Publications by authors named "Reza Valadan"

32 Publications

Protective efficacy by a novel multi-epitope vaccine, including MIC3, ROP8, and SAG1, against acute Toxoplasma gondii infection in BALB/c mice.

Microb Pathog 2021 Apr 3;153:104764. Epub 2021 Feb 3.

Toxoplasmosis Research Center, Communicable Diseases Institute, Mazandaran University of Medical Sciences, Sari, Iran. Electronic address:

Toxoplasma gondii is an intracellular apicomplexan parasite, which can cause a serious infectious disease in pregnant women and immunocompromised individuals. Therefore, the development of a polyvalent vaccine consisting of all stages of the parasite life cycle using the epitopes from tachyzoites, bradyzoites, and sporozoites is likely to be required for complete protective immunity. In this study, we designed protein vaccine candidate based on the prediction of specific epitopes (i.e., B cell and T cell) from three Toxoplasma gondii antigens. The MRS protein (MIC3: 30-180, ROP8: 85-185, and SAG1: 85-235) was expressed in Escherichia coli, and purification was performed using a HisTrap HP column and then we evaluated immunogenicity and protective property in BALB/c mice. Seventy-two mice were randomly divided into six groups, including three vaccinations (i.e., MRS, MRS-Freund, and MRS-Calcium Phosphate Nanoparticles (MRS-CaPNs)) and three control (i.e., Phosphate-buffered saline, Freund, and CaPNs) groups. All groups were immunized three times via subcutaneous injection within three-week intervals. In the vaccination groups, the BALB/c mice were injected with 20 μg of MRS protein for the first time and 10 μg of MRS for the next two times. Antibodies, cytokines, and splenocytes proliferation in the immunized mice were assayed using the enzyme-linked immunosorbent assay. Protective efficacy was analyzed by challenging the immunized mice with T. gondii of RH strain. Antibody, cytokine, and lymphocyte proliferation assays showed that the mice immunized with MRS induced stronger humoral and T helper type 1 cell-mediated immune responses, compared to the control mice. However, co-immunization with adjuvants (i.e., Freund and CaNPs) resulted in impaired immune responses. Effective protection against the parasite achieved an increase in survival time in the immunized mice, especially in the MRS-CaNPs group. The obtained results of the present study demonstrated that multi-epitope protein vaccination, MRS, is a potential strategy against toxoplasmosis infection. In addition, the vaccine co-delivered with CaPNs could provide an important key for vaccine candidate to control T. gondii infection.
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http://dx.doi.org/10.1016/j.micpath.2021.104764DOI Listing
April 2021

Designing a novel multiepitope peptide vaccine against melanoma using immunoinformatics approach.

J Biomol Struct Dyn 2020 Nov 23:1-13. Epub 2020 Nov 23.

Department of Immunology, Molecular and Cell Biology Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Malignant melanoma is the most aggressive and life-threaten skin cancer. Nowadays, the prevention and treatment of melanoma are challenging areas for researchers and physicians. Therefore, we implemented an based approach to design a multi-epitope peptide vaccine for melanoma. This approach consists of immunoinformatics, molecular docking, and dynamic stimulation assessments to identify potent targets. Three most immunogenic melanoma proteins; NEYSO-1, gp-100, and MART-1were considered to predict immunodominant B and T cell epitopes. The prioritized epitopes had significant potential to induce strong humoral and cellular immunity and INF-γ responses without the possibility of allergenicity. To enhance the immunogenic properties of the vaccine, we used adjuvants HBHA, the helper epitope of PADRE, and three segments of the helper epitope from TTFrC. To design the final vaccine construct, appropriate linkers are used to join immunogenicscreened-epitopes and also the adjuvants. The physicochemical and immunological properties of the vaccine were evaluated.The designed-vaccine construct was docked to TLR4 to visualize the complex affinity and then conformational dynamics simulation was used to analyze time-dependent interaction behavior. In silico cloning demonstrated that the vaccine can be efficiently expressed in . Therefore, the designed vaccine might have the ability to induce humoral and cellular immune responses against melanoma cancer antigens. This vaccine has a high-quality structure and suitable characteristics such as high stability, solubility, and a high potential for expression in a prokaryotic system. However, these results need the experimental study to ensure the immunogenicity and safety profile of the melanoma candidate vaccine construct.
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http://dx.doi.org/10.1080/07391102.2020.1846625DOI Listing
November 2020

Design an Efficient Multi-Epitope Peptide Vaccine Candidate Against SARS-CoV-2: An in silico Analysis.

Infect Drug Resist 2020 25;13:3007-3022. Epub 2020 Aug 25.

Department of Immunology, Molecular and Cell Biology Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Background: To date, no specific vaccine or drug has been proven to be effective against SARS-CoV-2 infection. Therefore, we implemented an immunoinformatic approach to design an efficient multi-epitopes vaccine against SARS-CoV-2.

Results: The designed-vaccine construct consists of several immunodominant epitopes from structural proteins of spike, nucleocapsid, membrane, and envelope. These peptides promote cellular and humoral immunity and interferon-gamma responses. Also, these epitopes have a high antigenic capacity and are not likely to cause allergies. To enhance the vaccine immunogenicity, we used three potent adjuvants: Flagellin of subsp. enterica serovar Dublin, a driven peptide from high mobility group box 1 as HP-91, and human beta-defensin 3 protein. The physicochemical and immunological properties of the vaccine structure were evaluated. The tertiary structure of the vaccine protein was predicted and refined by Phyre2 and Galaxi refine and validated using RAMPAGE and ERRAT. Results of ElliPro showed 246 sresidues from vaccine might be conformational B-cell epitopes. Docking of the vaccine with toll-like receptors (TLR) 3, 5, 8, and angiotensin-converting enzyme 2 approved an appropriate interaction between the vaccine and receptors. Prediction of mRNA secondary structure and in silico cloning demonstrated that the vaccine can be efficiently expressed in .

Conclusion: Our results demonstrated that the multi-epitope vaccine might be potentially antigenic and induce humoral and cellular immune responses against SARS-CoV-2. This vaccine can interact appropriately with the TLR3, 5, and 8. Also, it has a high-quality structure and suitable characteristics such as high stability and potential for expression in .
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http://dx.doi.org/10.2147/IDR.S264573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459237PMC
August 2020

Cold Atmospheric Plasma Is a Potent Tool to Improve Chemotherapy in Melanoma In Vitro and In Vivo.

Biomolecules 2020 07 8;10(7). Epub 2020 Jul 8.

Department of Immunology, Molecular and Cell Biology Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari 4847191971, Iran.

Malignant melanoma is a devastating disease. Because of its aggressiveness, it also serves as a model tumor for investigating novel therapeutic avenues. In recent years, scientific evidence has shown that cold atmospheric plasma (CAP) might be a promising modality in cancer therapy. In this study, we aimed to evaluate the effect of CAP generated by an argon plasma jet alone or in combination with dacarbazine (DAC) on melanoma cells in vitro and in vivo. The effects of the CAP on inducing lipid peroxidation and nitric oxide production were higher in B16 melanoma cells in comparison to non-malignant L929 cells. Assays on cell growth, apoptosis, and expression of genes related to, e.g., autophagic processes, showed CAP to have a substantial impact in melanoma cells while there were only minoreffects in L929 cells. In vivo, both CAP monotherapy and combination with DAC significantly decreased tumor growth. These results suggest that CAP not only selectively induces cell death in melanoma but also holds promises in combination with chemotherapy that might lead to improved tumor control.
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http://dx.doi.org/10.3390/biom10071011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407977PMC
July 2020

Tropolone alkaloids from Colchicum kurdicum (Bornm.) Stef. (Colchicaceae) as the potent novel antileishmanial compounds; purification, structure elucidation, antileishmanial activities and molecular docking studies.

Exp Parasitol 2020 Jun 27;213:107902. Epub 2020 Apr 27.

Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran.

Natural compounds played an important role for prevention and treatment of the disease as well as are the important compounds for the design of the new bioactive compounds. In this study, eight tropolone alkaloids were isolated from Colchicum kurdicum including colchicoside, 2-demethyl colchicine, 3-demethyl colchicine, demecolcine, colchifoline, N-deacetyl-N-formyl colchicine, colchicine and cornigerine by column and preparative thin layer chromatography. The chemical structures were identified by H NMR and C NMR spectroscopy. Moreover, the antileishmanial activity on Leishmania major, anti-inflammatory activity, iron chelating activity and toxicity studies including hemolytic activity, brine shrimp toxicity, cytotoxicity and acute toxicity and docking study of all isolated bioactive compounds were evaluated. As result, colchicoside and colchicine had potent leishmanicidal effects and N-deacetyl-N-formyl colchicine and cornigerine had the highest anti-inflammatory effects. All compounds had the significant iron chelating activity. According to toxicity studies, isolated compounds showed the low hemolytic activity and cytotoxicity, high LC, LC and LD. In the molecular docking study, colchicoside had the high dockscore. According to the study, with future studies all isolated compounds could be used for design the novel antileishmanial drugs.
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http://dx.doi.org/10.1016/j.exppara.2020.107902DOI Listing
June 2020

Emergence of Terbinafine Resistant in Iran, Harboring Mutations in the Squalene Epoxidase () Gene.

Infect Drug Resist 2020 13;13:845-850. Epub 2020 Mar 13.

Invasive Fungi Research Center, Department of Medical Mycology and Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Introduction: and are important causative agents of superficial mycoses, demonstrating emergent antifungal drug resistance. We studied the antifungal susceptibility profiles in Iranian isolates of these two species.

Methods: A total of 96 and 45 isolates were subjected to molecular typing by ribosomal ITS region. Antifungal susceptibility profiles for terbinafine, griseofulvin, clotrimazole, efinaconazole, luliconazole, amorolfine and ciclopirox were obtained by CLSI broth microdilution method. The squalene epoxidase () gene was subjected to sequencing for mutations, if any, in isolates exhibiting elevated MICs for terbinafine.

Results: Luliconazole and efinaconazole showed the lowest MIC values against and isolates. There were five isolates with terbinafine MICs ≥32 µg/mL in our sample. They belonged to type VIII and harbored two alternative gene sequence variants, leading to Phe397Leu and Ala448Thr or Leu393Ser and Ala448Thr substitutions in the enzyme. All terbinafine resistant strains could be inhibited by luliconazole and efinaconazole.

Conclusion: This study documented a step in the global spread of resistance mechanisms in . However, treatment alternatives for resistant isolates were available.
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http://dx.doi.org/10.2147/IDR.S246025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078656PMC
March 2020

Designing a potent L1 protein-based HPV peptide vaccine: A bioinformatics approach.

Comput Biol Chem 2020 Apr 17;85:107209. Epub 2020 Jan 17.

Molecular and Cell Biology Research Center, Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Background: Oncogenic human papilloma viruses (HPV) are the cause of various types of cancer, specifically cervical cancer. L1 protein is the main protein of HPV capsid which targeted in many vaccine-producing attempts. However, they have not enough coverage on the various high risk HPV types. Therefore, having a low cost potent HPV vaccine to protect against all members of the α-papillomaviridea family will be promising. In this study, L1 protein-based peptide vaccine was designed using immunoinformatics methods which provides physicochemical properties such as stability in room temperature, potential of antigenicity, non-allergic properties and no requirement with eukaryotic host system.

Results: The designed vaccine has two HPV conserved epitopes with lengths 18 and 27 amino acids in all members of α-papillomaviridea. These peptides promote humoral and cellular immunity and INF-γ responses. In order to ensure strong induction of immune responses, Flagellin, a Toll like receptor 5(TLR-5) agonist, and a short synthetic toll like receptor 4 (TLR-4) agonist were also joined to the epitopes. Structure of the designed- vaccine was validated using Rampage and ERRAT and a high quality 3D structure of the vaccine protein was provided. Docking studies demonstrated an appropriate and stable interaction between the vaccine and TLR-5.

Conclusions: The vaccine is expected to have a high quality structure and suitable properties including high stability, solubility and a high potential to be expressed in E.coli. High potentiality of the vaccine in inducing humoral and cellular immune responses, may be considered as an anti-tumor vaccine.
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http://dx.doi.org/10.1016/j.compbiolchem.2020.107209DOI Listing
April 2020

HRM-PCR is an accurate and sensitive technique for the diagnosis of cutaneous leishmaniasis as compared with conventional PCR.

Acta Parasitol 2020 Jun 17;65(2):310-316. Epub 2019 Dec 17.

Skin Research Center, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran.

Background: Cutaneous leishmaniasis (CL) is considered one of the main health problems in Iran. Therefore, it is required for control and therapeutic purposes, an accurate and fast tool for the diagnosis and identification of Leishmania species.

Methods: In the present study, three techniques, including microscopic examination, conventional PCR, and high-resolution melting (HRM)-PCR, have been evaluated, to find the most accurate and rapid test. In total, 105 skin scraping smears were taken from suspected dermal lesions of patients belonging to two known endemic CL areas, Gonbad and Bam districts, in Iran. Subsequently, the specimens were analyzed with microscopic, conventional PCR, and HRM-PCR techniques.

Results: Most positive samples (89.5%) were observed using HRM-PCR, and among the three techniques, HRM-PCR was the most sensitive (89%, 95% CI 81-94) technique. Microscopic examination test had the lowest sensitivity (57%, 95% CI 47-66%). The highest agreement among positive samples was observed between HRM-PCR and conventional PCR tests.

Discussion: Our results showed that the HRM-PCR technique is the most accurate and sensitive test for recognizing CL, and also a valuable alternative test for conventional PCR to detect various species.
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http://dx.doi.org/10.2478/s11686-019-00154-5DOI Listing
June 2020

Different types of adjuvants in prophylactic and therapeutic human papillomavirus vaccines in laboratory animals: a systematic review.

Arch Virol 2020 Feb 4;165(2):263-284. Epub 2019 Dec 4.

Department of Medical Laboratory Sciences, Student Research Committee, School of Allied Medical Sciences, Mazandaran University of Medical Sciences, Sari, Iran.

Human papillomavirus (HPV) causes cervical carcinoma, which and is the third most common cancer, accounting for 275,000 deaths annually worldwide. Adjuvants have a key role in promotion of vaccine efficacy; therefore, using prophylactic and therapeutic vaccines combined with adjuvant could be of great benefit in prevention and treatment of cervical cancer. There are different types of adjuvants, including MF59 adjuvants, RNA-based, JY (interleukin2/chitosan), cholera toxin (CT), heat-labile enterotoxin (LT), Freund's adjuvant, alum, SA-4-1BBL, λ-carrageenan (λ-CGN), heat shock proteins (HSPs), juzen-taiho-to (JTT) and hochu-ekki-to (HET), ISCOM and ISCOMATRIX™, very small size proteoliposomes (VSSPs), granulocyte macrophage colony-stimulating factor (GM-CSF), and Toll-like receptors (TLRs). Adjuvants have various functions, especially in therapeutic vaccines, and they lead to an increase in cytotoxic T lymphocytes (CTLs), so they are important in the design of vaccines. Here, we review the currently used adjuvants and their combinations with HPV protein vaccines in order to introduce an appropriate adjuvant for HPV vaccines.
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http://dx.doi.org/10.1007/s00705-019-04479-4DOI Listing
February 2020

Wnt-β-catenin Signaling Pathway, the Achilles' Heels of Cancer Multidrug Resistance.

Curr Pharm Des 2019 ;25(39):4192-4207

Pharmaceutical Sciences Research Center, Mazandaran University of Medical Sciences, Sari, Iran.

Background: Most of the anticancer chemotherapies are hampered via the development of multidrug resistance (MDR), which is the resistance of tumor cells against cytotoxic effects of multiple chemotherapeutic agents. Overexpression and/or over-activation of ATP-dependent drug efflux transporters is a key mechanism underlying MDR development. Moreover, enhancement of drug metabolism, changes in drug targets and aberrant activation of the main signaling pathways, including Wnt, Akt and NF-κB are also responsible for MDR.

Methods: In this study, we have reviewed the roles of Wnt signaling in MDR as well as its potential therapeutic significance. Pubmed and Scopus have been searched using Wnt, β-catenin, cancer, MDR and multidrug resistance as keywords. The last search was done in March 2019. Manuscripts investigating the roles of Wnt signaling in MDR or studying the modulation of MDR through the inhibition of Wnt signaling have been involved in the study. The main focus of the manuscript is regulation of MDR related transporters by canonical Wnt signaling pathway.

Result And Conclusion: Wnt signaling has been involved in several pathophysiological states, including carcinogenesis and embryonic development. Wnt signaling is linked to various aspects of MDR including P-glycoprotein and multidrug resistance protein 1 regulation through its canonical pathways. Aberrant activation of Wnt/β- catenin signaling leads to the induction of cancer MDR mainly through the overexpression and/or over-activation of MDR related transporters. Accordingly, Wnt/β-catenin signaling can be a potential target for modulating cancer MDR.
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http://dx.doi.org/10.2174/1381612825666191112142943DOI Listing
July 2020

Genetic diversity and antifungal susceptibility patterns of Aspergillus nidulans complex obtained from clinical and environmental sources.

Mycoses 2020 Jan 3;63(1):78-88. Epub 2019 Nov 3.

Medical Mycology Reference Laboratory, National Center for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

The molecular epidemiology and antifungal susceptibility of Aspergillus nidulans species complex has not been well studied. To evaluate the genetic diversity and antifungal susceptibility patterns of clinical and environmental isolates of A. nidulans complex. Sixty clinical and environmental isolates of Aspergillus section Nidulantes were collected from five countries (Iran, The Netherlands, Spain, Portugal and Greece). The species were molecularly identified by sequencing of β-tubulin gene. The genetic diversity of A nidulans complex isolates (n = 54) was determined with a microsatellite genotyping assay. Antifungal susceptibility profile was determined using EUCAST method. The isolates were classified as A nidulans (46.7%), A spinulosporus (26.6%), A quadrilineatus (10%), A pachycristatus (3.3%), A rugulosus (3.3%), A unguis (5%), A creber, (1.7%), A olivicola (1.7%) and A sydowii (1.7%). Thirty-four sequence types (STs) were identified among the 54 A nidulans complex isolates. A high level of genetic diversity was found among A nidulans sensu stricto strains but low diversity was found among A spinulosporus strains. Amphotericin B showed high MICs to all species. The most active azole was posaconazole (GM = 0.64 mg/L), while itraconazole showed the highest MICs among azoles (GM = 2.95 mg/L). A spinulosporus showed higher MICs than A nidulans sensu stricto for all antifungals except for micafungin and anidulafungin. Interspecies variations may result in differences in antifungal susceptibility patterns and challenge antifungal therapy in infections caused by A nidulans. Differences in the distribution of STs or persistence of multiple STs might be related to the sources of isolation and niche specialisation.
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http://dx.doi.org/10.1111/myc.13019DOI Listing
January 2020

Effects of Benzo(a)pyrene on the endometrial receptivity and embryo implantation in mice: An experimental study.

Int J Reprod Biomed 2018 Dec 28;16(12). Epub 2019 Jan 28.

Department of Immunology, Faculty of Medicine, Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran.

Background: Benzo(a)pyrene (BaP) as an environmental pollutant is ubiquitous in the environment and it has destructive effects on human health. So far, various studies have demonstrated that BaP can cause adverse effects on the female reproductive system, but the existing information is limited about the effects of BaP on the endometrial receptivity and embryo implantation.

Objective: The aim of this study was to investigate the effects of BaP on the endometrial receptivity and implantation in mice.

Materials And Methods: In this experimental study, 40 pregnant BALB/c mice were divided into 5 groups ( = 8/each) as follows: experimental groups received the doses of 100 µg/kg, 200 µg/kg, and 500 µg/kg BaP dissolved in corn oil, the control group received normal saline and sham group received corn oil. Pregnant mice administered these solutions from Day 1 to Day 5 of gestation by gavage. On Day 6, the mice were sacrificed. Then their embryos were counted and the hormonal, histomorphological and molecular analyses were performed on the mocusa of uterine tube.

Results: The data revealed that BaP reduces estrogen and progesterone levels, decreases the number of implantation site, endometrium thickness, uterine lumen diameter, stromal cells and endometrial glands, and blood vessels in the endometrium. However, the expression of Activin receptor-like kinase 5 and E-cadherin genes was not changed by BaP with different doses.

Conclusion: The finding of this study showed that BaP can change estrogen and progesterone levels, and endometrial morphology leads to impairing the endometrial receptivity and decreasing the number of implantation site.
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http://dx.doi.org/10.18502/ijrm.v16i12.3680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600280PMC
December 2018

Status of insecticide resistance and its biochemical and molecular mechanisms in Anopheles stephensi (Diptera: Culicidae) from Afghanistan.

Malar J 2019 Jul 26;18(1):249. Epub 2019 Jul 26.

Liverpool School of Tropical Medicine, Liverpool, UK.

Background: Insecticide resistance of Anopheles stephensi, the main malaria vector in eastern Afghanistan, has been reported previously. This study describes the biochemical and molecular mechanisms of resistance to facilitate effective vector control and insecticide resistance management.

Methods: Mosquito larvae were collected from the provinces of Kunar, Laghman and Nangarhar from 2014 to 2017. The susceptibility of the reared 3-4 days old adults was tested with deltamethrin 0.05%, bendiocarb 0.1%, malathion 5%, permethrin 0.75% and DDT 4%. Cytochrome P450 content and general esterase, glutathione S-transferase (GST) and acetylcholinesterase (AChE) activities were measured in the three field populations and the results were compared with those of the laboratory susceptible An. stephensi Beech strain. Two separate allele-specific PCR assays were used to identify L1014, L1014F and L1014S mutations in the voltage gated sodium channel gene of An. stephensi. Probit analysis, ANOVA and Hardy-Weinberg equilibrium were used to analyse bioassay, biochemical assay and gene frequency data respectively.

Results: The population of An. stephensi from Kunar was susceptible to bendiocarb, apart from this, all populations were resistant to all the other insecticides tested. The differences between all values for cytochrome P450s, general esterases, GSTs and AChE inhibition rates in the Kunar, Laghman and Nangarhar populations were statistically significant when compared to the Beech strain, excluding GST activities between Kunar and Beech due to the high standard deviation in Kunar. The three different sodium channel alleles [L1014 (wild type), L1014F (kdr west) and L1014S (kdr east)] were all segregated in the Afghan populations. The frequencies of kdr east mutation were 22.9%, 32.7% and 35% in Kunar, Laghman and Nangarhar populations respectively. Kdr west was at the lowest frequency of 4.44%.

Conclusions: Resistance to different groups of insecticides in the field populations of An. stephensi from Kunar, Laghman and Nangarhar Provinces of Afghanistan is caused by a range of metabolic and site insensitivity mechanisms, including esterases, cytochrome P450s and GSTs combined with AChE and sodium channel target site insensitivity. The intensity and frequency of these mechanisms are increasing in these populations, calling for urgent reorientation of vector control programmes and implementation of insecticide resistance management strategies.
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http://dx.doi.org/10.1186/s12936-019-2884-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660931PMC
July 2019

Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice.

Microb Pathog 2019 Sep 15;134:103601. Epub 2019 Jun 15.

Department of Medical Parasitology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; National Institutes for Medical Research Development (NIMAD), Tehran, Iran. Electronic address:

Toxoplasma gondii, the etiological agent of toxoplasmosis, can cause severe or lethal damages in both animals and man. So, tends to develop a more effective vaccine to prevent this disease is extremely needed and would be so prominent. The novel dense granule antigen 14 (GRA14) has been identified as a potential vaccine candidate against T. gondii infection. The aim of this study was evaluation of protective immunity induced by prime/boost vaccination strategy of GRA14 antigen with calcium phosphate (CaPNs) or Aluminum hydroxide (Alum) nano-adjuvants in BALB/c mice. The finding showed that immunization with the prime-boost strategy using plasmid DNA (pcGRA14) and recombinant protein (rGRA14) with nano-adjuvants significantly elicited levels of specific IgG antibodies and cytokines against T. gondii infection. Given that, there were the high levels of total IgG, IgG2a, IFN-γ in mice of rGRA14-CaPNs and pcGRA14 + rGRA14-CaPNs groups, which indicating a Th-1 type response. While immunization of mice with Alum based rGRA14 and pcGRA14 + rGRA14 elicited specific IgG1 and IL-4 levels, which was confirmed a Th-2 type response. Mice immunized with DNA prime-protein boost vaccine with nano-adjuvants produce more vigorous specific lymphoproliferative responses than mice immunized with other antigen formulations. In addition, the CaPNs-based prime-boost vaccine of pcGRA14 + rGRA14 showed the longest survival time in mice and the lowest parasitic load in their brain tissue compared to the other groups. The results obtained in this study show that the use of GRA14 based DNA prime-protein boost vaccination regime with CaPNs can dramatically enhanced both humoral and cellular immune responses. Therefore, this strategy can provide a promising approach to the development of an effective vaccine against T. gondii infection in the future.
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http://dx.doi.org/10.1016/j.micpath.2019.103601DOI Listing
September 2019

In silico analysis and expression of a novel chimeric antigen as a vaccine candidate against Toxoplasma gondii.

Microb Pathog 2019 Jul 9;132:275-281. Epub 2019 May 9.

Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran. Electronic address:

Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.
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http://dx.doi.org/10.1016/j.micpath.2019.05.013DOI Listing
July 2019

Novel Point Mutations in and Genes Associated with Itraconazole and Posaconazole Resistance in Isolates.

Microb Drug Resist 2019 Jun 18;25(5):652-662. Epub 2019 Jan 18.

1 Invasive Fungi Research Center (IFRC), School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

is a common environmental species known to cause occupational allergic disease in grain handlers. We have recently observed azole-resistant isolates of this fungus as a cause of onychomycosis. To further characterize the cause of resistance, the genes encoding 14 α-sterol demethylase enzyme ( and ) were characterized and analyzed in 9 ITC-susceptible isolates and 6 isolates with high minimum inhibitory concentrations (MICs) of clinical (nail and sputum) and environmental strains. We found that six isolates with itraconazole MIC >16 mg/L demonstrated nonsynonymous mutations, including V51I, L378P, E483K, and E506G, and synonymous mutations, including F53F, A186A, Q276Q, and H359H. Moreover, P486S was detected in five strains with ITR MIC >16 mg/L. One mutation, F324S, was detected in an isolate with posaconazole MIC >16 mg/L. The effect of E483K and P486S mutations of CYP51A on azole resistance was further investigated using homology modeling and molecular dynamics. We found that E483K and P486S mutations were located near the ligand access channel of CYP51A that could partly lead to narrowing the entry of the ligand access channels. Therefore, we concluded that E483K and P486S mutations may potentially contribute to the limited access of inhibitors to the binding pocket and therefore confer resistance to azole agents.
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http://dx.doi.org/10.1089/mdr.2018.0300DOI Listing
June 2019

Molecular Modeling and Structural Stability of Wild-Type and Mutant CYP51 from Leishmania major: In Vitro and In Silico Analysis of a Laboratory Strain.

Molecules 2018 Mar 19;23(3). Epub 2018 Mar 19.

Molecular and Cell Biology Research Center (MCBRC), Faculty of Medicine, Mazandaran University of Medical Sciences, Sari 48157-33971, Iran.

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. is the most common cause of cutaneous leishmaniasis. In the parasites, sterol 14α-demethylase (CYP51), which is involved in the biosynthesis of sterols, has been identified as an attractive target for development of new therapeutic agents. In this study, the sequence and structure of CYP51 in a laboratory strain (MRHO/IR/75/ER) of were determined and compared to the wild-type strain. The results showed 19 mutations including seven non-synonymous and 12 synonymous ones in the CYP51 sequence of strain MRHO/IR/75/ER. Importantly, an arginine to lysine substitution at position of 474 resulted in destabilization of CYP51 (ΔΔG = 1.17 kcal/mol) in the laboratory strain; however, when the overall effects of all substitutions were evaluated by 100 ns molecular dynamics simulation, the final structure did not show any significant changes (-value < 0.05) in stability parameter of the strain MRHO/IR/75/ER compared to the wild-type protein. The energy level for the CYP51 of wild-type and MRHO/IR/75/ER strain were -40,027.1 and -39,706.48 Kcal/mol respectively. The overall Root-mean-square deviation (RMSD) deviation between two proteins was less than 1 Å throughout the simulation and Root-mean-square fluctuation (RMSF) plot also showed no substantial differences between amino acids fluctuation of the both protein. The results also showed that, these mutations were located on the protein periphery that neither interferes with protein folding nor with substrate/inhibitor binding. Therefore, strain MRHO/IR/75/ER is suggested as a suitable laboratory model for studying biological role of CYP51 and inhibitory effects of sterol 14α-demethylase inhibitors.
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http://dx.doi.org/10.3390/molecules23030696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6017637PMC
March 2018

Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4) of Tachyzoite of RH Strain.

Iran J Parasitol 2017 Oct-Dec;12(4):498-505

Toxoplasmosis Research Center (TRC), Mazandaran University of Medical Sciences, Sari, Iran.

Background: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 () proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of in an appropriate expression vector and eukaryotic cell for production of recombinant protein.

Methods: RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on gene sequence with I and RI restriction sites at 5' end of forward and reverse primers, respectively. gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.

Results: Cloning of gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.

Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of gene from tachyzoites used as antigenic component for serological assay and vaccine development.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5756299PMC
January 2018

Association between interleukin-32 polymorphism and multiple sclerosis.

J Neurol Sci 2017 Aug 23;379:144-150. Epub 2017 May 23.

Molecular and Cell Biology Research Center, Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran.

Background And Aim: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Environmental and genetic factors play a key role in the development of the disease. Interleukin-32 (IL-32) is a cytokine inducing crucial inflammatory cytokines such as TNF-α, IL-6, IL-1β, and MIP-2. The present study was an attempt to reveal any association between IL-32 levels and C/T promoter SNP with susceptibility to MS.

Methods: This case control study recruited a total of 304 subjects including 132 MS patients and 172 sex- and age-matched healthy controls. Clinical and epidemiological characteristics of the RRMS, PPMS, and PPMS populations were assessed. Serum levels and C/T polymorphism of IL-32 were determined by ELISA and RFLP-PCR methods, respectively.

Results: Serum levels of IL-32 were significantly different between MS patients and controls. IL-32 was dramatically higher in the patients than that healthy controls (2297.4±280.2 ver. 712.9±90.2, p=0.001). C allele was prominent in MS patients than the controls and might increase the risk of MS up to 1.6 fold (95% CI; 1.02-2.4, p=0.038). In addition, the presence of C allele enhanced IL-32 production drastically.

Conclusion: This is the first study in which IL-32 gene promoter C/T polymorphism and its serum levels were investigated. The increase in serum levels of IL-32 in accordance with additive effect of the presence of C allele in MS patients might introduce IL-32 as a key player in MS pathogenesis or immunedysregulation.
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http://dx.doi.org/10.1016/j.jns.2017.05.045DOI Listing
August 2017

Avian gametologs as molecular tags for sex identification in birds of prey of Iran.

Zoo Biol 2017 Jul 30;36(4):289-293. Epub 2017 Jun 30.

Faculty of Science, Department of Biology, Ferdowsi University of Mashhad, Mashhad, Iran.

Global environmental change and rapid destruction of natural habitats necessitate the conservation of endangered and threatened birds of prey. Recently, molecular sex identification methods based on amplification of introns of chromodomain-helicase DNA binding protein1 (CHD1) have provided valuable tools for ecological study and conservation breeding programs of birds. These methods employ a primer pair flanking an intron which varies considerably in length between the avian gametologs CHD1Z and CHD1W. Herein, we test the applicability of CHD1Z and CHD1W as universal tags for molecular sex identification in birds of prey of Iran. We showed successful sex identification in 22 species of birds of prey using feathers as the source of DNA. The results suggest that the regions of CHD1W and CHD1Z amplified in this study are conserved among most of Falconiformes, enabling accurate sex identification in birds of prey.
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http://dx.doi.org/10.1002/zoo.21363DOI Listing
July 2017

Differential expression of glutamate transporters in cerebral cortex of paraoxon-treated rats.

Neurotoxicol Teratol 2017 Jul 8;62:20-26. Epub 2017 Jun 8.

Department of Physiology & Pharmacology, Molecular and Cell Biology Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. Electronic address:

Glutamatergic system is involved in pathological effects of organophosphorus (OP) compounds. We aimed to determine in vivo effects of paraoxon, the bioactive metabolite of parathion, on the expression of glutamate transporters as well as Bax and Bcl2 in rat cerebral cortex. Male Wistar rats received an intraperitoneal (i.p.) injection of one of three doses of paraoxon (0.3, 0.7, or 1mg/kg) or corn oil as vehicle (1ml/kg). After 4 or 18h, cerebral cortices were dissected out and used for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot assays to measure mRNA and protein levels, respectively. The cortical glial glutamate transporters (GLAST and GLT-1) were up-regulated in animals treated with 0.7mg/kg of paraoxon, but down-regulated in 1mg/kg group. Neuronal glutamate transporter (EAAC1) was unchanged in 0.7mg/kg treated rats, while reduced in 1mg/kg group. No significant difference was found in the mRNA and protein expression of EAAC1 in animals intoxicated with 0.3mg/kg of paraoxon. Paraoxon (1mg/kg) resulted in an up-regulation of Bax and down-regulation of Bcl2 mRNA levels in the rat cerebral cortex. These results indicate that paraoxon can differentially regulate expression of glutamate transporters at mRNA and protein levels in the cerebral cortex. Changes in the expression of glutamate transporters are closely related to paraoxon-induced seizure activity.
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http://dx.doi.org/10.1016/j.ntt.2017.06.001DOI Listing
July 2017

Enhancing immune responses to a DNA vaccine encoding Toxoplasma gondii GRA14 by calcium phosphate nanoparticles as an adjuvant.

Immunol Lett 2017 05 9;185:40-47. Epub 2017 Mar 9.

Toxoplasmosis Research Center, Mazandaran University of Medical Sciences, Sari, Iran. Electronic address:

Several approaches have been used to improve the immunogenicity of DNA vaccines. In the current study, we constructed the plasmid encoding T. gondii dense granule 14 (GRA14) and investigated the immunological properties of calcium phosphate nanoparticles (CaPNs) as nano-adjuvant to enhance the protective efficacy of pcGRA14. BALB/c mice intramuscularly injected three times at two-week intervals and the immune responses were evaluated using lymphocyte proliferation assay, cytokine and antibody measurements, survival times, and parasite load of mice challenged with the virulent T. gondii RH strain. The results showed that the immune responses were induced in mice receiving pcGRA14 DNA vaccine. Interestingly, pcGRA14 coated with nanoparticles led to statistically significant enhancements of cellular and humoral immune responses against Toxoplasma infection (P<0.05). After challenge with RH strain of T. gondii, immunized mice with pcGRA14 showed prolong survival time compared to control groups (P<0.05). In addition, pcGRA14 coated with nano-adjuvant exhibited the lowest parasitic load in the infected mice tissues. For the first time, our data indicate that the pcGRA14 coated with CaPN was more effective for stimulation of immune responses and should be considered as an adjuvant in the design of vaccines against toxoplasmosis.
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http://dx.doi.org/10.1016/j.imlet.2017.03.006DOI Listing
May 2017

Effects of moderate treadmill exercise and fluoxetine on behavioural and cognitive deficits, hypothalamic-pituitary-adrenal axis dysfunction and alternations in hippocampal BDNF and mRNA expression of apoptosis - related proteins in a rat model of post-traumatic stress disorder.

Neurobiol Learn Mem 2017 Mar 28;139:165-178. Epub 2017 Jan 28.

Laboratory of Learning and Memory, Research Center and Department of Physiology, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran. Electronic address:

Post-traumatic stress disorder (PTSD) is a condition that develops after an individual has experienced a major trauma. Currently, selective serotonin reuptake inhibitors (SSRIs) like fluoxetine are the first-line choice in PTSD drug treatment but their moderate response rates and side effects indicate an urgent need for the development of new treatment. Physical activity is known to improve symptoms of certain neuropsychiatric disorders. The present study investigated the effects of moderate treadmill exercise, the antidepressant fluoxetine and the combined treatment on behavioural deficits, and hypothalamic-pituitary-adrenal (HPA) axis dysfunction. We also examined alternations in hippocampal brain-derived neurotrophic factor (BDNF) and mRNA expression of apoptosis - related proteins in a rat model of PTSD: the single prolonged stress (SPS) model. Rats were exposed to SPS (restraint for 2h, forced swimming for 20min and ether anaesthesia) and were then kept undisturbed for 14days. After that, SPS rats were subjected to chronic treatment with fluoxetine (10mg/kg/day, for 4weeks), moderate treadmill running (4weeks, 5day per week) and the combined treatment (fluoxetine plus treadmill exercise), followed by behavioural, biochemical and apoptosis markers assessments. SPS rats exhibited increased anxiety levels in the elevated plus maze and light/dark box, impaired fear conditioning and extinction in inhibitory avoidance (IA) task, impaired spatial memory in a recognition location memory task and enhanced negative feedback on the HPA axis following a dexamethasone suppression test. SPS rats also showed reduced hippocampal BDNF and enhanced apoptosis. Moderate treadmill exercise, fluoxetine and the combined treatment alleviated the SPS-induced alterations in terms of anxiety levels, HPA axis inhibition, IA conditioning and extinction, hippocampal BDNF and apoptosis markers. Furthermore, the combined treatment was more effective than fluoxetine alone, but in most tests, the effects of the combined treatment were similar to those of exercise alone, suggesting that exercise is the main factor in the beneficial effects of the combined therapy in PTSD patients.
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http://dx.doi.org/10.1016/j.nlm.2017.01.009DOI Listing
March 2017

A systematic review and meta-analysis of seroprevalence of varicella zoster virus: A nationwide population-based study.

J Clin Virol 2017 02 6;87:49-59. Epub 2016 Dec 6.

SEC Faculty, Penrhyn Road, Kingston University London, Kingston upon Thames, KT1 2EE, UK. Electronic address:

Varicella zoster virus (VZV) causes chicken pox as a primary infection following which it becomes latent in neurons. It may then reactivate to cause shingles (herpes zoster). Severity of lesions and VZV pathogenicity are depended on the host's immune response and variant in VZV Dr Athina Myrto ChioniIdentification of VZV seroprevalance rate in general population may lead to develop new health strategic managements such as vaccination. Therefore, we aimed to provide a systematic review of the seroprevalence of VZV infection among Iranian population and estimate age- and gender- specific prevalence of VZV. Keywords "seroprevalence"; "varicella zoster virus" and "Iran"; were searched in international electronic databases and also in national Persian databases. Twenty two pooled studies among 262 total studies containing (240 published articles; 18 dissertations; and 4 proceedings abstracts) from 1992 to 2014 with total sample size of 7867 individuals were included in the final review. Data was analyzed using random effect method. The heterogeneity was calculated using I-square statistics The overall IgG seroprevalence rate of VZV infection in general population of Iran was 78.50% (95% CI; 77.74%-79.25%). There was significant heterogeneity among the studies (P<0.0001; I=99.4%). Furthermore, the relative risk of VZV infection is high in females (80.47%, 95% CI; 79.40%-81.54%) and older adults (95.30%, 95% CI; 94.11% -96.48%). Our results may represent a true background and estimation of VZV infection in Iran and generate the cost-benefits immunization program. Moreover, the ensuing data suggests further attention on disease seroprevalence in order to obtain efficient data for therapeutic intervention targeted against VZV.
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http://dx.doi.org/10.1016/j.jcv.2016.12.001DOI Listing
February 2017

Immunological evaluation of a DNA cocktail vaccine with co-delivery of calcium phosphate nanoparticles (CaPNs) against the Toxoplasma gondii RH strain in BALB/c mice.

Parasitol Res 2017 Feb 2;116(2):609-616. Epub 2016 Dec 2.

Toxoplasmosis Research Center (TRC), Mazandaran University of Medical Sciences, Sari, Iran.

Many recent studies have been conducted to evaluate protective immunity mediated by DNA vaccines against toxoplasmosis. Cocktail DNA vaccines showed better immune responses compared to single vaccines. The objective of the current study was to evaluate the protective efficacy of rhomboid 4 (ROM4) and cocktail DNA vaccines (ROM4 + GRA14) of the Toxoplasma gondii RH strain with or without coated calcium phosphate nanoparticles (CaPNs) as the adjuvant to improve the immunogenicity against the T. gondii RH strain in BALB/c mice. Cocktail DNA vaccines of pcROM4 + pcGRA14 of the T. gondii RH strain were constructed. CaPNs were synthesized and the cocktail DNA vaccine was coated with the adjuvant of CaPNs. Immunogenicity and the protective effects of cocktail DNA vaccines with or without CaPNs against lethal challenge were evaluated in BALB/c mice. pcROM4 and cocktail DNA vaccine coated with CaPNs significantly enhanced cellular and humoral immune responses against Toxoplasma compared to pcROM4 and cocktail DNA vaccine without CaPNs (p < 0.05). These findings indicate that the survival time of immunized mice after challenge with the RH strain of T. gondii was increased compared to that of controls and the DNA vaccine provided significant protection in mice (p < 0.05). The CaPN-based cocktail DNA vaccine of pcROM4 + pcGRA14 showed the longest survival time compared to the other groups. Co-immunization with CaPN-based cocktail DNA vaccine (pcROM4 + pcGRA14) boosted immune responses and increased the protective efficacy against acute toxoplasmosis in BALB/c mice compared to both single gene and bivalent DNA vaccine without nano-adjuvants.
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http://dx.doi.org/10.1007/s00436-016-5325-6DOI Listing
February 2017

Alterations in mRNA and protein expression of glutamate transporters in rat hippocampus after paraoxon exposure.

Neurotoxicology 2016 12 18;57:251-257. Epub 2016 Oct 18.

Department of Immunology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran; Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran. Electronic address:

Organophosphates affect brain function through a variety of mechanisms beyond their shared role as cholinesterase inhibitors. The aim of the current study was to investigate the changes in the expression of glial (GLAST and GLT-1) and neuronal (EAAC1) glutamate transporters at mRNA and protein levels in paraoxon-treated rat hippocampus. Adult male Wistar rats were intraperitoneally treated with either vehicle (corn oil) or one of three dosages of paraoxon (0.3, 0.7 or 1mg/kg). After 4 or 18h, both hippocampi of each rat were collected to detect mRNA and protein expression of glutamate transporters using the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting, respectively. Animals treated with 0.3mg/kg paraoxon showed no difference in mRNA and protein levels of the glutamate transporters when compared with control group. At 4h after exposure with 0.7 and 1mg/kg paraoxon, the expression of GLAST and GLT-1 increased at mRNA and protein levels and remained elevated after 18h. No difference in the expression of EAAC1 at mRNA and protein levels was observed in any paraoxon-treated groups compared with the control group. This study showed an increased expression of glial (GLAST and GLT-1), but not neuronal (EAAC1) glutamate transporters, in adult rat hippocampus following administration of convulsive dosages of paraoxon. These suggest a protective and compensatory adaptation for effective uptake of glutamate in hippocampus induced by paraoxon and thus attenuating seizure activity.
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http://dx.doi.org/10.1016/j.neuro.2016.10.009DOI Listing
December 2016

Comparison of leucine-rich repeat-containing G protein-coupled receptor 5 expression in different cancer and normal cell lines.

Biomed Rep 2016 Jul 20;5(1):130-132. Epub 2016 May 20.

Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari 48175-861, Iran.

Evaluating the expression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) may be useful for predicting the best models and achieving more accurate results in cancer research. Therefore, the aim of the present study was to analyze the LGR5 expression levels in different cell lines. Eight commonly used cell lines were assessed (COS-7, NIH3T3, HEK293, VERO, HeLa, BHK, HepG2 and AGS). All the cell lines were cultured in RPMI-1640 medium contain 10% fetal calf serum at 37°C in humidified conditions with 5% CO. According to the western blotting results, LGR5 was expressed in all cell lines. Densitometry results of LGR5 expression in the different cell lines showed that high LGR5 expression levels were apparent in BHK, AGS, VERO and NIH3T3 cell lines compared with the other cell lines. The results indicate that for the normal and cancer cell lines, BNK and AGS may be a better choice, respectively, for cancer studies.
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http://dx.doi.org/10.3892/br.2016.684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907073PMC
July 2016

Effect of First Line Gastric Cancer Chemotherapy Regime on the AGS Cell Line - MTT Assay Results.

Asian Pac J Cancer Prev 2016 ;17(1):131-3

Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences, Sari, Iran E-mail :

Background: Combination chemotherapy regimes are common treatments for cancer. The aim of this study was to evaluation the effect of individual chemotherapeutic agents in comparison with a first line chemotherapy regime treatment in the AGS gastric cancer cell line by MTT assay.

Materials And Methods: In this experimental study, AGS cells were grown in RPMI-1640 supplemented with 10% fetal calf serum and 100 IU/ml penicillin, and 10 μg/ml streptomycinin, under a humidified condition at 37° with 5% CO2. All cells were washed with PBS and detached with trypsin, centrifuged and 8000 cells re-plated on to 96- well plates. LD50 doses of Epirubicin, Cisplatin and 5-fluorouracil were added to each well in mono or triple therapy. Anti-proliferative activities were determined by MTT assay after 24, 48 or 72 h.

Results: Results of MTT assays showed that there were no significant differences among 3 drugs in monotherapy (p=0.088), but there was significant difference between combination therapy with epirubicin (P=0.031) and 5FU (p=0.013) on cell survival at 24 h. After 48 and 72 hours, cell viability showed significant differences between the 3 drugs (p=0.048 and P=0.000 for 48 and 72 h, respectively) and there was significant difference between combination therapy with epirubicin (P=0.035 and P=0.002 for 48 and 72 h, respectively).

Conclusions: The results showed no significant differences between these chemotherapy drugs each given alone, but combination therapy with 3 drugs had significant effects on cell viability in comparison with epirubicin alone.
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http://dx.doi.org/10.7314/apjcp.2016.17.1.131DOI Listing
December 2016

Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR.

Data Brief 2015 Sep 6;4:384-9. Epub 2015 Jul 6.

Molecular and Cell Biology Research Center (MCBRC), Department of Immunology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Mazandaran, Iran.

Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5' end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays.
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http://dx.doi.org/10.1016/j.dib.2015.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510554PMC
September 2015

VERO stable cell lines expressing full-length human epidermal growth factor receptors 2 and 3: platforms for subtractive phage display.

DNA Cell Biol 2015 Sep 29;34(9):573-8. Epub 2015 Jun 29.

1 Molecular and Cell Biology Research Center, Mazandaran University of Medical Sciences , Sari, Iran .

Cross-talk between human epidermal growth factor receptor 2 and 3 (HER2 and HER3) may potentially contribute to therapeutic resistance in human breast cancer. Subtractive phage display allows highly specific selection for antibody fragments directed against cells surface HER2 and HER3. The strategies to select conformation- and activation-specific antibodies against HER2 and HER3 require tightly regulated HER2 and HER3 expressing cells that allow controlled activation/inactivation of these receptors during panning procedures. To achieve this, first, we found that the VERO cell line is an appropriate cell line for heterogeneous expression of HER2 and HER3, and then we established a panel of VERO stable cell lines expressing high levels of HER2 and HER3 alone and in combination. We also showed that HER2 and HER3 expressed in VERO cells were biologically active and could form heterodimer following neuregulin1 treatment. The cell line established here not only provided platforms for phage display-based methods but also could be used in any HER-related studies.
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http://dx.doi.org/10.1089/dna.2015.2917DOI Listing
September 2015