially activate and reinforce immune escape mechanism of cancers. The suppression of CD25/IL-2 interaction would be useful against Treg cells activation and ultimately immune escape of cancer. Here, software, web servers and databases were used, at which designed small interfering RNAs (siRNAs), designed peptides and virtual screened small molecules against CD25 were introduced for the prospect of eliminating cancer immune escape and obtaining successful treatment. We obtained siRNAs with low off-target effects. Further, small molecules based on the binding homology search in ligand and receptor similarity were introduced. Finally, the critical amino acids on CD25 were targeted by a designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further and studies.
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http://dx.doi.org/10.1515/hsz-2020-0326DOI Listing
January 2021

Bioenergetic analysis of aged-phenotype skin in a rare syndromic cutis laxa.

J Cosmet Dermatol 2021 Feb 1. Epub 2021 Feb 1.

Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran.

Background: Skin aging is an inevitable phenomenon characterized by wrinkled skin and loss of elasticity. To date, several studies have been performed on skin aging to discover the underlying mechanisms and improve efficient preventive strategies and anti-aging therapeutics.

Aims: Here, we aimed to investigate the modifications of oxidative phosphorylation and glycolysis which are the critical determinants of aging in aged-phenotype skin.

Methods: Due to the complexity of the skin aging process, we performed bioenergetic measurements on aged-phenotype fibroblasts from an inherited cutis laxa syndrome which remarkably presents clinical features of normal aged skin. Bioenergetic analysis was performed on cutis laxa samples (n = 3) and healthy samples (n = 3) using Seahorse XFe24 Analyzer. We also compared the sensitivity of cultured aged-phenotype fibroblasts to normal cells in glucose withdrawal.

Results: Our results show a significant increase in oxidative phosphorylation parameters but not glycolysis in the patient fibroblast cells implying increased energy demand and preferential dependence on mitochondrial respiration in those cells. Interestingly, we found the patient cells demonstrate hypersensitivity to glucose starvation, supporting their enhanced energy consumption.

Conclusions: In summary, our work suggested increased energy demand and higher oxidative phosphorylation in aged-phenotype cells which can be considered in anti-skin aging therapeutic design.
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http://dx.doi.org/10.1111/jocd.13951DOI Listing
February 2021

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells.

BMC Biotechnol 2021 Jan 29;21(1). Epub 2021 Jan 29.

Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.

Background: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient.

Results: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis.

Conclusion: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.
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http://dx.doi.org/10.1186/s12896-020-00665-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7844963PMC
January 2021

Evaluation of Silencing Effect of miR-133a-5p Mimic on TIM-3 Expression in AML (HL-60) Cell Line.

Indian J Clin Biochem 2020 Jul 4;35(3):359-366. Epub 2019 Jun 4.

Department of Genetic and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Acute myelogenous leukemia (AML) is a complex blood malignancy leading to immature leukemic stem cells (LSCs) proliferation. T cell immunoglobulin mucin-3 (TIM-3) is known as a biomarker of AML LSCs. Several microRNAs (miRNAs) can affect gene expression in AML. In this study, the silencing effect of miR-133a-5p on TIM-3 expression in AML cell lineage (HL-60) was investigated. It's been hypothesized that miR-133a-5p may suppress the TIM-3 expression in AML cell line. Initially, miRNA-TIM-3 prediction, enrichment, and network analysis were done. Then, miR-133a-5p mimic was transfected into HL-60 cells. The TIM-3 protein and gene expression were measured by flow cytometry analysis and real-time PCR, respectively. MTT assay was also carried out. Based on the Bioinformatics predictions, miR-133a-5p was able to silence TIM-3 expression. Also, significant pathways pertained to miR-133a-5p were obtained using enrichment analysis. According to this, miR-133a-5p was mainly engaged in the MAPK signaling pathway and Nicotine addiction pathway using the KEGG database. The TIM-3 protein expression of the transfected cells was measured as 17.15 ± 8.87% ( = 0.001). A 52.48% significant gene silencing in mRNA level was obtained in comparison to the negative control. Despite of down regulation of TIM-3, HL-60 cell viability has not been significantly changed. It has been finally confirmed that miR-133a-5p could strongly suppress TIM-3 expression in AML cell line. Presumably, down regulation of TIM-3 could affect MAPK and Nicotine addiction signaling pathways.
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http://dx.doi.org/10.1007/s12291-019-00834-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7326904PMC
July 2020

DMLR: A toolkit for investigation of deoxyribozyme-mediated ligation based on real time PCR.

Biochem Biophys Res Commun 2020 04 30;524(2):405-410. Epub 2020 Jan 30.

Department of Cell and Molecular Biology & Microbiology, Faculty of Biological Science and Technology, University of Isfahan, HezarJarib Street, Isfahan, 81746-73441, Iran.

Deoxyribozymes or DNAzyme are identified as catalytic DNA sequences which catalyze different chemical reactions. Ligating deoxyribozymes catalyze the formation of branched and linear products. Due to the lack of efficient read-out systems, there is no report on in vivo application of ligating deoxyribozymes. To expand the biological application of branched-RNA forming deoxyribozymes, we performed our study in order to suggest a practical toolkit for measurement of in vivo real-time activity of ligating deoxyribozymes. Further in vitro studies were designed to analyze the effects of the location of branch site on reverse transcriptase (RT) interference. With this toolkit even the activity of RT was measured precisely. Our results indicate that the activity of RT enzyme significantly affected by a 17 nt branched adaptor synthesized by 10DM24 ligating deoxyribozyme. The RT stalls at or near the RNA branch point during both initiation and elongation phases. The DNA synthesis is decreased 4.3 and 2.7 fold during initiation and elongation phases respectively. In conclusion, we introduce a general and practical toolkit called "DMLR" which is based on Real-time PCR method. The use of DMLR precisely determines RT behavior when encountered with any backbone modification with the ability of stopping the enzyme activity.
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http://dx.doi.org/10.1016/j.bbrc.2020.01.075DOI Listing
April 2020

The Comparison Between the Mutated HuIFN-β 27-101 and the Wild Type Interferon β: the Comprehensive In Silico Study to Evaluate the Effect of Mutations on IFN-β.

Adv Pharm Bull 2019 Oct 24;9(4):640-648. Epub 2019 Oct 24.

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Postal Code: 81746-73441 Isfahan, Iran.

Interferon beta (IFN-β) is used to combat multiple sclerosis (MS) disease. Creating R27T and V101F mutations (mHuIFN-β-27 and mHuIFN-β-101) is one of the tasks performed to improve human interferon beta (HuIFN-β) half-life, function and expression. In this work, the impact of R27T and V101F mutations in recombinant IFN-β on its binding to interferon receptors were studied by molecular docking. This work was performed through in silico study. The simulation of mutation was performed using the online Rosetta Backrub software and checked using server verify3D. Comparison of access to the solvent of the amino acids in the structures created was performed using the asaview online server. Also, the effect of mutations on the fold of the protein was reviewed by the online HOPE server. The molecular docking was performed between HuIFN-β and the external region of IFNAR receptor using the online ClusPro2 protein-protein docking server. The comparison of the values of the negative binding energy (ΔGbind) obtained from protein-protein molecular docking between IFNAR receptor and HuIFN-β, mHuIFN-β-27, mHuIFN-β-101 and mHuIFN-β-27-101 ligands did not show a significant difference, and these differences do not see any meaningful relationship between them (P > 0.9999). Regarding these results, it can be concluded that these mutations do not have a negative effect on the composition of the complex rHuIFN-β/IFNAR. So, they do not interfere with the binding of the IFN-β to the receptor. It is concluded that the quality of the rHuIFN-β is improved by introducing these two mutations.
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http://dx.doi.org/10.15171/apb.2019.074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912172PMC
October 2019

Integral membrane protein expression of human CD25 on the cell surface of HEK293 cell line: the available cellular model of CD25 positive to facilitate in vitro developing assays.

Biomol Concepts 2019 Sep 21;10(1):150-159. Epub 2019 Sep 21.

Institute of Hematology, Peking Union Medical College, Beijing, China.

Typically, CD25 is expressed on the cellular surface of regulatory T (Treg) cells. These cells are significant in regulating the self-tolerance and also preventing the immune system from attacking a person's own tissues and cells. They promote the cancer progression by playing an important role in evading the immune system. Thus, the experimental procedures was aimed to clone and express human CD25 in HEK293 cell line, as the available cellular model, for the purpose of developing assays to facilitate and enhance the studies on an available CD25 positive cell. The secondary RNA structure of CD25 was evaluated by in silico analysis. Then, cDNA of human CD25 were synthesized from isolated total mRNA of cultured and stimulated PBMCs from blood donors. After cloning the cDNA of CD25 into a pcDNA3.1(+) plasmid, using the effective transfection of the recombinant pcDNA3.1(+) in HEK293, qRT-PCR and flow cytometry methods were used to quantitatively evaluate CD25 transcripts and protein level. There was a 4.8 fold increase in transcripts and a 76.2% increase in protein levels of CD25 when comparing the transfected and control cell lines. The genetically engineered HEK293 cell line expressing Treg cell surface marker of CD25 was introduced in this study for the first time. This cell line can be used to overcome the problematic issues for studying Treg cells including low population of Tregs in peripheral blood, low recovery methods for Treg isolation, time-consuming and non-cost benefit methods in the conditions of in vitro cell culture experiments for the studies focused on the binding of IL-2 to CD25.
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http://dx.doi.org/10.1515/bmc-2019-0018DOI Listing
September 2019

Expression Pattern of microRNAs, miR-21, miR-155 and miR-338 in Patients with Type 1 Diabetes.

Arch Med Res 2019 04 24;50(3):79-85. Epub 2019 Jul 24.

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran. Electronic address:

Background: Type 1 diabetes (T1D) is a multifactorial disease identified by a deficiency in the production of insulin. MicroRNAs (miRNAs) are identified as important epigenetic regulators in T1D. Many studies highlight the differential expression of these small non-coding molecules in the pathogenesis of T1D.

Aim Of The Study: In the present study, the expression pattern of miR-21, miR-155 and miR-338 were analyzed in the peripheral blood mononuclear cells (PBMCs) of T1D patients compared to healthy controls.

Methods: The expression levels of miR-21, miR-155 and miR-338 were measured in the PBMCs of 30 T1D patients and 11 healthy controls by real time PCR method. The final results were statistically analyzed and ROC curves were created for miRNAs with significant differential expression.

Results: Both miR-155 (p value: 0.021) and miR-21 (p value: 0.05) were upregulated in the PBMCs of T1D patients compared to healthy controls. There was no significant difference in the expression level of miR-338 between patients and controls. Furthermore, ROC curve analysis was performed for miR-21 (AUC: 0.65) and miR-155 (AUC: 0.73) which suggests the potential role of miR-155 as a biomarker in T1D patients. Using integrative computational analysis, a number of dysregulated miR155-mRNA and miR21-mRNA interactions were also suggested.

Conclusion: Our findings suggest the significant association between the expression levels of miR-21 and miR-155 with T1D. In addition, miR-155 (AUC: 0.73) could be considered as a possible biomarker to track disease in T1D patients.
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http://dx.doi.org/10.1016/j.arcmed.2019.07.002DOI Listing
April 2019

Targeting a viral DNA sequence with a deoxyribozyme in a preparative scale.

Biochimie 2019 Oct 1;165:161-169. Epub 2019 Aug 1.

Department of Biology, University of Isfahan, Isfahan, Iran.

Deoxyribozymes are synthetic and single stranded DNAs that are capable of catalysis of a variety of reactions, including cleavage of DNA substrates. Deoxyribozymes are usually characterized by analytical single-turnover kinetic assays, however, for many applications e.g. characterization of the reaction products, semi-preparative and preparative reactions are required. At such scales, there is a lack of comprehensive analysis and conditions that supports high amount of products in an appropriate time-scale are vaguely guessed by researchers. In this report, catalytic activity of an oxidizing DNA-cleaving deoxyribozyme, F-8(X), was comprehensively inspected in semi-preparative (10 μM substrate) scale. A 60 nucleotides long synthetic DNA sequence was selected as the target DNA for this study. The DNA sequence was originated from a single stranded DNA virus. Investigations revealed high yield in the presence of optimal concentration of oxidizing agents. The optimal conditions have been applied for scale-up of the reaction to preparative (40 μM substrate) and multi-turnover reactions to achieve highest amount of product in a cost-, time- and labor-effective manner. Such a comprehensive analysis of a deoxyribozyme's activity in semi-preparative scale provides a platform for expanded applications of DNA catalysts as a tool in chemical biology.
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http://dx.doi.org/10.1016/j.biochi.2019.07.022DOI Listing
October 2019

Genetic Variation in Intergenic and Exonic miRNA Sequence and Risk of Multiple Sclerosis in the Isfahan Patients.

Iran J Allergy Asthma Immunol 2018 Oct 14;17(5):477-484. Epub 2018 Oct 14.

Department of Biology, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran AND Department of Biology, Fars Science and Research Branch, Islamic Azad University, Shiraz, Iran.

MicroRNAs (miRNAs), have been documented to perform a key role in the pathogenesis of multiple sclerosis (MS), a chronic inflammatory and autoimmune disease. Recent studies have shown that single nucleotide polymorphism in the sequence of the miRNA may change their production and expression which can lead to miRNA dysfunction and pathogenicity. Some studies have reported the relationship between miRNA polymorphism and the increased risk of autoimmune disease. This study was conducted to investigate the association between mir155 rs767649, mir196a2 rs11614913 and mir23a rs3745453 polymorphism and the risk of multiple sclerosis in the Iranian MS patients in Isfahan. A population of 80 patients and the same number control were selected. After DNA extraction, genotyping was performed through tetra amplification refractory mutation system-PCR method (T ARMS PCR). The frequencies of TT, TC and CC genotypes of mir23a were 46, 35 and 20% in MS patients and 42, 14 and 24 in healthy subjects respectively. These results showed that individuals carrying the genotypes of rs3745453 TC had a 2.3-fold increased risk of MS (OR=2.3, p=0.048). There was no significant difference between genotypes and allele frequency of mir155 and mir196a2 in patients and healthy controls (p>0.05). Our findings specified that CT heterozygosity in mir23a gene significantly related with risk of MS. Unlike mir155 and mir196a2, mir23a rs3745453 may have contributed to the etiology of MS in Isfahan patients. However, extensive studies are required to gain more reliable and authentic results.
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October 2018

Increased expression of microRNAs, miR-20a and miR-326 in PBMCs of patients with type 1 diabetes.

Mol Biol Rep 2018 Dec 7;45(6):1973-1980. Epub 2018 Sep 7.

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

Type 1 diabetes (T1D) is an autoimmune disorder which is characterized by autoimmune attack on β cells of pancreas and lack of insulin. The involvement of microRNAs (miRNAs) in the development of immune system and their differential expression in various autoimmune diseases including T1D have been well established. In this study, the association between expression levels of miR-20a, miR-326 and T1D were evaluated. The expression levels of miR-20a and miR-326 were measured in the PBMCs of 21 T1D patients and 16 healthy controls using qPCR method. In silico analysis was also performed on targetome of miR-20a and miR-326. Both miR-20a (p value: 0.015) and miR-326 (p value: 0.005) were upregulated in the PBMCs of T1D patients compared to healthy controls. Furthermore, different dysregulated miR326-mRNA and miR20a-mRNA interactions were also suggested using integrative computational analysis. The expression level of miR-20a and miR-326 indicates significant association with T1D which suggests the possible regulatory effects of these non-coding RNAs in T1D.
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http://dx.doi.org/10.1007/s11033-018-4352-zDOI Listing
December 2018

Identification of Two Novel Mutations in KDM3A Regulatory Gene in Iranian Infertile Males

Iran Biomed J 2019 05 28;23(3):220-7. Epub 2018 Jul 28.

Isfahan Fertility and Infertility Center, Isfahan, Iran.

Background: KDM3A is a key epigenetic regulator expressed in the testis and is required for packaging and condensation of sperm chromatin. To this point, the association of the KDM3A gene with infertility has not been studied in human. The aim of this study was to screen any new mutation in KDM3A gene to explore more details of human male infertility.

Methods: In this work, 150 infertile men (oligozoospermia and azoospermia) and 150 normal healthy fathers were studied. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing were used to screen any mutation in exons 12, 22, and 24 of KDM3A.

Results: The infertile men showed various SSCP patterns for the exons 12 and 24, but not for exon 22. A transversion point mutation in exon 12 and a single nucleotide deletion in exon 24 were detected using sequencing analysis. The transversion mutation was located in the preceding exon of lysine-specific demethylase1 and Jumonji (Jmj)-C domain and the later one (deletion) in the cupin-like motif of KDM3A protein. Neither Y chromosome microdeletions nor partial azoospermia factor deletion was found in these patients.

Conclusion: The mutations found in infertile men with otherwise unexplained severe spermatogenic failure could be considered as the origin of their abnormalities.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6462298PMC
May 2019

In vitro and computational studies on the effects of ARE deletion and targeted mutations on the expression of interferon beta-1a in HEK293T cells.

Appl Microbiol Biotechnol 2018 Aug 21;102(16):7047-7059. Epub 2018 Jun 21.

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, 81746-73441, Iran.

Interferon beta (IFNβ) is transiently expressed in response to viral infections and widely used to treat relapsing-remitting multiple sclerosis (MS). We introduced mutations in the IFNβ gene (in the 27th and 101st codons and in the Kozak sequence, and also deletion of 3' and 5' unstable, untranslated region, UTR) with the aim of increasing the expression of IFNβ. Computational analyses of mutant and wild-type RNAs and proteins of IFNβ by RNAfold, ASAView, HOPE and Ramachandran plot, and iStable web servers showed that the mutations could decrease RNA stability, protein solvent accessibility, and protein stability but could not change correct folding. Two recombinant IFNβ101 and IFNβ101+27 constructs were designed by site-directed mutagenesis. The wild-type IFNβ gene also was used as a control. In vitro experiments by quantitative real-time PCR, dot blot, SDS-PAGE, and Western blot assays showed an increased expression level of recombinant IFNβs. 79.9-fold, 99.7-fold, and 190-fold elevations in the mRNA expression of IFNβw, IFNβ101, and IFNβ101+27 were seen, respectively, in comparison with the endogenous IFNβ mRNA in untransfected HEK293T cells. The IFNβ mRNA expression was increased 2.38-fold and 1.25-fold for 101+27 and 101 mutated forms, respectively, in comparison with the IFNβ wild-type construct. An elevation in IFNβ protein production was also clearly detected in the transfected HEK293T cell containing recombinant IFNβ101 and IFNβ101+27 constructs. Finally, these directed mutations in the IFNβ gene successfully elevated protein and mRNA production but in silico analyses of mutant mRNAs showed decreased mRNA stability, unlike previous studies, in comparison with the wild-type mRNA.
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http://dx.doi.org/10.1007/s00253-018-9162-7DOI Listing
August 2018

Reconstruction of the genome-scale co-expression network for the Hippo signaling pathway in colorectal cancer.

Comput Biol Med 2018 08 26;99:76-84. Epub 2018 May 26.

Laboratory of Systems Biology and Bioinformatics (LBB), Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. Electronic address:

The Hippo signaling pathway (HSP) has been identified as an essential and complex signaling pathway for tumor suppression that coordinates proliferation, differentiation, cell death, cell growth and stemness. In the present study, we conducted a genome-scale co-expression analysis to reconstruct the HSP in colorectal cancer (CRC). Five key modules were detected through network clustering, and a detailed discussion of two modules containing respectively 18 and 13 over and down-regulated members of HSP was provided. Our results suggest new potential regulatory factors in the HSP. The detected modules also suggest novel genes contributing to CRC. Moreover, differential expression analysis confirmed the differential expression pattern of HSP members and new suggested regulatory factors between tumor and normal samples. These findings can further reveal the importance of HSP in CRC.
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http://dx.doi.org/10.1016/j.compbiomed.2018.05.023DOI Listing
August 2018

Network-based expression analyses and experimental validations revealed high co-expression between Yap1 and stem cell markers compared to differentiated cells.

Genomics 2019 07 22;111(4):831-839. Epub 2018 May 22.

Laboratory of Systems Biology and Bioinformatics (LBB), Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran. Electronic address:

The Hippo signaling pathway is identified as a potential regulatory pathway which plays critical roles in differentiation and stem cell self-renewal. Yap1 is a primary transcriptional effector of this pathway. The importance of Yap1 in embryonic stem cells (ESCs) and differentiation procedure remains a challenging question, since two different observations have been reported. To answer this question we used co-expression network and differential co-expression analyses followed by experimental validations. Our results indicate that Yap1 is highly co-expressed with stem cell markers in ESCs but not in differentiated cells (DCs). The significant Yap1 down-regulation and also translocation of Yap1 into the cytoplasm during P19 differentiation was also detected. Moreover, our results suggest the E2f7, Lin28a and Dppa4 genes as possible regulatory nuclear factors of Hippo pathway in stem cells. The present findings are actively consistent with studies that suggested Yap1 as an essential factor for stem cell self-renewal.
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http://dx.doi.org/10.1016/j.ygeno.2018.05.007DOI Listing
July 2019

Cooverexpression of EpCAM and c-myc genes in malignant breast tumours.

J Genet 2017 Mar;96(1):109-118

Faculty of Sciences, Division of Genetics, Department of Biology, University of Isfahan, Isfahan 81746-73441, Iran.

The overexpression of epithelial cell adhesion molecule (EpCAM), a proto-oncogene, affects progression, treatment, and diagnosis of many adenocarcinomas. C-myc has been shown to be a downstream target of EpCAM and is also one of the most important proto-oncogenes routinely overexpressed in breast cancer. However, cooverexpression of EpCAM and c-myc genes has not been investigated in breast cancer tissues, particularly in Iranian population. The aim of this study was to assess the expression of EpCAM and c-myc genes in malignant breast cancer tissues using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) followed by analyses of the association between the outcomes. In this study, 122 fresh tissues, including 104 malignant and 18 benign samples, were disrupted by mortar and pestle, and then the RNA was isolated from the samples and converted to cDNA. The relative expression levels of EpCAM and c-myc genes were measured by 2 method using RT-qPCR. EpCAM protein level was also assessed in 66 cases using Western blot technique. Using RT-qPCR method, our results showed that EpCAM was overexpressed in 48% of malignant and 11.1% of benign samples. Evaluating EpCAM protein overexpression in a portion of samples depicted the fully concordance rate between Western blot and RT-qPCR techniques. C-myc expression was first evaluated by RT-qPCR method, showing the overexpression rate of 39% and 28% in malignant and benign samples, respectively. These data were also quite concordant with the clinically available immunohistochemistry reports of the same samples studied in this study. Importantly, overexpression of EpCAM and c-myc was significantly associated and showed an agreement of 57.3%. This study demonstrated the cooverexpression of EpCAM and c-myc in breast tumours collected from breast cancer patients of the Iranian population. EpCAM and c-myc positive cases were significantly associated with reduced and enhanced risk of ER/PR positivity respectively. However, both were associated with grade III of breast cancer.
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http://dx.doi.org/10.1007/s12041-017-0748-0DOI Listing
March 2017

Molecular Genetic and Epigenetic Basis of Multiple Sclerosis.

Authors:
Zohreh Hojati

Adv Exp Med Biol 2017 ;958:65-90

Department of Biology, Genetics Section, School of Sciences, University of Isfahan, Isfahan, Iran.

Multiple Sclerosis (MS) is a chronic immune-mediated disease of spinal cord and brain. The initial event in MS occurs when activated CD4 T cells in periphery exacerbates immune responses by stimulating immune cells such as B cells, CD8 cells, mast cells, granulocytes and monocytes. These proinflammatory cells pass blood brain barrier by secreting proinflammatory cytokines including TNF-α and INF- which activate adhesion factors. APCs (antigen-presenting cells) reactivate CD4 T cells after infiltrating the CNS and CD4 T cells produce cytokines and chemokines. These proinflammatory cytokines aggravate inflammation by inducing myelin phagocytosis through microglia and astrocytes activation. MS is believed to have a multifactorial origin that includes a combination of multiple genetic, environmental and stochastic factors. Although the exact component of MS risks that can be explained by these factors is difficult to determine, estimates based on genetic and epidemiological studies suggest that up to 60-70 % of the total risk of MS may be contribute to genetic factors. In continue, firstly we provide an overview of the current understanding of epigenetic mechanisms, and so present evidence of how the epigenetic modifications contribute to increased susceptibility of MS. We also explain how specified epigenetic modifications may influence the pathophysiology and key aspects of disease in MS (demyelination, remyelination, inflammation, and neurodegeneration). Finally, we tend to discuss how environmental factors and epigenetic mechanisms may interact to have an effect on MS risk and clinical outcome and recommend new therapeutic interventions that might modulate patients' epigenetic profiles.
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http://dx.doi.org/10.1007/978-3-319-47861-6_6DOI Listing
September 2017

Screening of Two Neighboring Mutations in Iranian Infertile Men with Non-Obstructive Azoospermia.

Int J Fertil Steril 2017 Jan-Mar;10(4):390-394. Epub 2016 Nov 1.

Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

The genetic association between cystic fibrosis transmembrane conductance regulator () gene mutations and male infertility due to congenital bilateral absence of vas deferens (CBAVD) is well established. Mutant , however may also be involved in the etiology of male infertility in non-CBAVD cases. The present study was conducted to estimate the frequency of ∆I507 and ∆F508 gene mutations in Iranian infertile males. We undertook the first study of association between these mutations and non-obstructive azoospermia in Iran. In this case-control study, 100 fertile healthy fathers and 100 non-obstructive azoospermia's men were recruited from Isfahan Infertility Center (IIC) and Sari Saint Mary's Infertility Center, between 2008 and 2009. Screening of F508del and I507del mutations was carried out by the multiplex-ARMS-PCR. Significance of differences in mutation frequencies between the patient and control groups was assessed by Fisher's exact test. The ΔF508 was detected in three patients. However there are no significant association was found between the presence of this mutated allele and infertility [OR=9.2 (allele-based) and 7.2 (individual-based), P=0.179]. None of the samples carried the ΔI507 mutation. Altogether, we show that neither ΔI507 nor ΔF508 is involved in this population of Iranian infertile males with non-obstructive azoospermia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5134755PMC
http://dx.doi.org/10.22074/ijfs.2016.4593DOI Listing
November 2016

Association between polymorphisms of exon 12 and exon 24 of JHDM2A gene and male infertility.

Int J Reprod Biomed 2016 Jun;14(6):389-96

Division of Genetics, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

Background: Some dynamic changes occurs during spermatogenesis such as histone removal and its replacement with transition nuclear protein and protamine. These proteins are required for packing and condensation of sperm chromatin. JHDM2A is a histone demethylase that directly binds to promoter regions of Tnp1 and Prm1 genes and controls their expression by removing H3K9 at their promoters.

Objective: The association between polymorphisms of exon 12 and exon 24 in JHDM2A gene and male infertility were evaluated for the first time.

Materials And Methods: In this experimental study, 400 infertile men (oligospermia and azoospermia) and normal healthy fathers were evaluated (n=200). Single Strand Conformation Polymorphism (SSCP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used for screening any polymorphisms that are exist in exon 12 and exon 24.

Results: Exon 24 PCR products were analyzed by RFLP but no polymorphism was found in this exon at the restriction site of EcoRV enzyme. Our monitoring along the whole nucleotides of exon 12 and exon 24 were continued using SSCP method, but we found no change along these exons.

Conclusion: Generally, this study evaluated the association between polymorphisms in exon 12 and exon 24 of JHDM2A gene and male infertility which suggests that polymorphisms of these exons may not be associated with the risk of male infertility.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4971551PMC
June 2016

Targeted mutations in Val101 and Arg27 interferon beta protein increase its transcriptional and translational activities.

Cytokine 2016 Feb 23;78:1-6. Epub 2015 Nov 23.

Genetics & Molecular Biology Department, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Interferon β (IFNβ) is the most prescribed drug that has been used frequently for the treatment of multiple sclerosis (MS) patients. The aim of this study is to improve the production of IFNβ by induction of site directed mutagenesis. Accordingly, recombinant constructs were designed in order to enhance the expression of IFNβ mRNA and protein. The recombinant plasmids were transfected to the CHO cell line, following RNA extractions and cDNA synthesis. The effects of recombinant constructs were analyzed by real time PCR, ELISA and MTT assay. Transfected samples with either IFNβ101 or IFNβ101+27 have shown 11.55 and 2.26 fold elevation and over-expression compare to the wild type construct respectively. Our data also indicated that the IFNβ101 and IFNβ101+27 constructs increase IFNβ protein expression more than 2.2 and 4.5 fold, respectively compared to the control group. It could be concluded that the substitution of Phe in the codon 101 position, which may increase the binding activity of IFNβ with its receptors and introduction of an additional N glycosylation site (Asn-X-Thr) in the position 27 of IFNβ protein may cause such an effect. The proliferative activity of transfected cells by a recombinant IFNβ101 decreases in comparison to the wild type, although it was not statistically significant. Over-expression of IFNβ in such a level is promising not only for the patients but also for the pharmaceutical industries.
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http://dx.doi.org/10.1016/j.cyto.2015.11.019DOI Listing
February 2016

The FimH Gene in Uropathogenic Escherichia coli Strains Isolated From Patients With Urinary Tract Infection.

Jundishapur J Microbiol 2015 Feb 3;8(2):e17520. Epub 2015 Feb 3.

Department of Microbiology and Immunology, Cellular and Molecular Research Center, Faculty of Medicine, Shahrekord University of Medical Sciences, Shahrekord, IR Iran.

Background: Urinary tract infections (UTIs) are one of main health problems caused by many microorganisms, including uropathogenic Escherichia coli (UPEC). UPEC strains are the most frequent pathogens responsible for 85% and 50% of community and hospital acquired UTIs, respectively. UPEC strains have special virulence factors, including type 1 fimbriae, which can result in worsening of UTIs.

Objectives: This study was performed to detect type 1 fimbriae (the FimH gene) among UPEC strains by molecular method.

Materials And Methods: A total of 140 isolated E. coli strains from patients with UTI were identified using biochemical tests and then evaluated for the FimH gene by polymerase chain reaction (PCR) analysis.

Results: The UPEC isolates were identified using biochemical tests and were screened by PCR. The fimH gene was amplified using specific primers and showed a band about 164 bp. The FimH gene was found in 130 isolates (92.8%) of the UPEC strains. Of 130 isolates positive for the FimH gene, 62 (47.7%) and 68 (52.3%) belonged to hospitalized patients and outpatients, respectively.

Conclusions: The results of this study indicated that more than 90% of E. coli isolates harbored the FimH gene. The high binding ability of FimH could result in the increased pathogenicity of E. coli; thus, FimH could be used as a possible diagnostic marker and/or vaccine candidate.
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http://dx.doi.org/10.5812/jjm.17520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4376967PMC
February 2015

Enhanced expression of bioactive recombinant VEGF-111 with insertion of intronic sequence in mammalian cell lines.

Appl Biochem Biotechnol 2015 Apr 18;175(8):3737-49. Epub 2015 Feb 18.

Division of Genetics, Biology Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran,

Among all VEGF-A isoforms, VEGF-111 is particularly important in molecular biology research owing to its potent angiogenic properties and its remarkable resistance to proteolysis. These features make it a potential candidate for therapeutic use in ischemic diseases. VEGF-111 is not expressed in normal cells, but expression is induced by UV-B irradiation and exposure to genotoxic agents. Here, to increase expression at the transcriptional and translational levels, we synthesized and cloned recombinant VEGF-111 cDNA. Two fragments encoding exons 1-4 and intron 4/5 plus exon 8a were amplified and cloned into the pBud.CE4.1 vector using a class IIs restriction enzyme-based method. The expression of VEGF-111 in CHO-dhfr - and HEK 293 cell lines was evaluated with real-time PCR, dot blotting, and ELISA. VEGF expression was increased about 10- and 18-fold in transfected CHO-dhfr - and HEK 293 cells, respectively. Dot blotting and ELISA confirmed successful production of VEGF-111 in both cell lines.
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http://dx.doi.org/10.1007/s12010-015-1541-2DOI Listing
April 2015

Effects of disruption of the nucleotide pattern in CRID element and Kozak sequence of interferon β on mRNA stability and protein production.

Autoimmunity 2015 23;48(5):336-43. Epub 2015 Mar 23.

Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University , Tehran , Iran .

Interferon β (IFNβ) is the most important drug that has been used frequently for multiple sclerosis treatment. This study has tried to improve the IFNβ production by introducing mutations in the coding region of IFNβ, while its amino acid sequence is intact. Two recombinant vectors IFNβ(K) and IFNβ(K+CRID )were designed by site-directed mutagenesis. The IFNβ(K) and IFNβ(K+CRID) have two substitutions in Kozak sequence and four substitutions in CRID sequence, respectively. The Chinese hamster ovary (CHO) cell codon usage optimization was also performed for both of them. They were transiently transfected to CHO-dhfr(-) cell line using Lipofectamine kit (Invitrogen, Grand Island, NY). The amount of mRNA and protein was determined by real time PCR and ELISA. The results of this study indicate that the amount of IFNβ protein produced by CHO cells containing IFNβ(K) has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβ(K+CRID) construct; more than 4.6-fold and 6-fold, respectively. It could be concluded that disruption of AT pattern in CRID element increase RNA and protein production, improve IFNβ mRNA stability and, may also enhance mRNA half-life. In a similar way, more proteins are produced by modification of Kozak sequence.
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http://dx.doi.org/10.3109/08916934.2015.1022164DOI Listing
April 2016

New Insights into VEGF-A Alternative Splicing: Key Regulatory Switching in the Pathological Process.

Avicenna J Med Biotechnol 2014 Oct;6(4):192-9

Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, Iran.

Vascular endothelial growth factor (VEGF-A) is one of the most important regulatory factors in pathological and physiological angiogenesis. Alternative splicing is a complicated molecular process in VEGF-A gene expression which adds complexity to VEGF-A biology. Among all VEGF-A exons, alternative splicing of exon 8 is the key determinant of isoform switching from pro-angio-genic VEGF-xxx to anti-angiogenic VEGF-xxxb. This is known as a key molecular switching in many pathological situations. In fact, the balance between VEGF-xxx and VEGF-xxxb isoforms is a critical controlling switch in both conditions of health and disease. Here, the properties of VEGF-xxx and VEGF-xxxb isoforms were discussed and their regulatory mechanism and their roles in certain pathological processes were evaluated. In summary, it was suggested that C-terminal VEGF-A alternative splicing can provide a new treatment opportunity in angiogenic diseases.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4224658PMC
October 2014

Comparative insight into expression of recombinant human VEGF111b, a newly identified anti-angiogenic isoform, in eukaryotic cell lines.

Gene 2014 Dec 5;553(1):57-62. Epub 2014 Oct 5.

Division of Genetics, Biology Department, Faculty of Sciences, University of Isfahan, Isfahan, Iran. Electronic address:

VEGF-A is a critical growth factor in tumor growth and progression. Two families of VEGF-A isoforms are produced through alternative splicing including VEGFxxx pro-angiogenic and VEGFxxxb anti-angiogenic isoforms. VEGF111b is a new member of the VEGFxxxb family that is induced by mitomycin C and doesn't express in normal conditions. The potent anti-angiogenic properties of VEGF-111b and its remarkable resistance to proteolysis make it an interesting alternative candidate for therapeutic use in all types of cancers. Here, the recombinant VEGF-111b cDNA with insertion of intronic sequence was constructed by using a class IIs restriction enzyme-based method. The recombinant pBud-VEGF111b was transfected into CHO dhfr(-) and HEK 293 cell lines which are currently the standard hosts for the production of candidate therapeutic proteins. Then, the VEGF-111b expression was evaluated in two cell lines using the Real-time PCR. The production of VEGF-111b protein was also investigated here by dot blotting. The VEGF expression was increased about 109 and 185-folds in transfected CHO-dhfr(-) and HEK 293 cells, respectively, in comparison with the un-transfected cells. Dot blotting approach confirmed that both cell lines have successfully produced the VEGF-111b protein.
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http://dx.doi.org/10.1016/j.gene.2014.10.002DOI Listing
December 2014

Determination of ctxAB expression in Vibrio cholerae Classical and El Tor strains using Real-Time PCR.

Int J Mol Cell Med 2013 ;2(1):9-13

Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran.

Cholera is an infection of the small intestines caused by the bacterium V. cholerae. It is a major cause of health threat and also a major cause of death worldwide and especially in developing countries. The major virulence factor produced by V. cholerae during infection is the cholera toxin. Total mRNA extraction and reverse transcription was performed for making ctxAB cDNA. Relative Real-Time PCR analysis showed unequal enterotoxin production in V. cholerae strains. The results showed that, classical strain produces cholera toxin more than El Tor strain.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3920520PMC
February 2014

The molecular study of IFNβ pleiotropic roles in MS treatment.

Iran J Neurol 2013 ;12(4):149-56

MSc Student, Department of Biology, School of Sciences, University of Isfahan, Isfahan, Iran.

Multiple sclerosis (MS) is one of the most important autoimmune diseases recognized by demyelination and axonal lesion. It is the most common cause of disability in the young population. Various immunomodulatory and immunosuppressive therapies, including different formulations of interferon beta (IFNβ), glatiramer acetate (GA), mitoxantrone, and natalizumab are available for this disease. However, interferon has been the best prescribed. Although the precise mechanism of IFNβ is unclear, many studies indicate some potential mechanism including blocking T cells activation, controlling pro- and anti-inflammatory cytokine secretion, preventing activated immune cell migration through BBB, and inducing repair activity of damaged nerve cells by differentiating neural stem cells into oligodendrocytes. These molecular mechanisms have significant roles in IFNβ therapy. More researches are required in order for us to comprehend the mechanism of action of IFNβ, and improve and develop drugs for more efficient MS treatment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3829308PMC
June 2014