F. Shariari, Sh. Moradi, M. Totonchi, L. Satarian, S.j. Mowla, H. Baharvand,
Volume 10, Issue 3 (9-2019)
Abstract
Aims: The retinal pigment epithelium cells (RPE) have crucial roles in the health and functionality of retina. Any damage or dysfunction of these cells can lead to severe retinopathies. Identification of signaling pathways and biological processes involved in RPE differentiation can be useful in devising more robust therapeutic approaches.
Materials and Methods: In the present study, we used the intersection of three online prediction databases and their ::union:: with one experimental database to select microRNAs gene targets. Next, by the intersect of the targeted genes with an increase in their expression in epithelial to mesenchymal transition (EMT) of RPE cells, we tried to build a microRNA-mRNA integrative network. Further, several pathway analyses tools were used to perform a more accurate and comprehensive analysis of the signaling pathways and biological processes being regulated by selected miRs in the EMT of the RPE cells.
Findings: Our study revealed that among the 3406 genes being upregulated over the course of EMT in RPE cells, adj p-value≤0.05, fold change≥1.5, 93 genes were miR-204-5p and miR 211-5p target genes. Further analysis of the obtained target gene list demonstrated that these two microRNAs are mostly involved in maintaining RPE cells from going through EMT via regulation of cell adhesion and secretion subnetworks and also MAPK and TGF-β1 signaling pathways while preserving cells from apoptosis and neuronal fates.
Conclusion: This study indicated that miR-204-5p and miR 211-5p are involved in protecting RPE cells from EMT and reinforce their epithelial cell identity.
S. Askari, S. Hasannia, R. Hasan Sajedi, V.r. Yassaee,
Volume 10, Issue 3 (9-2019)
Abstract
CEL I endonuclease pertaining to the S1 endonuclease family. The enzyme, with its high specificity, has the ability to identify different types of mutations and base replacement in the DNA molecule, which makes it important in commercial products to use in research and clinical laboratories. Although the enzyme exists in the celery plant, the extraction of the enzyme is a time-consuming process and not economical and the yield of the final product is low. In addition, due to its post-translational modifications to achieve the final active structure, no report has published to indicate the expression of the active form of this enzyme in the bacterial hosts yet. Therefore, one of the production sources of the active form of this enzyme is its cloning and expression in eukaryotic hosts, including yeast and mammalian cell lines. In this study, in order to express CEL I endonuclease, its gene sequence was optimized and synthesized in host eukaryotic HEK293T. CEL I was subcloned by double digest with KpnI and XhoI enzymes in the pBudCE4.1expression vector. The expression construct was transfected into the HEK293T cell line by lipofectamine transfection. Expression of the recombinant protein after transfection into HEK293T cells was confirmed by multiple methods including polyacrylamide gel electrophoresis, ELISA, RT-PCR, and western blot reaction. The analysis of SDS-PAGE and western blot data confirmed the molecular weight of approximately 30kDa. Purification was carried out with the Ni-NTA column and the amount of purified protein was determined to be about 0.2mg/ml. Finally, the activity of endonuclease enzyme was investigated on both normal and mutated heteroduplex DNA amplified by PCR. The results showed that the expression of this protein in HEK293T host had shown sufficient activity.
Faezeh Khatami, Farzaneh Najafi, Fataneh Yari, Ramazan Ali Khavari Nejad,
Volume 10, Issue 4 (12-2019)
Abstract
The high quality and quantity of extracted DNA is necessary for a variety of molecular biology studies. Low yields and poor quality of genomic DNA extracted from petal due to high levels of secondary metabolites. Carotenoids, anthocyanins, phenolic acids and flavonoids are the most effective secondary metabolites in petals, which are considered as contaminating compounds and could lead to interfere with DNA during extraction and purification. Considering that the basis of the most molecular research in genetic engineering and genomics is high-quality of DNA, therefore, it seems that finding an efficient method for reducing adverse effects of these contaminating compounds for the exteraction is essential. In this regard, iron-magnetic nanoparticles have been used to improve the exteraction of high yields and quality of DNA from rose petals in the present work. In the following, to compare the efficiency of DNA extraction, modified CTAB (Cetyl Trimethyl Ammonium Bromide) and rapid detection methods were used. The results showed that petal’s extracted DNA quantification and qualification by iron-magnetic nanoparticles procedure was much more reliable than two other methods. Inaddition, this method could extract optimal amount of DNA with the lowest amounts of samples within few minutes. Due to high qualification and quantification of DNA purification by iron-magnetic nanoparticles, the present procedure could be recommended as an efficient protocol for rose petal DNA extraction.
H. Rahmani, R. H.sajedi ,
Volume 10, Issue 4 (12-2019)
Abstract
Aims: Aequorin as a bioluminescence protein due to ease of use, non-toxic, and high capability of detecting has long been the interest of researchers. The aim of this study was to design a method for accurate and simple detection of important therapeutic agents using a bioluminescence inhibition based assay by using aequorin.
Materials & Methods: In this study, important drugs in therapeutic monitoring with structural similarity to Coelenterazine, were selected and their interaction with aequorin was investigated. Further, the conditions of the bioluminescence assay were optimized to achieve the lowest detection limit.
Findings: Among the drugs whose effects have been tested on aequorin, the only benserazide resulted in inhibition of the bioluminescence activity. This analyte can significantly reduce the bioluminescence of aequorin in a concentration-dependent manner. The best dose-response curve was obtained and IC50 of 0.26µM was calculated. The linear calibration curve was obtained in a range of about 100 to 1500nM with LOD and LOQ of 79 and 260nM, respectively. Furthermore, we demonstrated the application of the approach in human serum samples with a recovery of 97%. Guddem-Schild graph was plotted to determine the mechanism of inhibition which indicated that the IC50 of benserazide changed in the presence of different concentrations of Coelenterazine.
Conclusion: The proposed method can be used for measuring benserazide which can easily be applicable for real samples. Also, the results show that benserazide inhibits the bioluminescence activity of aequorin by competitive inhibition.
F.s. Hosseini, B. Mohamad Soltani, H. Baharvand , S. Hosseinkhani ,
Volume 10, Issue 4 (12-2019)
Abstract
The SPTBN4 gene, a part of the spectrin protein family, plays important roles in various cellular processes, including cell cycle, nerve cell development, and so on. Recently, a new miRNA has been found in this SPTBN4 gene, which was registered at the NCBI database. The aim of the present study was to investigate the expression of this miRNA, called SPTBN4-miR1, in the process of differentiation of human embryonal carcinoma cell line NT2 and also the overexpression effect of this miRNA on the differentiation of these cells. RT-qPCR results indicate that SPTBN4-miR1-5p and SPTBN4-miR1-3p show a significant increase in expression in the process of neural differentiation from day three until the 8th and 14th day of differentiation. Then, after overexpressing the SPTBN4-miR1 precursor in NT2 cells and retinoic acid treatment, the expression of pluripotent and differentiation revealed the role of SPTBN4-miR1-5p and SPTBN4-miR1-3p in promoting differentiation and exclusion from the pluripotent state. It seems that by making further studies and finding out the possible targets of these miRNAs, a distinctive marker can be achieved and used to improve the differentiation process.
M. Bahri , S. Hasannia, B. Dabirmanesh , H.h. Zadeh,
Volume 10, Issue 4 (12-2019)
Abstract
Introduction: Nowadays, bone tissue repair with increasing bone disorders and injuries have special importance. Bone tissue engineering provided specific solutions to these problems. The present study was conducted with the aim of purification of recombinant fusion peptide containing hydroxyapatite affinity tag using the ceramic chromatography column.
Material & methods: In this study, a fusion peptide was designed which at one side comprised the heparin-binding domain sequence, which can be attached to various types of growth factors involved in tissue repair and entrap these factors at the site of the lesion. On the other side, it contained a tag, which included a sequence derived from a laboratory study based on phage expression. The reason for keeping the sequence of this tag is to attach the peptide to the scaffold containing hydroxyapatite and purifying the recombinant peptide by the hydroxyapatite column. Therefore, the gene sequence was optimized and synthesized for expression in the prokaryotic host of E.coli strain BL21. Then the gene sequence was subcloned by double digestion with the SacI and BamHI enzymes into the expression vector of pET-21a(+). The expression of the recombinant peptide was investigated by SDS-PAGE and western blot. In order to optimize the purification conditions, two-step purification was carried out by applying fundamental changes in the main work method of the manufacturer company and was purified with acceptable purity. Finally, the existence of peptide assemblies was investigated by the SLD method.
Finding: The results of PCR cloning, enzymatic digestion using SacI and BamHI enzymes and sequencing indicated the accuracy of the cloning process. On the other hand, expression of the fusion peptide was confirmed by SDS-PAGE and Western blot techniques, and its migration onto the gel resulted in a band cleavage of about 12 kDa. Changes made to the manufacturer's workflow allowed the purification process to be optimized and the results of the DLS method showed the purity of the purified peptide.
Conclusion: The results indicate the desirable expression and remarkable purity of the fusion peptide designed in this study.
Nasrollah Saleh Gohari, Zahra Karami, Farbod Mohseni, Ali Karimzadeh, Katayoon Sedighi,
Volume 11, Issue 1 (3-2020)
Abstract
Breast cancer is a serious health concern for women. It contributes to about 23% of the cancer cases, and accounts for the second largest number of deaths among all cancers. Expensive and time consuming recognition methods currently available for recognition of breast cancer potentiates the need for improvement of novel, specific and ultrasensitive strategies. Biosensors are sensitive, specific and cost effective procedures. These also display the benefit of quick response due to direct calculation in physiological fluids (saliva, blood, serum, milk, urine etc.) in a non-invasive way. Aptamer-based biosensors for cancer cell recognition have shown advantages of rapidness, simplicity and cost-efficiency over traditional approaches. In this study, by linking DNAzyme and aptamer together, we established colorimetric biosensor for the detection of MCF7 breast cancer cells. MUC1 and PTK7 aptamers used as specific aptamers to binding to the breast cancer cells. This manner may also evade the modification of DNA and the use of labels, which can intensely rise the cost-efficiency and simplicity of cancer cell discovery. Our results indicated that these aptamers showed good activity for breast cancer cells detection in which in the control cells no activity was observed. Results also indicated that there is a good linear relationship between the cancer cell values and colorimetric signal. Finally, the obtained results indicated a cost effective and conveniently operated approach for cancer diagnosis in future.
Samira Ranjbar, Khosro Khajeh, Javad Mirnajafi-Zadeh, Bahareh Dabirmanesh, Shima Khodaverdian,
Volume 11, Issue 1 (3-2020)
Abstract
Electrical Kindling is one of the most popular epileptic model techniques that cause seizures such as temporal lobe epilepsy. So far, various therapies have been used to treatment of epilepsy. Among these treatments, low-frequency stimulation (LFS) has been widely considered for improving effect on drug-resistant epilepsy, but its mechanism is not well understood. Since calcium entering to the cytoplasm and increasing its concentration is one of the reasons for seizure, metabotropic glutamate receptor (mGluR1), dopamine receptor (D1) and ADPR cyclase (CD38), which increased calcium in the cytoplasm from different pathways, were selected. With this aim that by examining the change in the expression of these genes, we help to clarify the LFS improvement effect. In this study, the hippocampus of rats was used and the changes in genes expression were investigated using real-time PCR technique. The results showed that the expression of all selected genes increased significantly after kindling and then after the LFS the expression of all was returned to sham value. Hence, one of the ways in which LFS interferes may be related to the pathway for calcium entering to the cytoplasm.
Zahra Fathi, M. Mashhadi Akbar Boojar, Ehsan Dehnavi, Reza Hassan Sajedi,
Volume 11, Issue 1 (3-2020)
Abstract
Abnormal angiogenesis is associated with various diseases such as solid tumors and metastasis, retinopathies, and rheumatoid arthritis. VEGF-A is the most important mediator of angiogenesis among all growth factors. The bioactivity of VEGF is mediated by two tyrosine kinase receptors VEGFR-1 and VEGFR-2 present on endothelial cells. VEGF signaling through VEGFR-2 is the major angiogenic pathway that leads to stimulation of endothelial cell ingrowths into the tumor. It comprised of an extracellular portion, a cytoplasmic portion, and a short transmembrane domain. The extracellular portion consists of seven Ig-like domains (D1–D7), of which the 1st to 3rd domains function as ligand-binding sites. In the present work, a soluble recombinant extracellular domain 1-3 of VEGFR-2 was expressed in Pichia pastoris to inhibit the VEGF-induced angiogenesis. The 975 bp DNA fragment containing extracellular domain 1-3 kdr, was designed according to the nucleotide sequence at GenBank and protein sequence at Swiss-Prot. The recombinant secretory expression vector (pPinkαHC/KDR1-3) was constructed and transferred into yeast by electroporation. The high expression transformants were identified through complementation of adenine auxotrophy and cultured. KDR1-3 was expressed under the induction of %1 methanol and confirmed by using SDS-PAGE and western blot techniques. After being purified by affinity chromatography using Ni-NTA resins, binding of expressed product to hVEGF165 was proved by two direct ELISA and ELISA receptor binding assays. The data showed that human VEGFR-2 extracellular domain 1-3 with eukaryotic protein structure, that there is no reported paper about, was successfully expressed.
Parvin Moghaddam, Azadeh Zahmatkesh, Saeed Airian, Masomeh Bagheri, Homayoon Mahravani Behbahani, Khosrow Aghaiypour,
Volume 11, Issue 2 (6-2020)
Abstract
Foot and Mouth Disease (FMD) is a highly contagious and devastating disease that spreads rapidly and causes many economic damages. One of the important methods for detection of FMD and particularly differentiation of vaccinated from infected animals, is the use of non-structural proteins as antigens in ELISA kits. The purpose of this study was cloning of the gene sequence and expression of the antigenic regions of 3D nonstructural protein as one of the diagnostic options. For amplification of the antigenic regions of FMD virus 3D protein, specific primers containing NdeI and EcoRI restriction sites were designed and the polymerase chain reaction was performed. The sequences cut by these two enzymes, were inserted into PET21a+ vectors. The recombinant plasmids were then transformed into E. coli (DH5α). Colony-PCR tests and enzymatic digestions were performed on the resulting colonies and the presence of the target gene was confirmed. The gene sequence was further confirmed after sequencing. For production of recombinant antigens, the recombinant vector was transferred to the expression host of E. coli-BL21. The bacteria containing the recombinant gene were induced with IPTG and the expression of the recombinant protein was confirmed using the SDS-PAGE method. The molecular weight of the recombinant protein was about 24 kDa, and it can be used in the design of ELISA diagnostic kit.
Nooshin Gorjizadeh, Mohammad Bagher Shahsavani, Faezeh Moosavi-Movahedi, Reza Yousefi,
Volume 11, Issue 2 (6-2020)
Abstract
α-crystallin is a member of small heat shock protein family (sHSP) which shows both structural and chaperone functions. This protein plays important role in eye lens transparency and indicates protective function in the other tissues. The lenticular levels of copper ions significantly enhance in diabetic patients, aged and cataractous lenses. In eye lenses, the free copper ions induce ascorbic acid auto-oxidation, leading to formation of dehydroascorbic acid and other oxidative products as well as reactive oxygen species. The oxidized forms of ascorbic acid along with the reducing sugars enter into pathological reactions with the eye lens proteins, forming toxic advanced glycation end-products (AGEs). As one of the main components of eye lens antioxidant defense mechanism, glutathione could scavenge the copper ions, inhibiting the formation of reactive oxygen species in eye lenses.
In the current study, the structural and functional properties of human αB-crystallin were assessed using different spectroscopic methods. In the presence of copper ions, αB-crystallin exhibited important alterations in both structure and chaperone activity which upturned in the presence of glutathione. Moreover, incubation of human αB-crystallin with copper resulted in significant increase in the protein oligomeric size distribution which largely prevented upon simultaneous incubation with glutathione.
Overall, glutathione may scavenge free copper ions in the lenticular tissue, inhibiting their damaging effects on crystallin proteins and other redox-sensitive molecular targets such as ascorbic acid. Our results may introduce a new protective role for glutathione which is highly important in diabetic and aged lenses showing increased levels of copper ions.
Zeynab Rezaei, Zahra Abedi Kichi, Mehrdad Behmanesh,
Volume 11, Issue 2 (6-2020)
Abstract
Abstract: Hyperglycemia is a major cause of diabetes. Hyperglycemia-induced endothelial dysfunction is generally believed to be the basis of diabetic vascular complications such as retinopathy, nephropathy and cardiovascular diseases. The most important molecules in endothelial cells that can sense elevated level of glucose and transmit signals into the cell are G protein-coupled receptors (GPCRs).
In the present study, according to bioinformatics analysis of genomic sequences between healthy and patient individuals, two G proteins GPR182 and CALCRL were selected and their expression level were examined in hyperglycemic and normal conditions in HUVEC as a model of vascular endothelial cells at different glucose concentrations and various time intervals. In addition, the effects of hyperglycemia on cell viability and cell cytotoxicity were assessed by MTT and LDH assay respectively and also morphological changes by immunohistochemistry.
Overall our data reveal a probable role for GPR182 and CALCRL in hyperglycemia-induced endothelial dysfunction. Thus, they could be developed as a potential molecular targets for the endothelial dysfunction therapy.
Matinsadat Ghafelebashi, Parvaneh Maghami, Abdolhossin Shahverdi, Davoud Doranian, Marjan Sabbaghian,
Volume 11, Issue 3 (10-2020)
Abstract
According to the formation and evolution of life along with static magnetic fields,the permanent exposure has given adaptive ability to beings. Therapeutic magnetism is one of the branches of complementary medicine which uses the low intensity and non-harmful magnetic fields to the body. By studying in infertile couples (20% male factor), the only cause of infertility and in 50% of cases it is considered as an intermediate factor. One of the influential factors in infertility in men is sperm. In the present study, normal specimens in the magnetic field under the intensities of 1,6 and 12 millitesla and at 1,3 and 5 h intervals.
Sperm movement rate was evaluated by CASA, as well as sperm viability, by eosin staining of necrosin and morphology by staining Papanicula. The results of this step on normal sperm showed a significant reduction in the sperm movement ,which that was not affected by the field.
Morphological studies also show that sperm motility is not affected by magnetic field.. the survival rate of sperm was affected by the magnetic field was significantly reduced, and the sperm morphology remained unchanged
Seyedeh Fatemeh Sajjadi, Mohammad Ali Boroumand, Mehrdad Behmanesh,
Volume 11, Issue 3 (10-2020)
Abstract
Atherosclerosis is a chronic vascular disease and remains the leading cause of death and morbidity worldwide. Endothelial dysfunction is an important factor in the progression of atherosclerotic disease. Increased expression of cell adhesion index genes and decreased cell-binding proteins lead to abnormal endothelial function. These molecular changes are one of the most important indicators of endothelial cell dysfunction and the progression of atherosclerosis. CXCR3 is a G-protein-coupled chemokine receptor expressed by endothelial cells. The role of the receptor CXCR3 and its ligands in endothelial cells and heart disease is not yet fully understood. In this study, we evaluated the effect of CXCR3 downregulation on the expression level of adhesion (I-CAM-1, V-CAM-1), tight junction (TJP1), related to endothelial dysfunction.
In order to reduce the expression of the CXCR3 gene, the RNA-cleaving DNAzyme was used against the mRNA of the CXCR3 gene. DNAzyme was transfused into HUVEC cells by TurboFectTM. After confirmation of decreased CXCR3 gene expression, RNA extraction and cDNA synthesis were performed and then the expression of markers was evaluated by RT-qPCR technique.
Our result was showed the expression level of I-CAM-1 and V-CAM-1 were showed significant up-regulation in transfected cells compared with control cells, while the TJP1 gene was not showed significant change. It seems that reducing the CXCR3 gene expression could induce endothelial dysfunction through the change of adhesion markers genes expression. Therefore, this receptor can be considered as a potential molecular target for a better understanding of the mechanism of atherosclerosis.
Mahsa Tirmomenin, Farangis Ataei, Saman Hosseinkhani,
Volume 11, Issue 3 (10-2020)
Abstract
Inhibitor of apoptosis (IAP) are a family of proteins that block cell death through caspase activity. Survivin is smallest IAPs family member that overexpresses in different cancer types but not in normal tissue except embryonic tissue. Survivin may be used as a new marker to stratify cancer patients for more optimal treatment modalities. The aim of the current study was to investigate survivin DNA cloning into pET-28a and its expression in E.coli.
The sequence of survivin gene was amplified by PCR using specific primers and pcDNA-survivin temple. PCR product and pET-28a plasmid were digested by HindIII/NheI restriction enzymes and survivin was ligated into the digested vector. Then, the ligation product was transformed into the E.coli DH5a competent cells and screened by antibiotic selection marker (kanamycin). Positive colonies were selected by colony PCR and screened by double digestion of isolated plasmid. One positive colony was sequenced and confirmed. The recombinant plasmid was transformed into the expression strain of E.coli (BL21) by chemical method. The expression of survivin was induced in the different conditions and expression level investigated by SDS-PAGE.
The size of PCR product in agarose gel showed the correctness of amplification. The digested pET-28a plasmid also indicated the correctness of enzymatic reaction. The sequence of the cloned fragment revealed a 100% similarity to the human survivin. In expressing, adding IPTG increased the expression of survivin protein in all conditions, especially 37 ᵒC from 2 h after induction. At all conditions, most of survivin accumulated in the bacteria as inclusion body.
Seyedeh Maryam Hosseini, Sadegh Hasannia, Bijan Ranjbar,
Volume 11, Issue 3 (10-2020)
Abstract
Human alpha1-antitrypsin is a glycoprotein comprised of 394 amino acids and 52 kDa molecular weight, which is mostly synthesized and secreted by hepatocytes, diffuses to interstitial lung tissues, and has an essential function to protected tissues against neutrophil elastase. One of the significant challenges in dealing with alpha-1-antitrypsin is the structural instability of the folded form of protein and, consequently, the accumulation of polymers in lung tissue. This makes patients vulnerable to chronic obstructive pulmonary disease, severe asthma, and emphysema. Intravenous augmentation therapy of alpha 1-antitrypsin is one of the most prevalent therapies. Moreover, patients who are candidates for that have respiratory symptoms, and the use of bronchodilator (Salbutamol) is the first recommended standard treatment. In this study, protein purification was performed by using high-performance affinity chromatography and, its purity was confirmed by gel electrophoresis. The effect of different concentrations of salbutamol on heat-induced polymerization at 60 ° C was investigated by non-denaturing polyacrylamide gel electrophoresis, dynamic light scattering (DLS), and circular dichroism (CD) techniques. Protein activity was measured by trypsin inhibitor capacity (TIC) assay. The results indicated that salbutamol decreases the rate of polymerization by reducing the flexibility of the reactive center loop, thus decelerate protein activity reduction. Therefore, salbutamol can be an appropriate supplementary for alpha 1-antitrypsin and a proper option for the treatment of protein polymerization associated diseases.
Hamed Ghadiri, Sana Alavi, Bahareh Dabirmanesh, Khosro Khajeh,
Volume 12, Issue 1 (12-2020)
Abstract
The eukaryotic genome contains several replication Origins. Studies showed that the phenomenon and order of the origin activation is in a a particular discipline, called the “Replication Timing". Recent studies show that many factors are involved in regulating the timing of the replication process. One of the most important factors amongst them is the Rap1 interacting Factor 1 (Rif1) protein, which plays a key role in regulating the replication schedule in yeast and more advanced eukaryotes. Structure of this protein is mostly irregular and these properties prevent Rif1 from being expressed in a stable manner and makes it difficult to study.
The aim of the present study was to investigate the expression of recombinant C-terminal domain of mouse-Rif1(muRif1-CTD) protein in solution. For this purpose, the muRif1-CTD gene was extracted from eukaryotic constructs containing the complete Rif1 gene by PCR and was inserted into the pPAL7 expression vector comprising the Profinity eXact tag. Protein solubilization was carried out using different detergents and then detergent removal was performed by dialysis. In order to ensure that the soluble protein is active, the interaction analysis of the Rif1 protein with the G4 structures (previously reported to bind Rif1) was investigated using the gel shift assay. The results of this study showed the use of detergent for Rif1 solubilization without affecting its purification steps. But in the case of this protein, if the detergent is removed completely, it will not remain soluble.
Nasrin Kardan, Bahareh Dabirmanesh, Khosro Khajeh,
Volume 12, Issue 1 (12-2020)
Abstract
Protein deposition due to the process of accumulation inside or outside cells causes many neurological diseases such as Alzheimerchr('39')s, Huntingtonchr('39')s or Parkinsonchr('39')s seizures. Parkinsonchr('39')s disease is the second most common neurological disease after Alzheimerchr('39')s, in which patients develop disorders due to the accumulation of leprosy and the destruction of dopamine neurons. Alpha-synuclein protein contains 140 amino acids, the main protein known in lewy body accumulations. During the aggregation process, alpha-synuclein protein monomers bind together as oligomers and eventually become amyloid filaments. So far, there is no drug to stop or delay the progression of Parkinsonchr('39')s, but studies on the molecular mechanism of amyloid formation and the identification of inhibitors are increasing. For this purpose, in this study, the effect of BRICHOS domain resulting from BRI2, which can have various functions, including antimicrobial properties, on the process of alpha-synuclein accumulation as a model protein was investigated.The gene was first optimized and synthesized and then multiplied by PCR. The product was digested by enzymes Xho I and Nde1 and entered the expression vector pET28 a, which was transformed into E. coli bacteria.Finally, the peptide was purified by nickel chromatography. The alpha-synuclein gene was also expressed separately and purified.The anti-cumulative effect of BRICHOS domain on alpha-synuclein fibrillation was investigated using Toflavin T fluorescence method and TEM technique.
Fatemeh Asadi, Hamedreza Goodarzi, Javad Zahiri, Mojtaba Jaafarnia,
Volume 12, Issue 2 (1-2022)
Abstract
Introduction: The present study reports a case of familial episodic coma in which three girls manifesting refractory seizures followed by coma. Targeted gene panel of epilepsy using next-generation sequencing (NGS) technique was requested to identified disease -causing variant(s) in the patients.
Materials and Method: After obtaining a written informed consent from our patient, genomic DNA was extracted from venous blood, for identifying mutations in epilepsy genes, at first, coding regions as well as all intron–exon boundaries of the 72 genes were captured by Sure Select Target Enrichment System
V4 kit (Agilent, Santa Clara, CA),
then captured libraries were sequenced on an Illumina HiSeq 4000(Illumina Inc., San Diego, CA, USA), sequenced reads were aligned with a reference human genome and Picard tools was used to remove duplicated reads; variant calling was performed using the Genome Analysis Tool Kit (GATK). ANNOVAR was used to annotated variants, then
all variants were filtered out based on minor allele frequency (MAF) <1 % according to data bases of nucleotide including dpSNP, 1000 Genome project. In silico tools was performed to evaluated pathogenicity of variant(s).
Result: According to databases of pathogenicity prediction of gene, no specific mutation in epilepsy genes was found in our patients, but several polymorphisms were reported.
Conclusion: Given polymorphisms in genes related to epilepsy were found in our study, failed to provide us with an acceptable diagnosis of this condition, further research is needed to reveal the cause of the disease.
Key word: episodic coma, NGS, gene panel, epilepsy
Najme Dehghanbanadaki, Majid Taghdir, Reza Hassan Sajedi, Hossein Naderi-Manesh,
Volume 12, Issue 3 (9-2021)
Abstract
Abstract. FZD7 receptor is considered as an emerging target for the treatment of Wnt-βcatenin related cancers. This transmembrane receptor is overexpressed in many cancer types like breast cancer and ovarian carcinoma, and so selective targeting of this receptor has a great therapeutic capacity. On the other hand, one of the mechanisms proposed for the anticancer effect of Atrial natriuretic peptide (ANP) that known as a heart hormone at first, is Wnt-βcatenin inhibition through an FZD dependent manner but, the molecular mechanism of this inhibition is not clear. Here, using computational methods including molecular docking and molecular dynamics simulation, also designing a cellular system that enabled us to trace Wnt-βcatenin kinetics directly, we investigated the mechanism of the peptide inhibitory potential against the pathway. Our computational results show that ANP can directly interact with FZD7 and also, its binding site on FZD7 overlap to the binding region of the Wnt carboxyl-terminal domain (Wnt-CTD). The finding of the silencing experiments demonstrates the dependency of Wnt-βcatenin signaling of the cellular system to FZD7. The decrease of βcatenin in cells treated to ANP and Wnt is also significant to compare to the control experiments. Finally, our results show that ANP is a potential scaffold to design selective peptide against FZD7.
