Browsing by Author "Mehdizadeh Aghdam, Elnaz"

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Cloning and sequencing of riboswitches of Alishewanella tabrizica strain RCRI4 and comparative studies on similar riboswitches in other bacteria
(Tabriz University of Medical Sciences, Faculty of Pharmacy, 2016) Mehdizadeh Aghdam, Elnaz; Hartig, Jörg S.; Hejazi, Mohammad Saeid; Barzegar, Abolfazl
Introduction: Riboswitches, as cis acting non-coding RNA elements, regulate gene expression via specific binding of various small molecules. Their conserved structures are the key characteristics for binding specific molecules and gene regulation. Also, the contribution of riboswitches in antibiotic targeting is possible. In this study, we aimed first to find out the structural and functional level of similarity among riboswitches and rRNA structures including binding of aminoglycosides to riboswitches through computational tools. On the other side, riboswitches are rarely characterized in environmental bacteria. In this study, TPP riboswitch sourced from novel characterized specie Alishewanella tabrizica, was identified. Methods: After PDB structures’ selection, multiple sequence and structural alignment were carried out. Highly similar rRNA motifs with riboswitches (including “A site”) were sorted out. Subsequently, the probable interaction of riboswitches with aminoglycosides ere studied using docking studies. For identifying of TPP riboswitch of A. tabrizica, kinetic and affinity analysis of TPP binding to different TPP riboswitch aptamer domains sourced from A. tabrizica, Alishewanella aestuarii, E. coli, B. subtilis were studied and compared using In-line probing and Surface Plasmon Resonance (SPR) methods. Results: Docking analysis showed significant high binding energies between riboswitches-aminoglycosides. Accordingly, lysine, glycine and SAM-I riboswitches were recognized as the best RNA targets for all of the aminoglycosides. Docking results were also validated through rDock program and MD simulation. According to the KD values from experimental analysis, the affinity of TPP binding was the highest in A. tabrizica. In addition, the order of TPP binding affinity was TPP aptamer domains sourced from A. tabrizica > A. aestuarii > E. coli > B. subtilis. Conclusion: Taking together, it can be proposed that riboswitches have this potential to be the target of aminoglycosides. The observed variation TPP riboswitches affinity could be referred to the studied bacteria adaptation to diverse environmental conditions.
Effects of cytoplasmic recombinant protein expression (hll-2&mll-4) on hydrogen peroxide concentration and catalase activity in Escherichia coli
(Tabriz University of Medical Sciences, Faculty of Pharmacy, 2009) Mehdizadeh Aghdam, Elnaz; Hejazi, Mohammad Saeid
The expression of a foreign protein(s) in a recombinant host cell or organism often changes the biochemistry and physiology of the host and lower the amount of the target foreign protein. Identification of preventive factors helps to resolve their inhibitory effects and improve the product yield by increasing cell growth and recombinant proteins production from molecular cloning to bioprocess procedures.The Reactive oxygen species (ROS) is induced in the cells following various stresses. However, the effect of recombinant protein expression on ROS generation has not been studied yet. In this study, H2O2 concentration and catalase activity variations and their correlation with cell growth following cytoplasmic expression of human interleukin-2 (hIL-2) and mouse interleukin-4 (mIL-4) in Escherichia coli Bl21(DH3) were investigated. Additionally, the effect of recombinant protein expression under different conditions was compared to the effect of foreign DNA introduction on these factors. Plasmids pEThIL-2 and pETmIL-4 were used for expression of human interleukin-2 (hIL-2) and mouse interleukin-4 (mIL-4) inside the cytoplasm of the cells. Having confirmed protein expression, H2O2 concentration and catalase activity were measured at various ODs. Also, Hydrogen peroxide concentration was measured in interleukin-4 expressing and wild type cells which treated with different concentrations of IPTG. Empty vector introduction increased significantly H2O2 concentration of the cells. However, H2O2 concentration in hIL-2 and mIL-4 expressing cells was significantly higher than its concentration in empty vector transformed cells. Catalase activity was reduced in foreign DNA introduced cells. It was more lowered following expression of recombinant proteins. Results of this study revealed the relationship between foreign DNA introduction and protein expression with H2O2 elevation and catalase activity reduction. There was also correlation between H2O2 elevation and reduction in catalase activity with the cell growth depression and recombinant protein production amount.
Evaluating the effect of simultaneous K-Ras and Akt genes inhibition in changing the sensitivity to chemotherapy drugs in mutant and normal K-Ras colorectal cell lines
(Tabriz University of Medical Sciences, School of Pharmacy, 2023) Meraji, Amir; Heydari, Hamid Reza; Mehdizadeh Aghdam, Elnaz
Mutations in K-Ras genes drive about 30% of human cancers, causing continuous pathway activation. Targeting K-Ras alone often activates the AKT pathway. Thus, inhibiting both K-Ras and AKT shows promise, but current treatments lack effective dual-targeting strategies, requiring new approaches.Objectives:Our study aims to evaluate the efficacy of newly designed shRNAs in suppressing AKT and K-Ras protein expression in two cancer cell lines, CACO2 (wild type K-Ras) and AGS (K-Ras(G12D)). By doing so, we seek to determine the feasibility of these shRNAs as promising therapeutic targets.Methods:Plasmid vectors encoding the designed shRNAs were used to generate recombinant plasmids, which were then transfected into CACO2 (wild-type K-Ras) and AGS (K-Ras(G12D)) cancer cell lines. Cell viability was assessed using MTT assays, and apoptotic effects were measured via flow cytometry. The inhibitory impact of the shRNAs was confirmed through cobalt-induced treatment and RT-PCR analysis. Comparative analysis between wild-type and mutant K-Ras cell lines will be conducted to determine the outcomes. Results: The shRNAs reduced AKT and K-Ras protein expression in cancer cell lines, decreasing viability and inducing apoptosis. Cobalt treatment and RT-PCR confirmed these effects. Comparative analysis supported the approach’s efficacy.Conclusion: the designed shRNAs effectively targeted AKT and K-Ras in cancer cells, demonstrating promising potential as a therapeutic approach for colorectal cancer.
Identification of potential immunogenic regions in SARS-CoV-2 virus proteins using bioinformatics tools
(Tabriz University of Medical Sciences , School of Pharmacy, 2022) Golkar, Hossein; Hejazi, Mohammad Saeid; Molavi, Ommoleila; Heidari, Hamid Reza; Mehdizadeh Aghdam, Elnaz
SARS-CoV-2 was the cause of the recent global pandemic, COVID-19, which killed many people. Several vaccines were used to deal with this disease, but the side effects are one of the most critical challenges of the produced vaccines. Therefore, it is possible to produce vaccines with fewer side effects and more effective by using bioinformatics tools. Target : Identification of potential immunogenic regions in SARS-CoV-2 virus proteins using bioinformatics tools. Method :First, the sequences of individual SARS-CoV-2 proteins were obtained from the NCBI site. After submitting it to the IEDB database using the recommended method for MHC class II and MHC class I for all alleles presented in that database, possible epitopes were created. Then, epitopes with higher affinity were screened using Vaxijen, ToxinPred, and AllerTop servers in terms of immunogenicity and toxicity, and allergenicity, respectively. Also, the possible epitopes of linear B cells were analyzed by the IEDB server in the surface proteins of the virus. Results:Based on the analysis of the obtained data, in MHC class I alleles A*30:02, A*26:01, and A*32:01 and in MHC class II alleles DPA1*01:03/DPB1*04:01, DRB3 *02:02 and DPA1*01:03/DPB1*02:01 produced the highest number of epitopes with high affinity among all virus proteins. Spike, NSP12, and NSP3 proteins also produced the highest number of epitopes with high affinity for MHC class I and II.Conclusion:Based on immunoinformatics tools, proteins that produce the maximum number of high-affinity epitopes with the minimum amino acid sequence can be selected and used in vaccine design.
In vitro study of cytotoxic and apoptotic effects of inhibitory peptides against mutated K-Ras
(Tabriz University of Medical Sciences , School of Pharmacy, 2022) Motiei, Parinaz; Hejazi, Mohammad Saeid; Heidari, Hamid Reza; Mehdizadeh Aghdam, Elnaz; Molavi, Omleila
Ras genes mutations are responsible for approximately 30 % of human cancers. Of these, K-Ras is the most frequently mutated isoform as common mutations in K-Ras leads to the constant activation of downstream effector pathways. Though K-Ras is considered as an important target in some cancers, no anti-Ras therapy has succeeded in clinic and due to the disadvantages of other able therapeutic approaches for cancer therapy, discovering a new effective peptide seems necessary enough. Objective: In this study, we are going to examine cytotoxic effects of newly designed inhibitory peptides on three cancerous cell lines named CACO2 (wild type K-Ras), AGS (K-Ras(G12D)) and Miapaca2 (K-Ras(G12C)). Methods:Plasmid vectors containing the sequence which will lead cells to produce our designed peptides, were ordered and then a recombinant plasmid were obtained with our sequence that transformed to our cell lines. MTT assay has been used to assess cytotoxic effect of our newly designed peptides that their expression had induced using cobalt. the results will be compared between wild type K-Ras gene harboring cancerous cell lines and mutant K-Ras gene cell lines.Results: Newly designed peptides showed cytotoxic effect on cancerous cell lines which contains G12C and G12D mutated K-Ras Conclusion: Our results showed that the computational development based on the Molecular Dynamics simulations is in accordance with experimental results. Moreover, the two designed peptides PM1 and PM2 are capable of inhibiting the growth of mutant K-Ras harboring cancer cells.
Investigating the genetic diversity of aquatic bacteria of lake urmia in estuary of jighati (zarrine rood) and tatao (simine rood) rivers in the fall of 1400(2021)
(Tabriz University of Medical Sciences, School of Pharmacy, 2024) Khalafi, Armin; Mehdizadeh Aghdam, Elnaz; Tarhriz, Vahide; Hejazi, Mohammad Saeid; Montazersaheb, Soheila
Introduction: Lake Urmia is one of the saltiest lakes in the world, which faces the risk of dryness and excessive salinity. Aquatic microorganisms play an important role in the decomposition of substances, food chains and biochemical cycles and can also have functional effects. Therefore, the isolation and identification of bacteria is very necessary to better understand their function in an ecosystem like Urmia Lake, which is constantly changing.Objectives: The objectives of this study are to isolate and purify the bacteria in the water collected from the river mouth of Zarinerood and Siminerood rivers, and then to determine the 16sRNA sequence of the isolated bacteria to determine the identity of the bacteria and also to investigate the biochemical properties of gram, catalase, oxidase, and and motility of bacteria.Methodology: The population of bacteria was cultured in the form of single colonies during the growth process and successive isolations. After the DNA extraction stage and if the quality of the results of qualitative and quantitative investigation was suitable, the selected bacteria were sent for sequencing and also phenotypic tests were performed on the isolated bacteria.Findings: In this study, bacteria isolated from Lake Urmia were isolated, and finally 9 bacterial isolates were subjected to phenotypic studies. According to the results, the majority of these bacteria were gram positive, catalase positive, KOH negative, oxidase positive and amylase negative. DNA extracted from these bacteria was sent to determine the sequence of 16srRNA. The results of this study indicate the possibility of reducing the types of bacteria in this place.conclusion: According to the obtained results, the phenotypic results also do not show much diversity among the selected species and there is a possibility of reducing the bacterial species in this place.
Isolation and Characterization of aquatic Bacteria of Quri-Gol lake in 2021
(Tabriz University of Medical Sciences, School of Pharmacy, 2024) Shahin Doost, Amin; Hejazi, Mohammad saeid; Montazersaheb, Soheila; Mehdizadeh Aghdam, Elnaz; Tarhriz, Vahideh
Qurigol is one of the most important wetlands in East Azarbaijan province in terms of non-pathogenic bacterial population. To identify beneficial microorganisms, we referred to Qurigol place to collect, isolate and characterize the bacterial populations.Objectives: The objectives of this research were to isolate the bacteria present in Qurigol and then determine their identity with respect to related species and to investigate phenotypes such as gram, oxidase,Methodology: During the isolation process, the bacteria were cultured as single colonies separated from each other. Finally, after passing through the DNA extraction stage, phenotypic examination including gram test, oxidase, urease, catalase and motility was examined.Results: In this study, the bacteria isolated from the Qurigol pond were isolated and phenotypically investigated. After DNA isolation and extraction, 7 isolates were sent for 16srRNA sequencing due to having suitable optical absorption and the results of phenotypic tests were also reported. According to the results, the majority of these bacteria were gram positive, catalase positive, KOH negative, oxidase positive and amylase negative. Conclusion: Observing the state of the Qurigol showed that the dryness of the environment has damaged the normal flora, and the same can be concluded from the findings. We hope that in the coming years more importance will be given to valuable resources and measures will be taken to recover the area.
Modeling of biological network by using proteoinformatics analysis for identification of vaccine candidate antigen(s) of Mycobacterium tuberculosis
(Tabriz University of Medical Sciences, School of Pharmacy, 2021) Jamal Zabardast, Behrad; Pourseif, Mohammad Mostafa; Naghili, Behrouz; Mehdizadeh Aghdam, Elnaz; Omidi, Yadollah
According to WHO reports, tuberculosis is considered as one of the ten top causes of death worldwide. In 1921, Bacillus Calmette-Guerin (BCG) was introduced as a prophylactic vaccine against tuberculosis, which induces high protection in the children. On the other hand, the BCG showed questionable efficacy and variable levels of protection in adults, immunocompromised individuals, and inefficiency on drug-resistant strains. Therefore, the development of a new and more efficient vaccine against TB has the utmost importance. Using the latest techniques like next-generation sequencing (NGS) and virtual screening of proteins to find appropriate vaccine candidates can be a suitable solution for TB vaccine challenges. Aim: We aimed to utilize computational and immunoinformatic tools to identify new antigens as potential vaccine candidates for designing an epitope-based vaccine against Mycobacterium tuberculosis. Methods For generating a reliable and appropriate protein-protein interaction network, The STRING database was used to gather data for establishing a PPI network. Using topological analyzer tools facilitates identifying hub proteins of generated networks. Consequently, filtration tools have been applied to make a shortened vaccine antigen list by identifying hub proteins. We used concepts including subcellular localization, antigenicity, virulence factor, homology, allergenicity, and essentiality to achieve this goal. Result From 3993 proteins of M. tuberculosis's reference strain, 283 proteins have been considered as hub proteins. The results were filtered to reach eight antigen proteins for possible vaccine design using all in-silico methods mentioned above. Conclusion Our study confirmed the potency of topological analysis and immunoinformatic tools for effective antigen prediction and vaccine development.
Optimization of binding affinity and stability of anti- mutant KRAS peptides using Molecular Dynamics Simulation
(Tabriz University of Medical Sciences, School of Pharmacy, 2024) Moradnezhad Mahmudy, Amir Hossein; Barzegar, Abolfazl; Mehdizadeh Aghdam, Elnaz; hejazi, Mohammad Saeid
Background: RAS proteins play a central role in proliferation pathways, and mutations in them can lead to sustained RAS activation and developing cancer cells. Thera are some limitations regarding the inhibition of mutant RAS proteins by small molecules. Therefore, peptides are promising therapeutic targets for mutant RAS suppression. However, optimizing the stability and binding affinity of peptides is necessary. Objective: we aimed to optimize peptides with KRAS inhibition capability to achieve more potent and stable therapeutic candidates. Methods: In this study, an optimized peptide sequence, derived from Sos-αH motif in SOS-KRAS interaction, was used to D-amino acid mutation to increase the stability. Chimera software was used to mutate each L-amino acid into its D-amino acid form. MD simulations of KRAS-D peptide complexes were performed using “GROMACS” software by including and the D-amino acids topology. After simulating the system, supplementary analyzes were performed. We measured the affinity of peptides to KRAS using the umbrella sampling method. Furthermore, the hydrogen binding and β2 chain lengths of KRAS variants were calculated. The system snapshot was rendered by the software “CHIMERA UCSF”.Results: There are different approaches to select the best peptide candidates for K-Ras inhibition for example one is Based on the best peptide affinity to wild type/mutant KRAS proteins. Another way is the mechanistic reduction of β2 sheet size of wild type/mutant KRAS proteins. The D-sos peptide type D2E showed higher binding energies towards G12C-GCP and G12D-GCP and also acceptable binding threshold with the lowest binding affinity energy with K-ras wild type. Furthermore, they resulted in a decrease in the size of the β2 chain, indicating the ability of these peptides to reduce K-Ras activity.Conclusion: We suggested peptide type D2E as a promising mutant KRAS inhibitory peptides.
Screening and optimization of Halophilic bacteria producing phytase and beta-glucanases from center of Iran
(Tabriz University of Medical Sciences, Faculty of Pharmacy, 2016) Abdi, Peyman; Dilmaghani, Azita; Mehdizadeh Aghdam, Elnaz
In some areas, there are very difficult conditions for life that appear to be life-free at first. Those areas have very high salinity condition, while this salinity is a good environment for halophilic microorganisms, these microorganisms have the ability to produce hydrolytic enzymes. Some of these enzymes are Phytase and beta-glucanases. Phytase is an enzyme essential for bone health and digestive system, which is widely used in the paper industry, food industry, fruit juice industry, animal feed and poultry and medicine. Studies confirm that the use of this enzyme reduce the body needs to calcium and phosphate. Also, beta-glucanases is a very important enzyme as the body cannot make it itself. The capability of this enzyme to digest fiber helps to overcome gastrointestinal problems such as mal absorption. This enzyme has various uses in the pharmaceutical, food industry, and so forth. Objective: The aim of this study was finding halophilic bacteria isolated from halophilic soil of center of Iran producing Phytase and beta-glucanases enzymes and optimizing their growth. Materials and methods: At first, the halophilic bacteria were screened for producing of Phytase and beta-glucanases enzymes. Then, pH and temperature were optimized for the best growth of bacteria. Results: Results showed that two out of seven isolated halophilic bacteria were positive for beta-glucanases activity but none of them could produce phytase. The halophile producing beta-glucanases belong to Bacillus genus. Also, the best pH and temperature for the growth were 9 and 50 °C, respectively. Conclusion: Isolated Halophilic bacteria from the deserts of central Iran have the ability to produce hydrolytic enzymes and so that it can be the good source of hydrolytic enzymes such as phytase and beta-glucanases for future research and industrial work.
Screening of glutaminase and cellulase producing by halophilic bacteria from central districts of Iran
(Tabriz University of Medical Sciences, Faculty of Pharmacy, 2018) nejad Ali, Amin; Mehdizadeh Aghdam, Elnaz; Dilmaghani, Azita
Halophilic bacteria grow in a wide range of salt concentrations. According to Kouchner classification halophilic bacteria are divided in three groups: halotolerant bacteria, moderately halophilic bacteria and extreme halophilic bacteria. Halophilic bacteria can produce several enzymes such as cellulase and glutaminase which are important in industry and pharmacy. Objective: The aim of this study is isolation of halophilic bacteria producing cellulase and glutaminase from saline soils of the center of Iran. Moreover, the optimum pH and temperature for high activity and stability of the produced enzyme were identified. Materials and methods: At first, Halophilic bacteria isolated from center of Iran were cultured and then they were screened for producing of hydrolytic enzymes which include cellulase and glutaminase. Then, the best activity and stability of the produced enzymes were studied at various pH and temperatures. Results: Results showed that the isolated halophilice bacteria were able to produce hydrolytic enzymes. The halophilic bacteria which produce cellulase were belong to Bacillus. And also the halophilic bacteria produceing glutaminase were belong to Bacillus. Furthermore, the best pH and the best temperature for optimal activity of cellulase were 8 and 40 °C, respectively. Also, the best pH and the best temperature for cellulase stability were 8 and 60 ° C, respectively. Also, the best pH and the best temperature for optimal activity of glutaminase were 8 and 50 °C, respectively. Also, the best pH and the best temperature for Glutaminase stability were 8 and 60 °C, respectively. Conclusion: Isolated Halophilic bacteria from the deserts of central Iran have the ability to produce hydrolytic enzymes and so that it can be the good source of hydrolytic enzymes such as cellulase and glutaminase for future research and industrial work.
Structural mechanism study of KRpep2d anti-cancer peptide interaction with wild type and mutant KRAS using molecular dynamic simulation
(Tabriz University of Medical Sciences, Faculty of Pharmacy, 2020) Pashapour Anoosheh, Jeiran; Hejazi, Mohammad Saeid; Mehdizadeh Aghdam, Elnaz; Barzegar, Abolfazl
Background: Cancer is one of the major health concerns worldwide, and there is an ongoing effort to find novel treatment approaches. RAS Proteins play a pivotal role in the proliferation pathways, and their mutations can lead to persistent RAS activation and cancerous cells. In this regard, KRpep2d is an anticancer peptide against KRAS(G12D) mutant, but its exact mechanism of action is unknown. Objective: In this study, the molecular mechanism of KRpep2d on the inhibition of mutant KRAS via the MD simulation method is investigated. Methods: MD simulations of KRAS variants in the free and complex form with KRpep2d were carried out via "GROMACS" software. Supplementary analyses were performed after the simulation of each system. The distance variations of residues in different simulation systems, were conducted to define conformational changes. Moreover, the affinities of KRpep2d and the nucleotide to KRAS were measured by the "Umbrella Sampling" method. Also, residue interactions and β2-strand length of KRAS variants were calculated by "LigPlot plus" software and DSSP plugin of "GROMACS" software, respectively. System snapshots were represented by "CHIMERA UCSF" software. Results: It was shown that KRpep2d binding to the KRAS(G12D) makes the GTP move from the P-loop location. On the other hand, KRpep2d reduces the size of the effector binding region (β2-strand) of KRAS mutants. It was also observed that KRpep2d decreases and increases the Switch II and Switch I flexibility, respectively, in KRAS(G12D). All these results were exclusive for KRAS(G12D). Additionally, the parameters of β2-strand length and the affinity of KRpep2d were combined to measure relative activity. The acquired relative activity was the minimum for KRAS(G12D). Conclusion: KRpep2d shows anticancer activity on KRAS(G12D) by expelling the nucleotide from its location and reducing the β2-strand as the effector binding region of KRAS.