Sadeghi, N.,
Shirazi, N.,
Dehbashi, M.,
Maleki, B.,
Cho, W.C.,
Hojati najafabadi, Z. Practical Laboratory Medicine (23525517)42
Introduction: In response to the rapid spread of the SARS-CoV-2 virus, we developed a rapid molecular approach to diagnose COVID-19 without the need for RNA extraction. Methods: The study utilized two molecular methods, RT-qPCR and colorimetric RT-LAMP, to diagnose the RdRp and ORF8 genes, respectively, in oro-nasopharyngeal swabs. Due to the high sequence diversity of ORF8 in SARS-CoV and SARS-CoV-2, it has been identified as a suitable target for virus detection. The RT-LAMP method was also carried out directly on heat-treated swab samples. The strip tests were made using gold nanoparticles and combined with the RT-LAMP for further analysis. Results: The results showed that the isothermal amplification method had a sensitivity of 95 % (95 % C.I.: 86.08 %–98.96 %) and a specificity of 75 % (95 % C.I.: 19.41 %–99.37 %). The RT-LAMP-LFA method was able to distinguish positive and negative samples with 100 % sensitivity (95 % C.I.: 91.96–100) and 77.27 % specificity (95 % C.I.: 54.63–92.18). This method only required heating swab samples for 10 min at 65 °C before the RT-LAMP reaction. Conclusion: By utilizing the RT-LAMP in combination with the LFA, it is possible to diagnose SARS-CoV-2 rapidly without the need for RNA extraction. The entire process from sample collection to test interpretation takes only 75–90 min, and the results can be interpreted by untrained individuals with the naked eye. By employing the ORF8 gene as a diagnostic target and eliminating the need for RNA extraction, the direct RT-LAMP-LFA method achieves a significant breakthrough that was not previously reported. © 2024 The Authors
Helaoui, A.,
Sfar, S.,
Boudhiba, N.,
Dehghanian, F.,
Dehbashi, M.,
Bouchahda, H.,
Hojati najafabadi, Z.,
Kenani, A. Molecular Biology Reports (03014851)50(2)pp. 949-959
Background: Host genetic characteristics and environmental factors interactions may play a crucial role in cervical carcinogenesis. We investigated the impact of functional genetic variants of four xenobiotic-metabolizing genes (AhR, CYP1A1, GSTM1, and GSTT1) on cervical cancer development in Tunisian women. Methods: The AhR gene polymorphism was analyzed using the tetra-primer ARMS-PCR, whereas the CYP1A1 polymorphism genotypes were identified by PCR-RFLP. A multiplex ligation-dependent polymerase chain reaction approach was applied for the analysis of GSTM1 and GSTT1 polymorphisms. Results: The homozygous A/A genotype of the AhR gene (rs2066853) and the heterozygous T/C genotype of the CYP1A1 SNP (CYP1A1-MspI) appeared to be associated with an increased risk of cervical tumorigenesis (ORa = 2.81; ORa = 5.52, respectively). Furthermore, a significantly increased risk of cervical cancer was associated with the GSTT1 null genotype (ORa = 2.65). However, the null GSTM1 genotype showed any significant association with the risk of cervical cancer compared to the wild genotype (ORa = 1.18; p = 0.784). Considering the combined effect, we noted a significantly higher association with cancer risk for individuals with at least two high-risk genotypes of CYP1A1/GSTT1 (ORa = 4.2), individuals with at least two high-risk genotypes of CYP1A1/GSTT1/AhR (ORa = 11.3) and individuals with at least two high-risk genotypes of CYP1A1/GSTM1/GSTT1/AhR exploitation low-risk genotype as a reference. Conclusion: This study indicated that the single-gene contribution and the combined effect of xenobiotic-metabolizing gene polymorphisms (AhR, CYP1A1-MspI, GSTM1, and GSTT1) may have a considerable association with increased cervical cancer risk. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
Molecular Biology Reports (03014851)50(3)pp. 2183-2194
Background: As an available cell line, mouse pluripotent P19 has been widely employed for neuronal differentiation studies. In this research, by applying the in vitro differentiation of this cell line into neuron-like cells through retinoic acid (RA) treatment, the roles of some genes including DNMT3B, ICAM1, IRX3, JAK2, LHX1, SOX9, TBX3 and THY1 in neural differentiation was investigated. Methods and results: Bioinformatics, microscopic, and transcriptional studies were conducted in a time-dependent manner after RA-induced neural differentiation. According to bioinformatics studies, we determined the engagement of the metabolic and developmental super-pathways and pathways in neural cell differentiation, particularly focusing on the considered genes. According to our qRT-PCR analyses, JAK2, SOX9, TBX3, LHX1 and IRX3 genes were found to be significantly overexpressed in a time-dependent manner (p < 0.05). In addition, the significant downregulation of THY1, DNMT3B and ICAM1 genes was observed during the experiment (p < 0.05). The optical microscopic investigation showed that the specialized extensions of the neuron-like cells were revealed on day 8 after RA treatment. Conclusion: Accordingly, the neural differentiation of P19 cell line and the role of the considered genes during the differentiation were proved. However, our results warrant further in vivo studies. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.
European Journal of Clinical Investigation (00142972)52(11)
Background: Diagnosis is one of the main strategies to deal with infectious and deadly diseases such as coronavirus disease 2019 (COVID-19). The global pandemic of COVID-19 has led to an immediate need to expand rapid diagnostic techniques. New isothermal-based methods are being developed for COVID-19 detection aiming to resolve the limitations related to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method through immediate samples processing and minimizing false-negative or ambiguous results. Advances in nucleic acid amplification techniques (NAATs) can provide affordable and easy-to-use diagnostic platforms with high sensitivity and specificity in order to be available to the public as approved commercial kits. Aims: The development of point-of-care (POC) testing can assist in rapid clinical decision-making and mitigate burdens on health care facilities. Finally, we discussed the different diagnostic methods based on NAATs for COVID-19 in detail. Comparative parameters are addressed for all assays and Emergency Use Authorizations (EUA)-approved commercial tests are cited. Conclusions: Isothermal-coupled methods and LAMP-based molecular methods have been suggested as suitable portable tests with high diagnostic speed for use in POC testing. © 2022 Stichting European Society for Clinical Investigation Journal Foundation. Published by John Wiley & Sons Ltd.
Differentiation (14320436)126pp. 1-9
Neural differentiation as a major process during neural cell therapy is one of the main issues that is not fully characterized. This study focuses on the major deconstruction of the transcriptional networks that regulate cell fate determination during neural differentiation under the influence of RA signalling. In our studies, we used four different microarray datasets containing a total of 15,660 genes to determine which genes were differentially expressed during neural differentiation from pluripotent stem cells (P19), among the 17 samples from four different datasets that were integrated via meta-analysis approaches. Of the 15,660 gene expression in our data integration, 443 DEGs are induced during neural differentiation. Upstream dissection of these 443 DEGs revealed a network of protein-protein interactions (PPIs) from TFs and kinases, as well as intermediate proteins between them, which are indicated by three (POU51, NANOG, and FOXO1) down-expression genes and one PAX6 up-expression gene playing roles in up-stream of these 443 induced DEGs during neural differentiation. The constructed network from the PPIs database revealed that four novel sub-networks play major roles in neuron differentiation in cluster 3, retinol metabolism in cluster 4, Rap1 signalling pathways in cluster 2, and axonogenesis in cluster 6. These four clusters have revealed very useful information about how neural characterization will be created from pluripotent stem cells. This research reveals a plethora of information on the neural differentiation process, including cell commitment and neural differentiation, and lays the groundwork for future research into particular pathways involving protein-protein interactions in neurogenesis. © 2022 International Society of Differentiation
Scientific Reports (20452322)12(1)
The phenylpropanoid pathway serves as a rich source of metabolites in plants, and it is considered as a starting point for the production of many other important compounds such as the flavonoids, flavonols, coumarins, and lignans. Scrophularia striata is a member of the Lamiaceae family with some biological activities similar to flavonoid compounds such as antioxidant, antibacterial, anti-inflammatory and analgesic activities. Cinnamate 4-hydroxylase (C4H) and Chalcone synthase (CHS) are key enzymes of the phenylpropanoid pathway, leading to the biosynthesis of several secondary metabolites. In this study, two S. striata CHS and C4H were isolated and then analyzed. The investigation of the expression of these genes was performed under the effects of three salicylic acid (SA), jasmonic acid (JA), and gibberellic acid (GA) at concentrations of 100 and 300 ppm with a completely randomized design at the transcript level using Real Time PCR method. These have different expression patterns at developmental stages. Moreover, these genes present different sensitivities to hormonal treatment. Considering the total results, it was found that the amount of expression of these genes during the reproductive phase is higher than that of the vegetative phase. Additionally, the treatment of 300 ppm SA in the reproductive phase is the most effective treatment on increasing the corresponding phenylpropanoid compounds. A correlation analysis was performed between the phenylpropanoid compounds content and both CHS and C4H expression values at different phenological development stages. The results indicate that the expression variations of both CHS and C4H are significantly related to the changes in total phenolic content. We believe that the isolation of CHS and C4H can be helpful in better understanding phenylpropanoid metabolis. © 2022, The Author(s).
Dehbashi, M.,
Hojati najafabadi, Z.,
Bashi naeini, M.M.,
Ganjalikhany, M.R.,
Cho, W.C.,
Shimosaka, A.,
Navabi, P.,
Ganjalikhani-hakemi, M. Frontiers in Oncology (2234943X)11
For many years, high-affinity subunit of IL-2 receptor (CD25) has been considered as a promising therapeutic target for different pathologic conditions like allograft rejection, autoimmunity, and cancers. Although CD25 is transiently expressed by newly-activated T cells, it is the hallmark of regulatory T (Treg) cells which are the most important immunosuppressive elements in tumor microenvironment. Thus, Tregs can be considered as a potential target for chimeric antigen receptor (CAR)-based therapeutic approaches. On the other hand, due to some profound adverse effects pertaining to the use of CAR T cells, CAR NK cells have caught researchers’ attention as a safer choice. Based on these, the aim of this study was to design and develop a CAR NK cell against CD25 as the most prominent biomarker of Tregs with the prospect of overcoming immune escape mechanism in solid and liquid cancers. In the current study, an anti-CD25 CAR was designed and evaluated by comprehensive in silico analyses. Then, using lentiviral transduction system, NK-92 cell line was engineered to express this anti-CD25 CAR construct. In vitro functional analyses of anti-CD25 CAR for its reactivity against CD25 antigen as well as for cytotoxicity and cytokine production assays against CD25 bearing Jurkat cell line were done. In silico analyses demonstrated that the anti-CD25 CAR transcript and scFv protein structures were stable and had proper interaction with the target. Also, in vitro analyses showed that the anti-CD25 CAR-engineered NK-92 cells were able to specifically detect and lyse target cells with an appropriate cytokine production and cytotoxic activity. To conclude, the results showed that this novel CAR NK cell is functional and warrant further investigations. © Copyright © 2021 Dehbashi, Hojati, Motovali-bashi, Ganjalikhany, Cho, Shimosaka, Navabi and Ganjalikhani-Hakemi.
Journal of Cosmetic Dermatology (14732165)20(9)pp. 2999-3006
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. © 2021 Wiley Periodicals LLC
Dehbashi, M.,
Hojati najafabadi, Z.,
Bashi naeini, M.M.,
Ganjalikhani-hakemi, M.,
Shimosaka, A.,
Cho, W.C. Biological Chemistry (14316730)402(2)pp. 167-178
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 in silico designed small interfering RNAs (siRNAs), de novo 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 de novo designed peptide with disulfide bond. Hence we introduced computational-based antagonists to lay a foundation for further in vitro and in vivo studies. © 2020 Walter de Gruyter GmbH, Berlin/Boston 2020.
Bmc Biotechnology (14726750)21(1)
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. © 2021, The Author(s).
Imani, S.Z.H.,
Hojati najafabadi, Z.,
Khalilian, S.,
Dehghanian, F.,
Kheirollahi, M.,
Khorrami, M.,
Shaygannejad, V.,
Mirmosayyeb, O. Scientific Reports (20452322)11(1)
Multiple sclerosis (MS) is a chronic inflammatory and autoimmune disorder of the central nervous system characterized by myelin loss and axonal dysfunction. Increased production of inflammatory factors such as cytokines has been implicated in axon destruction. In the present study, we compared the expression level of IL7R, NFATc2, and RNF213 genes in the peripheral blood of 72 MS patients (37 familial MS, 35 sporadic MS) and 74 healthy controls (34 individuals with a family history of the disease, 40 healthy controls without a family history) via Real-time PCR. Our results showed that the expression level of IL7R was decreased in the sporadic patients in comparison with other groups. Additionally, there was an increased NFATc2 expression level in MS patients versus healthy controls. Increased expression of NFATc2 in sporadic and familial groups compared to the controls, and familial group versus FDR was also seen. Our results also represented an increased expression level of RNF213 in familial patients as compared to the control group. The similar RNF213 expression between sporadic and control group, as well as FDR and familial group was also seen. Diagnostic evaluation was performed by receiver operating characteristic (ROC) curve analysis and area under the curve (AUC) calculation. The correlation of clinical parameters including onset age and Expanded Disability Status Scale (EDSS) with our gene expression levels were also assessed. Overall, decreased expression level of IL7R in the sporadic cases and increased expression level of NFATc2 may be associated with the pathogenesis of MS disease. Confirmation of the effects of differential expression of RNF213 gene requires further studies in the wider statistical populations. © 2021, The Author(s).
Khalilian, S.,
Hojati najafabadi, Z.,
Dehghanian, F.,
Shaygannejad, V.,
Imani, S.Z.H.,
Kheirollahi, M.,
Khorrami, M.,
Mirmosayyeb, O. Scientific Reports (20452322)11(1)
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. © 2021, The Author(s).
Cell Journal (Yakhteh) (22285806)23(2)pp. 211-217
Objective: Alzheimer's disease (AD) is a type of dementia. Currently, there are not any existing and reliable methods for the prognosis or diagnosis of AD. Hence, finding a diagnostic/prognostic biomarker for AD helps physicians to prescribe the treatments and methods preventing disease progression. Circulating microRNAs (miRNAs) are the most promising biomarkers due to their non-invasive and easily accessible for diagnosis and prognosis of AD. The aim of current study is to evaluate expression levels of two unwell-known circulating miRNAs including hsa-miR-324-3p and hsa-miR-331-3p in serums of AD patients and to understand their roles in AD physiopathogenesis by in silico analysis. Materials and Methods: In this case and control study, to get the gene targets related to these two miRNAs, TargetScan, miRTargetLink Human and mirDIP web servers were applied. In addition, gene networks and gene ontology enrichment analysis were performed by STRING 10.5, KEGG and ShinyGO v0.41. Experimentally, expression levels of these two miRNAs in the serum of 21 patients with AD and 23 healthy individuals were compared using the quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. Results: The pathophysiological pathways associated with these two miRNAs were nucleotide metabolism and cellular response to stress pathway. Furthermore, the upregulated expression levels of hsa-miR-324-3p and hsa-miR-331-3p in comparison with the healthy control serums were not statistically significant (P>0.05). Conclusion: Non-significant results were obtained from the expression levels of AD patients and two significant pathways were obtained by networks and gene enrichment analysis. © 2021 Royan Institute (ACECR). All rights reserved.
Cell Transplantation (09636897)30
Cardiovascular disease is one of the most common causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources in cell-based therapies in heart regeneration. There are several methods to differentiate MSCs into cardiac-like cells, such as gene induction. Moreover, using a three-dimensional (3D) culture, such as hydrogels increases efficiency of differentiation. In the current study, mouse adipose-derived MSCs were co-transduced with lentiviruses containing microRNA-1 (miR-1) and Myocardin (Myocd). Then, expression of cardiac markers, such as NK2 homeobox 5(Nkx2-5), GATA binding protein 4 (Gata4), and troponin T type 2 (Tnnt2) was investigated, at both gene and protein levels in two-dimensional (2D) culture and chitosan/collagen hydrogel (CS/CO) as a 3D culture. Additionally, after induction of myocardial infarction (MI) in rats, a patch containing the encapsulated induced cardiomyocytes (iCM/P) was implanted to MI zone. Subsequently, 30 days after MI induction, echocardiography, immunohistochemistry staining, and histological examination were performed to evaluate cardiac function. The results of quantitative real -time polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by 3D culture of transduced cells increased expression of cardiac markers. Besides, results of in vivo study implicated that heart function was improved in MI model of rats in iCM/P-treated group. The results suggested that miR-1/Myocd induction combined with encapsulation of transduced cells in CS/CO hydrogel increased efficiency of MSCs differentiation into iCMs and could improve heart function in MI model of rats after implantation. © The Author(s) 2021.
Dehbashi, M.,
Hojati najafabadi, Z.,
Bashi naeini, M.M.,
Cho, W.C.,
Shimosaka, A.,
Ganjalikhani-hakemi, M. Gene Reports (24520144)24
The regulatory T cells function in the immune homeostasis, these cells express high level of CD25. It is known that CD25 expression levels are various among different cancers. Thus, we intended to dissect systems biology of a lncRNA pertained to CD25 and CD25 protein interactors-targeting miRNAs. Using RNA-seq data, co-expression analyses of the lncRNA pertained to some cancers were performed. Our analysis was done for protein interactors of CD25 by STRING 11.0, ShinyGO v0.60 and KEGG web servers were used for enrichment and network analysis of CD25. TargetScan 7.2, miRTargetLink Human and mirDIP were applied for determining the CD25 and CD25 interactors-targeting miRNAs. To find the lncRNA-miRNA and lncRNA-protein interactions, starBase v3.0, LncBase Predicted v.2 and SFPEL-LPI were utilized, respectively. Also, using Co-LncRNA, the co-expressed lncRNA analysis and the relative signaling pathways in some cancers including bladder, breast, head and neck, kidney, liver, lung, prostate and thyroid cancers using RNA-seq data were achieved. OIP5-AS1 was shown to have the interaction with CD25 and CD25 protein interactors-targeting miRNAs. In addition, the co-expression of OIP5-AS1 in cancers and their signaling pathways was identified. In conclusion, OIP5-AS1 can effect on CD25 expression in all relative signaling pathways of these cancers. © 2021 Elsevier Inc.
Journal of Isfahan Medical School (10277595)39(612)pp. 66-75
Background: Clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout of primary T cell has several limitations for clinical applications. Direct delivery of recombinant Cas9 protein and synthetic gRNA, as a pre-assembled ribonucleoprotein (RNP) complex, has become a potent approach to introduce highly efficient gene editing in primary T cells. In this study, we employed Cas9 RNP-based delivery system for targeted T Cell receptor alpha constant (TRAC) and β2 microglobulin (B2M) genes knockout in human primary T cells. Methods: Specific gRNAs were designed to target the first exons of TRAC and B2M genes. Cas9 protein and respective synthetic gRNAs were then mixed separately, and electroporated into human primary T cells isolated from peripheral blood mononuclear cells (PBMCs). The gene editing efficiency was quantified using tracking of indels by decomposition (TIDE) analysis and flow cytometry. Findings: Three days after electroporation of primary T cells with the TRAC and B2M targeting RNP complexes, TIDE analysis revealed the knockout efficiency of 13-60 percent for the TRAC-targeting gRNAs and 21-53 percent for B2M-targeting gRNAs. Flow cytometry analysis confirmed ~76% and ~27% complete loss of expression for the most efficient gRNAs targeting TRAC (TRAC-gRNA3) and B2M (B2M-gRNA2), respectively. Conclusion: Our results demonstrate that Cas9 RNP system can be efficiently delivered into primary T cells and result in targeted gene knockout. The protocol described here enables a streamlined and highly efficient solution for maximizing editing efficiency in primary T cells, and simplifies the gene editing process for next-generation immunotherapies. © 2021 Isfahan University of Medical Sciences(IUMS). All rights reserved.
Indian Journal of Clinical Biochemistry (09740422)36(2)pp. 159-166
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 Mycobacterium tuberculosis 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, p < 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. © 2020, Association of Clinical Biochemists of India.
Iranian Biomedical Journal (2008823X)25(1)pp. 62-67
Background: Among different roles of miRNAs in AD pathogenesis, hsa-miR-494-3p and hsa-miR-661 functions are poorly understood. Methods: To obtain the gene targets, gene networks, gene ontology, and enrichment analysis of the two miRNAs, some web servers were utilized. Furthermore, the expressions of these miRNAs were analyzed by qRT-PCR in 36 blood sera, including 18 Alzheimer’s patients and 18 healthy individuals. Results: The in silico analysis demonstrated the highlighted roles of metabolic and cellular response to stress pathways engaged in circulating hsa-miR-494-3p and hsa-miR-661 in AD. The qRT-PCR analysis showed that the downregulated expression level of hsa-miR-661 was statistically significant (p < 0.05). Also, the ROC curve of hsa-miR-661 displayed the significant AUC (p = 0.01). Conclusion: Based on our findings, the metabolic and cellular responses to stress pathways are closely connected to these two miRNAs functions. Besides, the qRT-PCR and Roc curve determined hsa-miR-661 could be as a biomarker for diagnosis or prognosis of AD patients. © 2021, Pasteur Institute of Iran. All rights reserved.
Biochemical and Biophysical Research Communications (0006291X)524(2)pp. 405-410
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. © 2020 Elsevier Inc.
Hojati najafabadi, Z.,
Ganjalikhani-hakemi, M.,
Ameri, M.,
Alimohammadi-jelodar, S.F.,
Dehbashi, M.,
Mohammad ganji, M.,
Homayouni, V.,
Khanahmad, H. Indian Journal of Clinical Biochemistry (09740422)35(3)pp. 359-366
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% (p = 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. © 2019, Association of Clinical Biochemists of India.
Journal of Horticultural Science and Biotechnology (14620316)95(2)pp. 183-191
Male flower induction and androgenesis in three genotypes of cucumber (Cucumis sativus), two Beth alpha F1 hybrids (BT1 and BT2, gynoecious) and Dastgerdi (DTG, monoecious), were evaluated. Induction of male flowers in gynoecious cucumber cultivars was assessed by single and combinatorial spraying of AgNO3 (3 mM), GA3 (1.5 mM) and CoCl2 (3 mM) after the first true leaf emergence. After 6 weeks from the beginning of treatments, the average numbers of male flowers induced by spraying AgNO3, GA3, and CoCl2 were 42, 14.5, and 0, respectively. Androgenesis was performed in two gynoecious cultivars, in which the donor plants were treated with AgNO3 (3 mM) or GA3 (1.5 mM) and the DTG cultivar without treatment. Among the three cultivars, the DTG showed the highest percentage of embryogenic calli formation (62.2%) and number of embryos per anther (1.81). In the gynoecious cultivars, the percentage of embryogenic calli formation (27.1%) and number of embryos per anther (0.23) in the anthers of GA3 treated plants were significantly higher than those treated by AgNO3. Cytological and SSR marker analysis of regenerants showed that 16 plants were spontaneous doubled haploid, 2 plants were diploid and one plant was tetraploid. © 2019, © 2019 The Journal of Horticultural Science & Biotechnology Trust.
Archives of Medical Research (01884409)50(3)pp. 79-85
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. © 2019 IMSS
Iranian Biomedical Journal (2008823X)23(3)pp. 220-227
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. © 2019, Pasteur Institute of Iran. All rights reserved.
Biomolecular Concepts (1868503X)10(1)pp. 150-159
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. © 2019 Moein Dehbashi et al.
Genomics (08887543)111(4)pp. 831-839
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. © 2018 Elsevier Inc.
Biochimie (03009084)165pp. 161-169
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. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
Advanced Pharmaceutical Bulletin (22517308)9(4)pp. 640-648
Purpose: 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. Methods: 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. Results: 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). Conclusion: 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. © 2019 The Author (s).
Iranian Journal Of Allergy, Asthma And Immunology (17355249)17(5)pp. 477-484
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. Copyright © October 2018, Iran J Allergy Asthma Immunol. All rights reserved.
Applied Microbiology And Biotechnology (14320614)102(16)pp. 7047-7059
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. © 2018, Springer-Verlag GmbH Germany, part of Springer Nature.
Molecular Biology Reports (03014851)45(6)pp. 1973-1980
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. © 2018, Springer Nature B.V.
Multiple sclerosis (MS) is an autoimmune disease that affects the human central nervous system and generally leads to permanent neurologic disability in young adults. Pathophysiology of MS contains two distinctive but interrelated and interacting arms—inflammatory demyelination and neurodegeneration. The massive immune activation in MS pathophysiology is the product of interactions among various proinflammatory and antiinflammatory cytokines as well as other players such as matrix metalloproteinases. Interferon-gamma and beta-interferons are heavily involved in inflammatory cascade of MS. In this study, the potential molecular mechanisms of IFNβ and IFNγ in MS, their antithetical effects, and the possible controversial roles of IFNγ in MS treatment will be described. © 2018 Elsevier Inc. All rights reserved.
Computers in Biology and Medicine (00104825)99pp. 76-84
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. © 2018 Elsevier Ltd
Journal of Genetics (09737731)96(1)pp. 109-118
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 −ΔΔCt 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. © 2017, Indian Academy of Sciences.
Journal of Mazandaran University of Medical Sciences (17359260)26(145)pp. 20-28
Background and purpose: Endothelial growth factor type b with anti-angiogenic activity and inhibition of tumor growth are considered as new anticancer drugs. The aim of this research was to study the expression of vegf111b in HEK293 human cells. Materials and methods: In this experimental study, HEK293 cells were transfected by pBUD.VEGF111b vector containing the VEGF111B gene through lipofectamine method. The mRNA of transfected cells and control cells were extracted and cDNA was built over it. Then, the expression levels of vegf111b were measured using Real time- PCR. Results: Transfection of HEK293 cells was successfully done and 48 hours after transfection of HEK293 cells, ct of the vegf111b expression in transfected cells was 23.17 and ct of the GAPDH control gene expression in these cells was 21.11. In the control (untransfected) cells the ct of GAPDH was 21.09 and there was no expression of vegf111b in these cells. Conclusion: Expression of Vegf111b recombinant protein in HEK293 cells is the first step for further research on this protein. Current study has provided the possibility of using this product for future research on angiogenesis and cancer treatments. © 2017, Mazandaran University of Medical Sciences. All rights reserved.
Advances in Experimental Medicine and Biology (00652598)958pp. 65-90
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-γ 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. © Springer International Publishing Switzerland 2017.
International Journal Of Fertility And Sterility (2008076X)10(4)pp. 390-394
The genetic association between cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and male infertility due to congenital bilateral absence of vas deferens (CBAVD) is well established. Mutant CFTR, 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 ΔI507 and ΔF508 CFTR gene mutations in Iranian infertile males. We undertook the first study of association between these CFTR 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. No significant association was however not 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 delta I507 nor delta F508 is involved in this population of Iranian infertile males with non-obstructive azoospermia. © 2016 Royan Institute (ACECR). All rights reserved.
International Journal Of Reproductive Biomedicine (24764108)14(6)pp. 389-396
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. © 2016, Research and Clinical Center for Infertitlity. All rights reserved.
Journal of Isfahan Medical School (10277595)33(367)pp. 2407-2416
Background: Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), is a fast, sensitive and reliable method of gene expression comparison that is prone to a lot of technical errors. On the other hand, historical reference (housekeeping) genes are not suitable for all tissues. Herein, we have tried to identify and evaluate the best reference gene for testis tissues for further qRT-PCR experiments. Methods: Testis tissues of 15 men with non-obstructive (NOA) and 15 men with obstructive (OA) azoospermia (as control individuals) were collected. Primer designing and verification of four candidate reference genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L37 (RPL37), ring finger protein 1 (RING1) and eukaryotic translation elongation factor 2 (eEF2) were performed using Beacon designer 8.1 software. PCR pre-optimization for reverse transcriptase input RNA and best primer concentration were included. Melt curve analysis was drawn and values of quantitation cycle (Cq) were extracted. Mean Cq analysis was calculated using BestKeeper v1 software and suitable reference genes were selected afterward. Findings: Comparing the mean Cq values between the NOA and OA groups declared that RPL37 and GAPDH showed the lowest standard deviations of 1.39 and 1.67 among the other candidates. GAPDH and RPL37 were selected as the best reference genes in testis tissues with their r values of 0.959 and 0.927, respectively. Conclusion: The results of this study show that the best reference genes for normalization of qRT-PCR data of testis tissues are GAPDH and RPL37. © 2016, Isfahan University of Medical Sciences(IUMS). All rights reserved.
Andrology (20472927)4(3)pp. 492-499
Summary: To evaluate the predictive value of histone demethylase KDM3A to protamine 1 (PRM1) mRNA expression ratio as a reliable marker of sperm retrieval in men with obstructive and non-obstructive azoospermia (NOA). Fifty eight azoospermic men, including 44 with NOA and 14 with obstructive azoospermia (OA). Testis tissue samples were collected from azoospermic men who have been referred for testicular sperm extraction (TESE) and micro-TESE. Relative expression ratio of KDM3A to PRM1 was analyzed after selection of approved reference genes. Histological classification of testis biopsies was performed. Sperm retrieval following TESE and micro-TESE was evaluated. A sperm retrieval prediction sensitivity of 95% was established when the Cq of PRM1 became smaller than the Cq of both KDM3A and GAPDH genes. However, azoospermic men with down-regulated KDM3A and decreased expression of PRM1 mRNA showed very low success for sperm retrieval (<25%), even after micro-TESE surgery. The KDM3A to PRM1 mRNA expression ratio can be used as a reliable marker of successful testicular sperm extraction in men with obstructive and non-obstructive azoospermia with 95% sensitivity. © 2016 American Society of Andrology and European Academy of Andrology.
Interferon beta (INFβ) is a therapeutic immunomodulatory agent, widely used in the treatment of multiple sclerosis (MS). The biological roles of INFβ and its mechanisms of action in MS are complex, multifactorial, and incompletely understood, but this chapter attempts to clarify the newest mechanisms of action of INFβ. INFβ suppresses inflammatory responses through different mechanisms in MS patients, including controlling the secretion of pro and anti-inflammatory cytokines, suppressing T cell activation, inducing differentiation of neural stem cells to oligodendrocytes which results in repair of damaged nerve cells, preventing the migration of activated immune cells through the blood-brain barrier, and some other mechanisms. Scientists have move toward new fields of study including individualized personalized medicine, and roles of micro RNA and vitamin D in MS patients who receive INFβ treatment. In summary, new advances in understanding of INFβ mechanisms of action will be mainly crucial and will in turn aid progression in MS management. © 2016 Elsevier Inc. All rights reserved.
Cytokine (10960023)78pp. 1-6
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. © 2015 Elsevier Ltd.
Journal of Isfahan Medical School (10277595)33(354)pp. 1691-1700
Background: Interferon beta (IFNβ) is one of the important cytokines expressed in response to stimulating factors such as antigens and plays roles in immunity and inflammatory process. In present study, the expression level of IFNβ-1a was examined in HEK293 cell line using real-time polymerase chain reaction (Real-Time PCR). Methods: IFNβ gene sequence was amplified using specific primers contained KpnI and BglII restriction site from pSVMdhfr-IFNβ plasmid as template. It was cloned in similarly digested pBud.CE4.1 linear vector. Construction of recombinant plasmid was verified via restriction fragment length polymorphism (RFLP) analysis, colony PCR and gene sequencing. Recombinant plasmid was transformed into competent Escherichia coli Top10 cells finally. After amplification, recombinant plasmid was purified and transfected into HEK293. At last, RNA extraction, cDNA synthesis and analysis of expression level of gene were performed using Real-Time PCR method. Findings: IFNβ gene was expressed under eEf1a promoter in HEK293 successfully. The expression level of target gene was increased 79.9 times in comparison with the control via transfection. Transfection of null vector showed 2.87 times elevation of target gene expression in response to the alien genome entered into the cell. Conclusion: The proteins produced in prokaryotic systems were non-glycosylated thus they had different physicochemical properties in comparison with the natural form. So, the production of IFNβ protein in human cell line under strong promoter of selected vector is one of the advantages of this research. Protein studies in this field are targeted for the future studies. © 2015, Isfahan University of Medical Sciences(IUMS). Allrights reserved.
Jundishapur Journal Of Microbiology (20084161)8(2)
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. © 2015, Ahvaz Jundishapur University of Medical Sciences.
Autoimmunity (08916934)48(5)pp. 336-343
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 IFNβK and IFNβK+CRID were designed by site-directed mutagenesis. The IFNβK and IFNβK+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 IFNβK has been elevated up to 3.5-fold. On the other hand, enormous amounts of IFNβ mRNA and protein were produced by cells containing IFNβK+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. © 2015 © 2015 Informa UK Ltd. All rights reserved: reproduction in whole or part not permitted.
Applied Biochemistry and Biotechnology (02732289)175(8)pp. 3737-3749
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. © 2015, Springer Science+Business Media New York.
International Journal of Integrative Biology (discontinued) (09738363)15(2)pp. 31-35
Vascular endothelial growth factors (VEGF-A) are a family of growth factors that stimulate angiogenesis in tumor growth and progression. The VEGF-A isoforms have pro-angiogenic effect. Another family of VEGF-A isoforms termed VEGFxxxb isoforms which contain antiangiogenic effects. Here we designed and expressed a new member of VEGFxxxb family, termed VEGFlllb. VEGFlllb was designed and cloned in pBUDce4.l expression vector to transfect HEK293 cells. Transiently transfected cells were examined by both real time PCR and western blot, to assess the VEGFlllb expression. Data were eveident that the recombinant VEGFlllb was successfully expressed in HEK293 cells. There was a significant difference in expression of VEGF gene in the transfected group than in the control group (l.3±0.l, P=0.03). Collectively there is no VEGFlllb primial expression in HEK293 cells and these cells are able to produce the recombinant VEFGlllb which is accounted for trapeutic applications. © OmicsVista Group, All rights reserved.
Gene (18790038)553(1)pp. 57-62
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. © 2014 Elsevier B.V.
Avicenna Journal Of Medical Biotechnology (20084625)6(4)pp. 192-199
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-angiogenic 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 VEGFxxxb 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. © 2014, Avicenna Journal of Medical Biotechnology. All rights reserved.
Symbiosis (03345114)62(3)pp. 151-155
The symbiosis of Medicago truncatula-Sinorhizobium meliloti is affected by phosphate (P) deficiency in the environment. Quorum sensing (QS) is a regulatory pathway in S. meliloti that controls various functions of freeliving and symbiotic bacteria in response to phosphate availability and regulation is mediated by a periplasmic protein PstS, and also bacterial density. The quorum sensing pathway of S. meliloti, involves three genes named sinI, sinR and expR and also some bacterial auto-inducers such as N-acyl homoserine lactones (AHLs). In the current study, the expression of the different genes of quorum sensing and pstS were evaluated under 0.1, 0.5 and 2 mM P. The qRT-PCR results showed an increased expression of pstS and also the quorum sensing genes sinI and sinR but not expR, following phosphate starvation. Indeed, the enhanced level of sinR induces the expressionof sinI that is responsible for the N-acyl homoserine lactones (AHL) production in S. meliloti. The different response of expR may be due to its negative control on sinR expression. In the symbiosis of M. truncatula-S. meliloti, it was shown that the concentration of phosphate in the medium alters the effective inoculating bacterial quorum (density). By increasing the phosphate concentration in the medium from 0.1 to 0.5 and 2 mM, considering the optimal plant growth and pink nodule (nitrogen-fixing) formation, the effective inoculating bacterial densities were 105, 107 and 109 CFU ml-1, respectively. Therefore, low phosphate concentrations can compensate for a low bacterial density by inducing the quorum sensing pathway and establishing a symbiosis. Conversely, bacterial density plays the main role in the formation of symbiosis at high phosphate concentrations. © Springer Science+Business Media Dordrecht 2014.
Gene (18790038)531(1)pp. 39-43
Purpose: Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes. Methods: Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2-δδCt method. Results: 23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%. Conclusion: Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique. © 2013 Elsevier B.V.
Journal Of Kerman University Of Medical Sciences (20082843)20(5)pp. 460-469
Background & Aims: The most significant cause of infertility in men is the genetic deletion in the azoospermia factor (AZF) region that is caused by the process of intra- and inter-chromosomal homologous recombination in amplicons. Homologous recombination could also result in partial deletions in AZF region. The aim of this research was to determine the association between the partial AZFc deletions and infertility. Methods: The blood samples were taken from 100 infertile men' who referred to the Infertility Center of Isfahan' Iran. 100 healthy matched people were also selected as the control group. The five markers of sY1201' sY1206' sY1161' sY1291, and sY1191 were applied in order to study partial deletions. Partial deletions were analyzed in AZF region using the Multiplex-STS-PCR technique. The chi-square test was conducted to check the difference between pretest and posttest. Differences were considered significant if P < 0.05. Results: 9% of studied persons showed gr/gr deletion (in the patient group). Only one case of gr/gr deletion was observed in the control group. Five patients showed b2/b3 deletion. One b2/b3 deletion was seen in the control group. The b2/b4 deletion was observed in 3 patients. In conclusion' partial deletions were observed in 14% of the patients. The statistical analysis of the gr/gr deletion in the study indicates a meaningful difference, but b2/b3 deletion does not represent a meaningful difference. Conclusion: Our results suggest that gr/gr deletions are associated with spermatogenic failure, and there is no association between b2/b3 deletion and infertility.
Seifi, T.,
Ghaedi, K.,
Salamian, A.,
Tanhaei, S.,
Safari, F.,
Hojati najafabadi, Z.,
Tavassoli, M.,
Baharvand, H.,
Esfahani, M.N. Avicenna Journal Of Medical Biotechnology (20084625)4(4)pp. 206-209
Background: Recently, we have shown that peroxisomal protein expression was induced upon retinoic acid treatment in mouse embryonic stem cells during the process of neurogenesis. Thus, characterization of the respective promoter could elucidate the molecular aspects of transcriptional regulation of this gene. Methods: Using the conventional software programs for promoter prediction, a putative promoter region was identified approximately 561 bp upstream of the peroxisomal protein coding sequence. In order to clone this region with a GC-content of 71.01%, a cocktail of ammonium sulfate buffer supplied with two additive components, betaine and dimethyl sulfoxide, and a high concentration of MgCl2 was used. Results: The modulated polymerase chain reaction composition significantly improved the amplification of GC-rich DNA target sequences. Improved amplification of this region was due to reduction in the formation of secondary structures by the GC-rich region. Conclusion: Therefore, this polymerase chain reaction composition could be generally used to facilitate the amplification of other GC-rich DNA sequences as verified by amplification of different GC rich regions. © 2012, Avicenna Journal of Medical Biotechnology.
Iranian Journal of Reproductive Medicine (20082177)10(4)pp. 315-320
Background: About 10% of infertilities with obstructive azoospermia are congenital and caused by CF gene mutations. M469I mutation was observed for the first time in Taiwanese patients. This mutation not only causes CF, but also may be the origin of infertility too. Objective: In this study, we aimed in designing a rapid, reliable RFLP-PCR procedure for detection of M469I mutation. The correlation and association between M469I mutation with infertility was investigated in this study. Materials and Methods: one hundred ten patients (90 non obstructive and 20 obstructive) and 60 normal individuals were considered in this study. M469I mutation was detected using RFLP-PCR. This technique was completely designed for M469I genotyping, for the first time in our study. Amplification of the region surrounding the mutation in exon 10 of CFTR gene was then performed. RFLP analysis was carried out using the NdeI restriction enzyme. Results: All genomic DNA samples were genotyped successfully. M469I mutation was observed only in patients group. Therefore, genotype containing mutant allele (GT) has been detected only in the patients group. There was no significant correlation between GT and TT genotypes with infertility (p=0.437). Conclusion: The M469I mutation has only been observed in Exon 10 CFTR gene of infertile patients, not in the control group. This mutation causes congenital bilateral absence of vaz deferens and finally infertility. This indicates a strong association between the M469I mutation and male infertility. Therefore, this is a CF-causing CFTR mutation that could be considered as a cause of infertility.
Gene (18790038)497(2)pp. 237-242
Introduction: The amplification status of proto oncogene HER-2/neu is one of the major molecular prognosis markers in breast cancer and recent adjuvant treatment with Trastuzumab has increased a request for the evaluation of HER-2/neu status in breast cancer. The aim of our study was the evaluation of HER-2/neu amplification status in malignant breast cancer by PCR techniques such as differential PCR and real time PCR and comparison of results of two methods together and with IHC results in some specimens. Methods: 86 malignant breast cancer tissue specimens were analysed initially by differential PCR and then by real time PCR. Sections from paraffin-embedded or fresh tissue samples were homogenized by squash and then DNA extraction was performed on cell suspension. A standard curve was initially plotted using BioEasy SYBR Green I for using 2 -ddct method. A 98bp fragment of HER-2/neu gene was co-amplified in the same reaction tube with a 150bp fragment of INFγ gene for differential PCR. Results: The IHC results existed only for 27 of 83 assessed samples by dPCR and for 30 of 86 assessed samples by real time PCR. 29 out of 83 (35%) samples tested by dPCR and 28 out of 86 (32.5%) samples tested by real time PCR have HER-2/neu gene amplification. Conclusion: There was concordance between the results of real time PCR and differential PCR in 61 of 83 specimens (73.5%) tested by both method. Furthermore, in comparison of IHC results with these two methods, 70% concordance between IHC and differential PCR, 63% concordance between IHC and real time PCR and 55.5% concordance between three methods were observed. © 2012 Elsevier B.V.
Journal Of Research In Medical Sciences (17357136)17(SUPPL.2)
BACKGROUND: Lung cancer has remained the most prevalent malignancy worldwide. It is the fifth leading cause of cancer death in Iran. Nevertheless, during last few years a gradual permanent increase in its incidence has been reported. Although the crucial role of tobacco smoke in lung cancer initiation has long been established, it is tempting to hypothesize that genetic polymorphisms may contribute to lung cancer predisposition. CYP1A1 gene encodes the main enzyme responsible for metabolic activation of several tobacco carcinogens. CYP1A1 MspI (6235T→C) polymorphism is the most studied variation within the CYP1A1, impacts on the basal levels of metabolism and is believed to be associated with elevated lung cancer risk, mainly in Asian population. METHODS: We investigated the frequency of this genetic variation in Iranian lung cancer patients through a cross-sectional study. 65 lung cancer cases and 80 healthy controls were recruited. RESULTS: The present findings confirmed the low frequency of the variant CYP1A1*2A allele in the control group. A significant increased risk for lung cancer was observed among those who possessed heterozygous (*1/*2A) genotype (Odds ratio = 2.79, 95% CI: 1.01-7.65). Adenocarcinoma was more frequent in non-smoker group (p = 0.00064); however, no significant increased risk was observed for squamous cell carcinoma and small cell carcinoma with respect to smoking. CONCLUSIONS: heterozygous (*1/*2A) genotype may increase the risk of lung cancer.
Journal Of Research In Medical Sciences (17357136)17(10)pp. 962-966
Background: Matrix metalloproteinases comprise a family of enzyme degrade components of extra cellular matrix. There are single nucleotide polymorphisms in the promoter regions of several genes with ability to influence cancer susceptibility. The aim of this study was to analyze association between MMP3 promoter polymorphisms and colorectal cancer occurrence and progression. Materials and Methods: In this case-control study 120 colorectal cancer patients and 100 controls were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the genomic deoxyribonucleic acid (DNA). The patients group was divided into different subgroups: a subgroup without metastatic activity (M-) and a subgroup that had developed metastasis (M+). Results: There was a significant difference in frequency of the MMP-3 genotype between cases and controls (χ2 =16.17; P=0.0003). The 5A homozygote in patients and controls was significantly different. The frequency of the 5A allele among affected patients (67.91%) was significantly higher than among the healthy controls (49%; χ2 =16.17, P=0.00005). At the time of diagnosis, individual who was carrying the 5A allele was more represented in the M+ subgroup than in M- subgroup (χ2 =7.49; P=0.006, OR=3.86; 95% CI, 1.43-10.33). The difference between M- and controls did not observe statistically significant (χ2 =0.009; P=0.92). Conclusions: Our results suggest that the presence of 5A polymorphism at the MMP-3 promoter region may favor the growth and the metastasis process in colorectal cancer patients and could be looked at as a risk factor for a worse prognosis.
Journal of Isfahan Medical School (10277595)29(142)
Background: Infertility is one of the important human problems. One of the main genetic factors of infertility is the deletions in the chromosome Y' which is reported in 10-15% of men with severe azoospermia and oligospermia. Three regions in azoospermia factor (AZFa), (AZFb) and (AZFc) are specified as the spermatogenetic regions. In this study we investigated the effect of changes in Yq11.223 (DAZ) region in chromosome Y on infertility. Methods: In this study the blood samples of 100 infertile men who referred to the Infertility Center of Isfahan and 100 healthy people, as the control group were taken. The genomic DNA was extracted from blood samples. The analyses were performed by applying the polymerase chain reaction (PCR) technique in AZF region of Yq11.223. Finding: In this study 70 azoospermia and 30 oligospermia patients were investigated. The deletions in AZF region of Yq11.223 were recognized in 7 azoospermia patients but there were not detected in oligospermia patients and the control group. It was also observed that the intensity of bands were changed comparing between azoospermia patients. Conclusion: Deletion frequency in the region of Yq11.223 was observed in 7 of 100 (7%) infertile Oligospermia and Azoospermia men. Our results confirm the effect of DAZ in the man's infertility.
Journal of Mazandaran University of Medical Sciences (17359260)21(84)pp. 23-31
Background and purpose: Polymerase chain reaction (PCR) is a rather quick and accurate method employed for gene detection and isolation. Primer designing is an important issue in this technique and plays a critical role in considering both the genome properties and cloning of the isolated genes. Streptomycin antibiotic is produced by Streptomyces griseus using str gene cluster with more than 25 genes. This gene cluster contains StrR gene encoding a specific protein regulator of this cluster. The pathway specific transcriptional activator then induces transcription of other genes in the str gene cluster. In this study, the researchers aimed at isolating promoterless StrR2 gene and then cloning it. Materials and methods: To isolate promoterless StrR gene, a set of primers (StrR2) was designed. One pair of these primers (St Nes) detected the StrR from the genome. Moreover, Nested-PCR was then used to detect amplified StrR2 gene. Sites for BamHI and XbaI were designed in other primers (Str nP1and Str nP2). These primers not only amplified the StrR2 gene but also created restriction enzyme sites in the amplified fragments. Results: Using PCR, the promoterless StrR2 gene was amplified and its structure was confirmed. This gene was successfully cloned, too. The correct structure of the recombinant plasmids was confirmed using different techniques such as gel electrophoresis, PCR and restriction digestion analysis. Conclusion: Using this vector, one can subclone the promoterless StrR2 gene in the Streptomyces expression vectors containing inducible promoters.
Cell Journal (Yakhteh) (22285806)13(3)pp. 179-186
Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay.
Tissue Antigens (13990039)76(1)pp. 60-63
Genomewide screen analysis has shown the close association of the human leukocyte antigen (HLA)-DRB1 region with susceptibility to multiple sclerosis and a number of autoimmune diseases. Using bioinformatics software, several potential short tandem repeat (STR) markers have been introduced in this region in the major histocompatibility complex data base (dbMHC). In this study, the identity and characteristics of two putative STR markers, D6S2879 and D6S2806, in this region were examined in Iranian population. The loci were genotyped in 85 individuals using polymerase chain reaction followed by polyacrylamide gel electrophoresis and sequencing. Analysis of the allelic frequency showed the presence of six and four alleles for D6S2806 and D6S2879, respectively. Analysis of deviations from Hardy-Weinberg equilibrium (HWE) showed that D6S2806 was in equilibrium (P > 0.05). However, the D6S2879 locus showed a significant deviation from HWE (P < 0.05). Therefore, the D6S2806 locus could be suggested as a marker for linkage analysis and disease-susceptibility investigations in the MHC-DRB1 gene region. © 2010 John Wiley & Sons A/S.
Korbekandi, H.,
Darkhal, P.,
Hojati najafabadi, Z.,
Abedi, D.,
Hamedi, J.,
Pourhosein, M. Iranian Journal Of Pharmaceutical Research (17350328)9(2)pp. 177-181
Clavulanic acid is produced industrially by fermentation of Streptomyces clavuligerus and researches have increased its production by strain improvement, recombinant DNA technology, and media composition and growth condition optimization. The main objective of this study was to increase the level of clavulanic acid production from Streptomyces clavuligerus (DSM 738), using UV irradiation. After incubation, the spores and aerial mycelia were scraped off the agar plate by a sterile loop. After passing through a cotton wool, the serially diluted spore suspension was spread on GYM- agar containing caffeine. The plates were irradiated with UV light, wrapped in aluminum foil and incubated. The colonies were sub-cultured again to express the mutations. An aliquot of the spore suspension prepared from the resulted culture was poured in GYM agar plates and incubated. The plates were overlaid with nutrient-agar containing penicillin G and Klebsiela pneumoniae, and incubated. The inhibition zone diameter was measured and compared with the wild type colony. Repeating this procedure, the overproducer mutants were selected. Concentration of clavulanic acid was determined by HPLC analysis. It was concluded that secondary metabolites, mainly antibiotics containing clavulanic acid, were produced about 6-7 days after the growth, and concentration of clavulanic acid was increased up to two-folds after UV mutagenesis. © 2010 by School of Pharmacy.
Iranian Journal Of Biotechnology (23222921)7(2)pp. 112
The claR of Streptomyces clavuligerus in the clavulanic acid gene cluster encodes a transcriptional regulator that controls clavulanic acid biosynthesis. The main goal of this study was isolation and molecular detection of the claR gene and its cloning in the Streptomyces specific vector (pMA:: hyg). By cinsideration of the claR gene's start codon, the specific primers were designed. After genomic DNA extraction from S. clavuligerus, the claR gene was amplified by Polymerase Chain Reaction (PCR). The structure of the amplified claR was confirmed by nested-PCR, PCR-restriction fragment length polymorphism (PCR-RFLP), and sequencing. A ligation mixture was prepared with the isolated claR gene and cut pMA::hyg vector. Escherichia coli competent cells were finally transformed with the ligation mixture. Presence of the recombinant vector in the transformed colonies was then confirmed by the colony-PCR procedure. The claR gene was also isolated from S. clavuligerus DSM41826, cloned and sequenced in the same manner. The pMA::hyg vector is a shuttle vector, which exists as a multicopy plasmid in E. coli, and as an integrative plasmid in Streptomyces. Therefore, the newly constructed vectors of this study can be regarded as an appropriate tool for site-directed mutagenesis and gene replacement strategies in S. clavuligerus.
International Journal of Integrative Biology (discontinued) (09738363)6(1)pp. 33-37
Matrix metalloproteinase-9 (MMP-9) gene plays an important role in several cancers development and progression including breast cancer. A single nucleotide substitution (C→T) in the MMP-9 promoter results in high expression of this gene and so affects susceptibility of tumor invasion and metastasis in some cancers. The aim of this study was to investigate the influence of C/T polymorphism on the occurrence and progression of breast cancer. This case-control study included 180 breast cancer patients and 100 healthy age-matched controls. Polymorphism in the promoter region (C-1562T) was genotyped by polymerase chain reaction-restriction fragment length polymorphism assay and sequencing. After data analyzing, a correlation was observed between the T allele (OR=3.27; P=0.004) and breast cancer occurrence. And also a high significant association was found between the occurrence of the T allele and progression and invasion of breast cancer (OR=5/85; P=0.000). The results suggest that the T allele of the C/T MMP-9 promoter polymorphism can be associated with the tumor development, progression and invasive phenotype of breast cancer, therefore it could be considered as a progression marker in this disease. © IJIB, All rights reserved.
Cancer Investigation (07357907)26(8)pp. 836-842
Interstitial collagenas-1 degrades a variety of extracellular matrix components. A single Guanine insertion polymorphism in the promoter has been found that influences on the transcription and expression level of the gene. It is suggested that this polymorphism may enhance susceptibility to some types of cancer. Therefore, this case-control study evaluated the association of this genotype polymorphism with susceptibility to initiation and invasion of colorectal cancer. For this reason, whole blood samples were obtained from 150 CRC patients and 100 control subjects in Tehran. Genomic DNA was extracted and genotyped by PCR-RFLP method. We showed that 2G allele and 2G/2G genotype had higher frequencies in patients (60% and 39%, respectively) than in controls (47% and 23%, respectively). The CRC patients were divided into two groups: with metastasis (M+) and without metastasis (M-) groups. The 2G allele was more frequent in M+ group compared with control group. However, no significantly difference was observed between M-group and control (χ2 = 0.48, P = 0.78 for 2G/2G genotype). Further stratification analyses showed that only gender (OR = 2.58, 95% CI = 0.89-7.52 for women and OR = 4.12, 95% CI = 1.62-10.42 for men) and smoking (OR = 3.03, 95% CI = 1.28-7.16 for non-smokers and OR = 4.09, 95% CI = 1.18-4.15 for smoker) may modify the risk of colorectal invasion related to 2G/2G genotype. Furthermore, individual with 2G/2G genotype seems to spread metastasis, 3 years earlier than those who were 1G/1G and 1G/2G. In conclusion, to our knowledge, the present epidemiological study for the first time indicates the relationship of 2G/2G genotype polymorphism with invasion risk of colorectal cancer in subgroups of gender and smoking, especially in smoker men. Copyright © Informa Healthcare USA, Inc.
Iranian Journal Of Biotechnology (23222921)6(1)pp. 45-49
In the human genome, chromosome 11 contains a cluster of matrix metalloproteinase (MMP) genes. Single nucleotide polymorphisms in the promoter region of MMP genes are important for MMP expression. A common adenine deletion polymorphism (5A) at position -1171 of the MMP-3 gene promoter (5′-AAAAAACCAT-3′ change to 5′-AAAAACCAT-3′) facilitates transcriptional factor binding and MMP-3 promoter activity. A case-control study was performed including 120 breast cancer patients (60 patients with metastatic activity and 60 patients without metastatic activity); and 60 healthy controls. Whole blood samples were obtained from patients and healthy controls. Genomic DNA was extracted from samples and the MMP-3 5A/6A genotypes were determined using PCR-RFLP. MMP-3 genotype distributions between patients and controls were similar (OR= 0.89, 95%CI, 0.43-1.84, P= 0.047). It was observed that the 5A allele was more frequent among patients with metastatic activity than controls (OR= 2.9, 95%CI, 0.94-8.9, P= 0.074). Therefore, the 5A polymorphism in the MMP-3 promoter showed correlation with the metastasis group than patients without metastasis; both at the time of diagnosis. However our results do not show evidence for correlation between 5A/6A polymorphism and breast cancer susceptibility.
Pakistan Journal of Biological Sciences (18125735)10(18)pp. 3079-3084
The genetics of streptomycin production is well characterized in Streptomyces griseus. More than 25 clustered genes encode proteins involved in biosynthesis, regulation and transport functions. StrR, the pathway specific transcriptional activator or regulator that located in this cluster, then induces transcription of other streptomycin production genes by binding multiple sites in the gene cluster. We aim to put strR gene in to different multicopy and integrated expression vector specifically designed for Streptomyces. To start with, the isolated strR gene was ligated into pBluescript (pBs) vector and transformed into different strains of Escherichia coli. The correct structure of the recombinant plasmid, isolated from transformed E. coli, was confirmed using gel electrophoresis, PCR and double digested with restriction enzymes BamHI and EcoRI. Finally the plasmid map, named pFDstrR. This unique vector has a much expanded Multiple Cloning Site (MCS), which makes it suitable for different purposes of gene cloning and also site directed mutagenesis or gene targeting. This gene will be lifted up and transfer into different varieties of Streptomyces specific vectors in order to make different transgenic or genetically manipulated Streptomyces. © 2007 Asian Network for Scientific Information.
Journal of Biological Sciences (discontinued) (18125719)7(7)pp. 1092-1101
Homologous recombination repair starts with Double-strand Breaks (DSBs) followed by crossing-over and recombination. The expected frequency of meiotic chromosomal exchange in the region of chromosome XII encoding ribosomal DNA in Saccharomyces cerevisiae is 3.5 to 5 events per cell per meiosis. However interchromosomal meiotic recombination in the rDNA gene is very rare, suggesting repression of DSB and crossing-over. On the other band, mitotic events such as intrachromosomal recombination producing 3 μm rDNA circles (which accumulate with cellular age) and unequal sister chromatid exchanges appear to be quite common. This study looked at the rDNA breakage in the strain ORD 1181, a rad50S mutant with SK1 background, which does a relatively fast and near synchronous meiosis. The fine analysis of the rDNA array was performed using restriction endonuclease enzymes that do not cleave within the rDNA array. The results suggest that there are at least two hot regions for chromosome breakage within the rDNA array. According to our previous studies we suggest that the DSB hot regions are in one homologue. However, there is possibility that other homologue is involving in DSB too. © 2007 Asian Network for Scientific Information.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis (18793592)564(2)pp. 129-137
In the yeast Saccharomyces cerevisiae the nucleolar organiser region (NOR) is located on chromosome XII. It contains 100-200 copies of rDNA - a minimum of 20 rDNA genes in tandem - and is termed the RDN locus. Yeast cells may exist in either haploid or diploid form. There are two forms of life cycle: haploid and diploid cells double by mitosis, and diploid cells are reduced to the haploid state by meiosis. Diploid cells have two homologous chromosomes for each of the 16 chromosomes. They are usually of the same size. However, in this study it is shown that homologous chromosomes XII can become different in size due to unequal sister chromatid exchange during mitosis in 'old' cells. © 2004 Elsevier B.V. All rights reserved.
