zohreh hojati

Cardiovascular disease is one of the leading causes of death worldwide. Mesenchymal stem cells (MSCs) are one of the most common sources of cell-based therapies in heart regeneration. There are several approaches to differentiate MSCs into cardiac-like cells, such as genetic modification. In addition, using of 3D culture, such as hydrogels, increases the efficiency of differentiation.

In the present study, lentiviruses containing microRNA 1 (miR- 1) and myocardium (Myocd) were co-transducted to mouse adipose-derived MSCs. Three days after, transduced MSCs were transferred to a hydrogel containing chitosan and collagen. After 21 days, the differentiation of encapsulated cells was evaluated. In this regard, the expression of cardiac markers such as NK2 homeobox 5 (Nkx2-5), GATA binding protein 4 (Gata4) and troponin T type 2 (Tnnt2) at the level of gene and protein were investigated.

The results of real-time quantitative polymerase chain reaction (qRT-PCR) and immunocytochemistry showed that co-induction of miR-1 and Myocd in MSCs followed by transfer to composite hydrogel increased the expression of cardiac markers.

The use of 3D culture such as chitosan/collagen hydrogel improves the differentiation of MSCs and subsequently obtains more mature cells for use in cell-based regenerative medicine

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.

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.

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).

Non-significant results were obtained from the expression levels of AD patients and two significant pathways were obtained by networks and gene enrichment analysis.

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.

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.

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.

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

In vitro organogenesis of cucumber (Cucumis sativus L.) was evaluated using the cotyledonary explants of “Beth Alpha” and “Dastgerdi” cultivars. Organogenesis was examined on the MS medium containing 2 mgl-1 Benzylaminopurine (BAP) along with eight combinations of abscisic acid (ABA) (0 and 1 mgl-1), silver nitrate (0 and 5 mgl-1), and cefotaxime (0 and 200 mgl-1). The results indicated the significant effect of ABA on increasing the percent of shoot induction and number of regenerated shoots. In the absence of ABA, silver nitrate had a significant effect on shoot induction and number of shoots. Not only the negative effects of cefotaxime was not observed, but also the number of shoots increased in one case. Root induction in the regenerated shoots was also studied using 5 different media including PGR-free MS medium and the media containing indolebutyric acid (IBA) (0.1 and 1 mgl-1) or naphthaleneacetic acid (NAA) (0.1 and 1 mgl-1). The highest number of roots per shoot in “Beth Alpha” (9.4) and “Dastgerdi” (8.6) were observed in the MS medium supplemented with 0.1 mgl-1 IBA. The genetic stability among 10 randomly selected clones of “Beth Alpha” cultivar was evaluated using AFLP markers. Besides that, cytogenetic assessment of 10 clones from each cultivar was implemented. In total 293 monomorphic bands were amplified using 9 primer combinations of AFLP marker, indicating the absence of somaclonal variation. The chromosome counting of root meristematic cells revealed the stability of ploidy level among regenerated clones.

Osteoporosis is one of the main causes of bone fractures in old age. The examination of osteoporosis in the elderly is very complicated due to the heterogeneity of the aging process. This study aimed to investigate the correlation of the TNC gene in the patients with an inherited and very rare osteoporosis syndrome. The importance of this study was the identification of a specific biomarker for the osteoporosis process in a monogenic disease.

In this case-control study, the patients with Osteogenesis Imperfecta were used as a simple and monogenic model to investigate the possibility of TNC gene role in osteoporosis. For this purpose, TNC protein network and its related biological pathways were firstly evaluated by bioinformatic analysis. Then, skin biopsies were taken from 3 patients with inherited osteoporosis syndrome called Osteogenesis Imperfecta and two healthy individuals. After culturing the biopsy, fibroblast cells were isolated from keratinocyte cells. Then, the total RNA of each sample was extracted from two different passages and cDNA was synthesized. Subsequently, the expression of TNC gene was measured by Real-time PCR in healthy and patient cells using various replicates.

TNC network proteins are significantly associated with the biological pathways involved in the ossification process. The expression of the TNC gene was assessed in wild type and patient cells. Finally, our results confirmed a significant increase in the expression of TNC in the patients’ compared to wild types’ cells by using technical and biological replicates (p:0.005, p:0.007) and Student t-test. In this study, Excel and GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA) were used.

Increased expression of TNC gene in a monogenic osteoporosis syndrome can indicate the potential role of this gene in the process of bone loss and introduce this gen as a new biomarker for osteoporosis. The study on rare Osteogenesis Imperfecta syndrome is a simple and monogenic model to investigate the heterogeneous osteoporosis. Therefore, the expression of the candidate gene needs to be evaluated and confirmed in an appropriate statistical population composed of patients with osteoporosis.

Among different roles of miRNAs in AD pathogenesis, hsa-miR-494-3p and hsa-miR-661 functions are poorly understood.

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.

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).

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.

Multiple sclerosis is an autoimmune inflammatory disease that causes the destruction of myelin and axonal degeneration. Given that multiple sclerosis is one of the most common neurological diseases, especially in young people, this disease is one of the main public health concerns. Therefore, it is important to understand the molecular mechanisms involved in the development of MS. Many molecular mechanisms control the process of myelination in the nervous system, and any changes in these regulatory mechanisms lead to impaired myelination. Current therapies have focus on controling the immune mechanisms involved in the disease process and are therefore only effective in the early stages of the disease and have no effect on axonal remyelination. But, therapies that reinforce the remyelination process may delay or prevent disability. Some recent studies have investigated the role of key genes of the Hippo signaling pathway in the myelination process of myelinating glial cells. According to these studies, the activity of YAP1/TAZ and its negative regulation in the Hippo pathway via CRB3, a cellular polarization factor, regulates myelin sheath synthesis. Thus, dysregulation of these genes can play a major role in the pathogenesis of many diseases related to the nervous system. Because there is no comprehensive review of the relationship between the hippo signaling pathway and multiple sclerosis; in this study, in addition to a narrative review of MS and its predisposing factors, the Hippo pathway as a pathway implicated in the defect of the myelination process in MS was investigated.

HER2 status testing in breast cancer is crucial for the detection of eligible patients for trastuzumab therapy. In this study, the relative copy number of HER2 gene, in patients with breast cancer, was determined by fluorescence in situ hybridization (FISH) and the results were compared with those of immunohistochemistry (IHC) to obtain the concordance rate between these two methods.

HER2 status of 31 invasive breast cancer samples was compared using IHC and FISH techniques. The ratio of HER2/CEP17 was used to determine the amplification of the HER2 gene. If the ratio of HER2/CEP17 is greater than 2.2, HER2 gene amplification has occurred in the cancer cells. Then, a comparative analysis is performed to estimate the concordance rate between FISH and IHC results.

The gene amplification of HER2 was observed in 26% of cases by FISH. The IHC and FISH results showed 100%, 36.36%, and 85.71% concordance rates for cases with IHC scores of 3+, 2+, and 0/+1, respectively. The overall concordance between the two methods was 80%. Based on statistical analysis, HER2 status showed a considerable correlation with tumor grade (P= 0.02). No correlation was observed between HER2 gene status and the size and type of tumor, characteristics of lymph node, and patients’ age.

The data suggested that IHC results are reliable for HER2 status testing in cases with IHC scores 0/+1 and 3+. However, in patients with an IHC score of +2, it is necessary to perform a complimentary test to evaluate HER2 status to avoid haphazard treatment with trastuzumab in negative cases and identifying positive cases for suitable treatment.

Tumor-infiltrating regulatory T (TI-Treg) cells perform the significant function in cancer immune escape. In this study, the third generation CAR construct was designed against human CD25 antigen, the significant cell surface biomarker of TI-Tregs.

Initially, the construct of anti-CD25 CAR was designed. Using RNAfold web server, the RNA secondary structure was evaluated. Also, utilizing lentiviral vectors, NK-92 cell line was transduced. Afterward, the expression level of anti-CD25 CAR RNA was assessed by qRT-PCR in NK-92 cells transduced with CAR and mock transfer vectors and also untreated cells.

The RNA secondary structure was stable. Also, the expression level of anti-CD25 CAR RNA in transduced NK-92 cells by pCDH-513B-1-anti-CD25 CAR transfer vector was significantly higher than transduced NK-92 cells by mock transfer vector and untreated cells (p˂0.0001).

The present study on anti-CD25 CAR RNA showed that this type of CAR transcripts were stable and expressed at high level. In fact, this type of CAR can be further studied in the future as a tool to remove the cancer immune escape in all types of solid and liquid cancers.

Interferon beta (IFN-β) is used to combat multiple sclerosis (MS) disease. CreatingR27T and V101F mutations (mHuIFN-β-27 and mHuIFN-β-101) is one of the tasks performedto 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 interferonreceptors were studied by molecular docking.

This work was performed through in silico study. The simulation of mutation wasperformed 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 performedusing the asaview online server. Also, the effect of mutations on the fold of the protein wasreviewed by the online HOPE server. The molecular docking was performed between HuIFN-βand the external region of IFNAR receptor using the online ClusPro2 protein-protein dockingserver.

The comparison of the values of the negative binding energy (ΔGbind) obtained fromprotein-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 thesedifferences do not see any meaningful relationship between them (P > 0.9999).

Regarding these results, it can be concluded that these mutations do not have anegative effect on the composition of the complex rHuIFN-β/IFNAR. So, they do not interferewith the binding of the IFN-β to the receptor. It is concluded that the quality of the rHuIFN-β isimproved by introducing these two mutations.

Yes-associated protein (YAP1), the downstream effector of the Hippo pathway, plays important roles in the regulation of tissue reconstruction, stem cell proliferation, and development of different cancers. The regulation of YAP1 phosphorylation, YAP1 expression level, and its cellular localization have been considered in cancer development. There are different experimental evidences that indicate that YAP1 activation results in tumorigenesis, tumor progression, and metastasis. YAP1 is a transcription co-activator, and its dysregulation has been suggested in various cancers including colorectal cancer (CRC). The localization of YAP1 in the nucleus results in YAP1 interactions with different transcription factors to promote the expression of genes involved in cell proliferation, metastasis, and stem cell maintenance. However, a number of studies have been reported the tumor suppressor role of YAP1 in CRC. Therefore, a better understanding of the YAP1 regulation could be helpful for prevention, diagnosis, and treatment of CRC. In this review, we will discuss different roles of YAP1 in CRC progression through the regulatory roles of long non-coding RNAs (LncRNAs), microRNAs (miRNAs) and circular RNAs (CircRNAs) in YAP1 regulation.

KDM3A is a key epigenetic regulator that is expressed in the testis and is required for packaging and condensation of sperm chromatin. To this point, the association of the KDM3A gene and 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. 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 for any mutation in exons 12, 22, and 24 of KDM3A.   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. The mutations found in infertile men with otherwise unexplained severe spermatogenic failure could be considered as the origin of their abnormalities.

Interferon beta is one of the members of type I interferons. Creating R27T and V101F mutations is one of the important researches performed to improve function, decrease immunogenicity, increase expression and increase half-life of interferon beta. In this study, the effects of R27T and V101F mutations on interferon beta binding to interferon receptors were studied by molecular docking. This study was performed through Bioinformatics methods. The required crystal structures were provided by the RCSB server. The simulations of R27T and V101F mutations were performed using online Rosetta Backrub software. Comparison of access to the solvent for the amino acids in the created structures was performed using asaview online server. Also, the effect of mutations on the structure and protein folding was investigated by the online Hope server and SPDBV software. The molecular docking between HuIFN-β and the external region of IFNAR receptor was performed using the online ClusPro2 protein-protein docking server. The comparison of the values of the negative binding energy (ΔGbind) obtained from protein-protein molecular docking between IFNAR receptor and HuIFN-β, mHuIFN-β-27, mHuIFN-β-101 and mHuIFN-β-27-101 ligands did not show a significant difference (P> 0.99). Regarding these results, it can be concluded that the produced mutations do not have a negative effect on the forming of rHuIFN-β/IFNAR complex and does not interfere with the binding of the interferon beta to the receptor and thus improves the quality of the produced rHuIFN-β.

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

Multiple Sclerosis (MS) is a major autoimmune disease of the central nervous system. Today it is specified that changes in the sequence of single nucleotides or SNPs are associated with disease or resistance to disease. In recent studies, the association of micro-RNA polymorphism has been reported with diseases such as cancer and autoimmune diseases. Micro RNA is a group of non-coding RNA that affects a variety of biological processes such as growth, cell division, and immunity.
blood samples were collected from 71 MS patients and 71 healthy subjects, and DNA was extracted. Genotyping analysis was performed using TARMS PCR technique. The relationship between rs3745453 polymorphism and MS was evaluated by statistical tests.
TT, TC and CC genotypic frequencies are 34, 24 and 13 in patients and 39, 11 and 21% in control group respectively. In both groups, the TT genotype was dominant and heterozygote frequencies were higher in patients than control group. Odd ratio (OR) in TC genotype was 2.7. In other words, heterozygote individuals have a 2.7 times higher chance of MS than others
This study was the first report of the relationship between mir23a polymorphism and MS disease. According to the results, we can say that mir 23a rs3745453 polymorphism is associated with Multiple sclerosis (P = 0.029). It is suggested that further studies should be done with more samples.

Type 1 diabetes (T1D) is caused by cell-mediated autoimmune attack on pancreatic beta-cells. Previous studies highlight the role of microRNAs (miRNAs) in the pathogenesis of T1D. MiRNAs are small non-coding RNAs involved in the regulation of gene expression post-transcriptionally. In this work, miR-18b was chosen and the differential expression of it was measured between T1D patients and healthy controls from Isfahan population.
MiR-18b was selected using Bioinformatics studies by miRWalk software. 22 T1D patients and 18 healthy controls from Isfahan population were enrolled in this study. Total RNA of the peripheral blood mononuclear cells (PBMCs) samples were extracted. After cDNA synthesis, the expression profile of miR-18b quantified by means of qPCR method in patients and controls. Finally the results were statistically analyzed.
In this study despite our hypothesis, the expression levels of miR-18b didn’t show any significant difference between T1D patients and healthy controls (p value: 0.145).
Due to the results of our experimental analysis, it seems that miR-18b doesn’t have any association with T1D disease in Isfahan population.

Interferons are some kind of natural cytokines which express in response to a variety of antigens including viral RNA, bacterial products, and tumor proteins. Interferon beta is used in the treatment of autoimmune diseases such as multiple sclerosis. Moreover, this drug inhibits cellular proliferation as well as angiogenesis and as a result, helps to cure cancer. In this research, in addition to cloning the interferon-beta gene along with conserved kozak sequence after the strong eEf1a promoter in the pBud.CE4.1 vector, the expression of this recombinant gene was compared to basal expression in HEK293T cell line using real-time PCR, SDS PAGE and western blot tests.
In the beginning, the interferon-beta gene was amplified from the pSVM dhfr vector containing the gene, using primers including BglII and KpnI restriction sites as well as conserved kozak sequence. Then the duplicated gene was digested and inserted in the linear pBud.CE4.1 vector. After ensuring entry of the gene using RFLP, colony PCR and sequencing, the recombinant vector was transfected into E. coli TOP10 competent bacteria. After that, amplified recombinant vector was extracted and transfected into HEK293 cell line.
The expression of interferon-beta cloned in pBud.CE4.1 vector showed a 79.9-fold increase, in comparison with the basal expression in HEK293T cell line. Moreover, non-recombinant vector transfection has increased the expression of interferon-beta up to 2.87 times in the cell line that is probably due to the existence of the viral promoter in the vector.
Real-time PCR and protein test results showed that recombinant beta interferon gene had been successfully expressed in HEK293T cell line. In order to produce more of this protein, optimization of various conditions are required for the HEK293T cell line.

VEGF111b is a new isoform of vascular endothelial growth factor (VEGF) recently considered as a new anticancer drug. The aim of this study was to evaluate the VEGF111b secretion from HEK293 cell wall in order to commercial production of this recombinant factor.
After the design of VEGF111b sequence using OLIGO software and NCBI gene bank data, it was cloned into the pBUD.cE4.1 vector. The pBUD.VEGF111b recombinant vector was transfected into HEK293 cells using lipofectamine kit. Forty-eight hours after the transfection the production of VEGF111b was estimated by Western blotting and Human anti VEGF antibody. The VEGF111b secretion into cell culture and cell lysate extract was measured using ELISA.
The correct cloning of VEGF111b into pBUD.cE4.1vector was confirmed using enzymatic digestion and gel electrophoresis. The observed production of recombinant peptide in HEK293 was confirmed with 12KDa band in cell lysate extract of Western blotting. The ELISA results at 450 nanometer absorbance for cell culture media and cell lysate extract were 19.20±2.81 pg/ml and 32.87±7.42 pg/ml, respectively. However, no VEGF111b expression was observed in negative controls.
The findings of this study indicate the powerful ability of transformation and secretion of VEGF111b from HEK293 cell wall to cell culture media with no breaking and proteolytic digestion. It seems that the commercial production and purification of this therapeutic peptide from HEK293 cell culture would be possible with high efficiency.

Background and 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.
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.
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.
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.

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 ∆I507 and ∆F508 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. However there are no significant association was found between the presence of this mutated allele and infertility [OR=9.2 (allele-based) and 7.2 (individual-based), P=0.179]. None of the samples carried the ΔI507 mutation. Altogether, we show that neither ΔI507 nor ΔF508 is involved in this population of Iranian infertile males with non-obstructive azoospermia.

DNA catalysts (Deoxyribozymes, DNA enzymes; DNAzymes) are catalytic artificial single-stranded DNA molecules. Using the technique of in vitro selection, individual DNA catalysts have been identified that catalyze RNA cleavage, RNA ligation and a wide range of other chemical reactions. DNA catalysts have been used in vitro as biochemical tools, analytical tools and sensors. DNA catalysts have also been utilized as in vivo therapeutic agents to target specific mRNA. Although many conceptual and practical challenges remain to be addressed, DNA catalysts make new promise for applications both in vitro and in vivo. In this review, we are focusing on definition and different types of DNA catalysts, in vitro Selection of DNA catalysts and their therapeutic applications. This review also describes the challenges and approaches to overcome obstacles involved in using DNA catalysts in the treatment of different diseases including cancers, viral and bacterial infection.

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.

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

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.

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.

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.

Urinary tract infections are one of the most frequent health problems and Uropathogenic Escherichia coli is the major pathogen resulting UTIs. The severity of UTIs is caused by the expression of a large range of virulence factors.In this study, we evaluated the allelic frequency fimH gene, in UPECs isolated from patients with UTIs. This study also aimed to determine the roles of C640T and T591A SNPs of the fimH gene in the ability of UPEC to cause UTIs.
Material and A total of 140 UPEC strains isolated from patients with UTIs were screened by PCR-RFLP for determining the prevalence of the C640T and T591A SNPs of fimH gene in UPEC strains isolated from patients referred to educational hospitals of Shahrekord. The genotyping of C640T and T591A SNPs was performed using Bme1390I and BseNI restriction enzymes, respectively through PCR-RFLP method.
There were no meaningful association between C640T and T591A SNPs of fimH gene and the ability of UPEC fimH variants to cause UTIs in the studied E. coli isolates.
FimH is one of the most major virulence factors among UPECs which is confirmed in most E. coli isolates. Further studies are required to determine the association between different fimH gene SNPs of isolated UPECs from UTIs patients and the ability of UPEC fimH variants to cause UTIs.

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.
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.
The results of this study show that the best reference genes for normalization of qRT-PCR data of testis tissues are GAPDH and RPL37.

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).

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.

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.

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.