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Abstract Background Aedes aegypti is the main vector of arboviral diseases worldwide. The species invaded and became established in southern Iran in 2020. Insecticide-based interventions are primarily used for its control. With insecticide resistance widespread, knowledge of resistance mechanisms is vital for informed deployment of insecticidal interventions, but information from Iranian Ae. aegypti is lacking. Methods Fifty-six Ae. aegypti specimens were collected from the port city of Bandar Lengeh in Hormozgan Province in the South of Iran in 2020 and screened for kdr mutations. The most common kdr mutations in Latin America and Asia (V410L, S989P, V1016G/I and F1534C), especially when present in combinations, are highly predictive of DDT and pyrethroid resistance were detected. Phylogenetic analyses based on the diversity of S989P and V1016G/I mutations were undertaken to assess the phylogeography of these kdr mutations. Results Genotyping all four kdr positions of V410L, S989P, V1016G/I and F1534C revealed that only 16 out of the 56 (28.57%) specimens were homozygous wild type for all kdr mutation sites. Six haplotypes including VSVF (0.537), VSVC (0.107), LSVF (0.016), LSIF (0.071), VPGC (0.257) and LPGC (0.011) were detected in this study. For the first time, 11 specimens harbouring the V410L mutation, and 8 samples with V1016I mutation were found. V410L and V1016I were coincided in 8 specimens. Also, six specimens contained 1016G/I double mutation which was not reported before. Conclusions The relatively high frequency of these kdr mutations in Iranian Ae. aegypti indicates a population exhibiting substantial resistance to pyrethroid insecticides, which are used widely in control operations and household formulations. The detection of the 410L/1016I kdr mutant haplotype in Iranian Ae. aegypti suggests possible convergence of invasive populations from West Africa or Latin America. However, as Iran has very limited maritime/air connections with those African countries, a Latin American origin for the invasive Ae. aegypti in Iran is more plausible. Graphical abstract

Background and Objectives: During the coronavirus pandemic, the overuse of antibiotics to reduce coinfections and mortality may be contributing to the rise of antimicrobial resistance. In this study, we aim to investigate the antibiotic resistance changes of Acinetobacter baumannii post-COVID-19 pandemic in Northern Iran. Materials and Methods: The current study is a cross-sectional study. Between 2022 and 2023, 2190 clinical samples were collected from patients with healthcare-associated infections (HAIs) at four hospitals in Sari, which served as corona centers after the COVID-19 pandemic. Antimicrobial sensitivity was determined using standard broth macro-dilution, and resistance genes were detected using multiplex PCR. Results: Based on the results co-amoxiclav had a resistance rate of 100%, while piperacillin/tazobactam showed the least resistance rate of 29.82%. In terms of GM MIC values, colistin was the most potent against multi-drug resistant isolates. The frequency of blaOXA-51, ampC, aphA6, and blaNDM genes were 100%, 99.12%, 90.35%, and 69.30% respectively. Conclusion: Our study revealed high multi-drug resistance rates. Piperacillin/tazobactam recommended for treating multidrug resistant Acinetobacter baumannii infections in Northern Iran.

Background and objectivesThis study aimed to investigate the possible prognostic significance of interferon alpha–beta receptor subunit 2 (IFNAR2) and tyrosine kinase 2 (TYK2) expressions.MethodsWe conducted a retrospective study including COVID-19 adult patients. All blood samples were collected before any interventions. The expressions of IFNAR2 and TYK2 were assessed using real-time PCR in venous blood samples of 54 cases and 56 controls. The transcript quantities of IFNAR2 and TYK2 genes were assessed using a Delta-Ct method.ResultsOur findings show no significant differences in gene expression levels for IFNAR2 and TYK2 between patients who required oxygen (O2) therapy and those who did not (p-value = 0.732 and p-value = 0.629, respectively). Likewise, there were no significant differences in IFNAR2 and TYK2 expressions between patients hospitalized for less than 7 days and those hospitalized for 7 days or more (p-value = 0.455 and p-value = 0.626, respectively). We also observed a weak correlation between IFNAR2 expression and CRP (p-value = 0.045, r = 0.192). There was a negative correlation between the expression levels of IFNAR2 and TYK2 transcripts in COVID-19 patients (p-value = 0.044; partial correlation coefficient = -0.283). Additionally, IFNAR2 and TYK2 were significantly downregulated in the COVID-19 group compared to healthy subjects (p-value = 0.002 and p-value = 0.028, respectively). However, neither IFNAR2 nor TYK2 expression was significantly different between the case subgroups based on COVID-19 severity. The IFNAR2 ΔΔCt (B = -0.184, 95% CI: -0.524–0.157, p-value = 0.275) and the TYK2 ΔΔCt (B = 0.114, 95% CI: -0.268–0.496, p-value = 0.543) were not found to be significant predictors of hospitalization duration. The area under the curve (AUC) for IFNAR2 expression is 0.655 (p-value = 0.005, 95% CI: 0.554–0.757), suggesting its poor discriminative value.ConclusionWe were unable to comment definitively on the prognostic power of IFNAR2 and TYK2 expressions in COVID-19 patients, and larger-scale studies are needed. The principal limitations of this study included the lack of longitudinal analysis and limited sample size.

Objective(s): Exhausted CD8+ T-cells over-express immune checkpoint receptors (ICRs), which interact with their ligands on malignant cells. However, some ICRs have been reported to be expressed on both T-cells and tumor cells, including V-domain immunoglobulin suppressor of T cell activation (VISTA), Galectin-9, and T-cell immunoglobulin mucin-3 (TIM-3). We aimed to evaluate the mRNA expression of VISTA, Galectin-9, and TIM-3 on CD8+ T-cells and leukemic cells in B-cell acute lymphoblastic leukemia (B-ALL).Materials and Methods: Samples were obtained from 26 untreated B-ALL patients and 25 control subjects. CD8+ T-cells were isolated using Magnetic Activated Cell Sorting (MACS). Relative gene expression was then evaluated by qRT-PCR with specific primers for VISTA, Galectin-9, and TIM-3. Also, the mRNA expression profile and clinical data of 154 B-ALL patients were obtained from the TARGET.Results: mRNA expression of Galectin-9 on CD8+ T-cells in B-ALL patients was significantly lower than those in the control group (P=0.043), while VISTA expression was not significantly different between the two study groups (P=0.259). Besides, TIM-3 expression was significantly higher in B-ALL patients than in the control group (P

Background and purpose: Breast cancer is one of the most common cancers among women. Abnormal expression of microRNAs is associated with cancer. Therefore, this study aimed to evaluate the expression of miRNA-210 in the serum of breast cancer patients. Materials and methods: The studied population included 49 breast cancer patients and 55 healthy individuals, and the samples were evaluated using Real Time PCR. Results: The data analysis of this study revealed that the expression level of miR-210 in the blood of breast cancer patients is significantly higher compared to the blood of healthy people. Also, there is a negative correlation between the age of breast cancer patients and the expression of miR-210 (r=-0.309, P=0.031) and a significant positive correlation between the increase in the expression of mir-210 and the expression of the Ki-67 marker (r=0.412, P = 0.004).

Background: Thymocyte selection-associated high mobility group box protein (TOX) and members of the nuclear receptor 4A (NR4A) are known as transcription factors involved in T cell exhaustion.Objective: To evaluate the mRNA expression of TOX and NR4A1-3 in CD8+ T cells in acute leukemia.Methods: Blood samples were obtained from 21 ALL and 6 AML patients as well as 20 control subjects. CD8+ T cells were isolated using MACS. Relative gene expression of TOX and NR4A1-3 was then evaluated using qRT-PCR.Results: Comparison of mRNA expression of TOX in CD8+ T cells showed no significant difference among the study groups (p>0.05), while the expression of NR4A1 was significantly lower in AML patients than in the control group (p=0.0006). Also, the expression of NR4A2 and NR4A3 was significantly lower in both ALL (p=0.0049 and p=0.0005, respectively) and AML (p=0.0019 and p=0.0055, respectively) patients.Conclusion: NR4As expressions were found to be lower in CD8+ T cells from patients with AML and ALL compared to controls, whereas the mRNA expression of TOX showed no significant difference. Although TOX and NR4As are associated with CD8+ T cell exhaustion in solid tumors, they might play different roles in acute leukemia, which requires further investigation.

Background and purpose: Pseudomonas aeruginosa is a significant contributor to hospital-acquired infections, including urinary infections, bacteremia, pneumonia, and burn wound infections. Due to the significant role of carbapenems in treating infections that are resistant to conventional antibiotics, it is necessary to identify the prevalence and resistance patterns of pathogens that cause hospital infections. This will help to prevent the spread of microbial resistance, and subsequently, reduce the death rate caused by such infections. Therefore, the aim of the present study is to determine the occurrence of metallo-beta-lactamase genes in Pseudomonas aeruginosa isolates in Sari hospitals. Materials and methods: This cross-sectional study included 150 isolates of Pseudomonas aeruginosa as clinical samples that were collected from patients from the burn department of Zare Hospital, Sari city. To determine the antibiotic sensitivity, the disk diffusion method was used. Additionally, PCR method was employed to examine the prevalence of beta-lactamase genes blaVIM-1 and blaIMP-1 in the isolates. Results: Based on the antibiotic sensitivity results, it was found that the highest resistance was observed to gentamicin, ceftazidime, imipenem, ciprofloxacin, amikacin, meropenem, cefepime, ceftriaxone, and calcitin. The data analysis also revealed that the frequencies of blaVIM-1 and bla IMP-1 genes were detected in isolates of 45% and 55%, respectively. Conclusion: The frequency of Pseudomonas aeruginosa strains carrying the MBLs genes is increasing in Zare Hospital in Sari. The findings of the study strongly suggest that there is necessary to prevent the experimental and indiscriminate administration of carbapenems in intensive care and burn departments of hospitals.

Candida auris, an emerging non-albicans multidrug-resistant yeast, has become a significant cause of invasive candidiasis in healthcare settings. So far, data on the metabolites of C. auris in different clades are minimal, and no studies have focused on clade V metabolites. Therefore, Gas chromatography–mass spectrometry (GC-MS) was used for the metabolomic profiling of clade I C. auris compared with fluconazole-resistant and susceptible C. auris in clade V strains. GC-MS chromatography revealed 28, 22, and 30 compounds in methanolic extracts of the fluconazole-susceptible and fluconazole-resistant C. auris clade V and C. auris clade I strain, respectively. Some compounds, such as acetamide and metaraminol, were found in fluconazole-susceptible and resistant C. auris clade V and clade I. N-methyl-ethanamine and bis(2-ethylhexyl) phthalate metabolites were found in both fluconazole -susceptible and resistant C. auris clade V, as well as 3-methyl-4-isopropylphenol, 3,5-bis(1,1-dimethyl)-1,2-benzenediol, and diisostyl phthalate metabolites in both fluconazole resistant C. auris clade V and I. Identifying these metabolites contributes to understanding the morphogenesis and pathogenesis of C. auris, highlighting their potential role in antifungal drug resistance and the control of fungal growth. However, further experiments are warranted to fully comprehend the identified metabolites’ regulatory responses, and there may be potential challenges in translating these findings into clinical applications.

Background: According to COVID-19 mutation and no defined treatment, it is necessary to find effective treatment. Chlorpromazine, a phenothiazine antipsychotic drug, has been shown in animal studies to have antiviral effects by inhibiting clathrin-mediated endocytosis. The aim of this study was to evaluate the effectiveness of adding chlorpromazine to the atazanavir/ritonavir regimen in the treatment of moderate COVID-19 patients. Methods: In this randomized double-blind clinical trial, sixty hospitalized patients with moderate COVID-19 confirmed by CT findings or polymerase chain reaction (PCR) were enrolled. All patients received atazanavir/ritonavir 300mg/100mg once daily. In two parallel groups, chlorpromazine 25 mg three times a day or a placebo was administered for up to 14 days. Complete blood count with differential, C-reactive protein (CRP), liver enzymes, and erythrocyte sedimentation rate was measured on days 1, 3, 5, 7, and 10. The primary outcome was the improvement of oxygen saturation and the secondary outcome was the duration of hospitalization and conversion of PCR test results. Results: Oxygen saturation during the hospitalization was not different among the two groups. The mean duration of hospitalization in the chlorpromazine group was 7.4±2.7 days and in the placebo was 8.2±3 days (P=0.2). Compared to baseline, both groups showed an increase in white blood cell count (P=0.04) and polymorphonuclear cells (P=0.04) but lymphocyte count decreased. At the end of the study, the PCR test was negative in 100% of patients in the chlorpromazine group and 95% of patients in the placebo group. Conclusion: In adult hospitalized patients with moderate symptomatic COVID-19, adding chlorpromazine to the atazanavir/ritonavir regimen did not improve outcomes.

Background: We aimed to design a B and T cell recombinant protein vaccine of Toxoplasma gondii with in silico approach. MIC13 plays an important role in spreading the parasite in the host body. GRA1 causes the persistence of the parasite in the parasitophorous vacuole. SAG1 plays a role in host-cell adhesion and cell invasion. Methods: Amino acid positions 73-272 from MIC13, 71-190 from GRA1, and 101-300 from SAG1 were selected and joined with linker A(EAAAK)A. The structures, antigenicity, allergenicity, physicochemical properties, as well as codon optimization and mRNA structure of this recombinant protein called MGS1, were predicted using bioinformatics servers. The designed structure was synthesized and then cloned in pET28a (+) plasmid and transformed into Escherichia coli BL21. Results: The number of amino acids in this antigen was 555, and its antigenicity was estimated to be 0.6340. SDS-PAGE and Western blotting confirmed gene expression and successful production of the protein with a molecular weight of 59.56kDa. This protein will be used in our future studies as an anti-Toxoplasma vaccine candidate in animal models Conclusion: In silico methods are efficient for understanding information about proteins, selecting immunogenic epitopes, and finally producing recombinant proteins, as well as reducing the time and cost of vaccine design.

Background and purpose: The mature form of brain-derived neurotrophic factor (BDNF) binds to BDNF/NT-3 growth factors receptor (Trk-B). This binding leads to activation of Ras–MAPK pathway which is integrated with cell growth and proliferation. The BDNF deficiency is correlated with various diseases and affects aging and miscellaneous. In the present study we aimed to design a chimeric LAMP-BDNF protein and assess its suitability to target neuronal cells employed in silico approaches. Materials and methods: The specific binding between the BDNF and Trk-B would be used to guide the drug loaded lysosomes in to brain tissue. So, the structure of the chimeric BDNF/LAMP2 protein was predicted and its quality was confirmed using bioinformatics tools. Then, the interaction between the structures of BDNF/LAMP2 protein and Trk-B was analyzed by protein-protein docking. The orientation of the interaction between these proteins was compared to orientation of Trk-B interaction with its specific antibody using structural superimposition. Results: Findings showed that the chimeric BDNF/LAMP2 protein could preserve the original structure of the BDNF molecule. Moreover, the chimeric BDNF/LAMP2 protein was found to be capable of interacting with Trk-B. The interaction orientation and binding affinity for BDNF/LAMP2 protein and Trk-B were similar to the orientation and binding affinity of interaction between Trk-B and its specific antibody. Conclusion: The designed chimeric BDNF/LAMP2b protein would be capable of targeting the exosomes toward neural cells and provides better drug delivery to brain tissue.

Abstract COVID-19 currently is the main cause of the severe acute respiratory disease and fatal outcomes in human beings worldwide. Several genes are used as targets for the detection of SARS-CoV-2, including the RDRP, N, and E genes. The present study aimed to determine the RDRP, N, and E genes expressions of SARS-CoV- 2 in clinical samples. For this purpose, 100 SARS-CoV-2 positive samples were collected from diagnostic laboratories of Mazandaran province, Iran. After RNA extraction, the real-time reverse transcription PCR (real-time RT-PCR) assay was performed for differential gene expressions’ analysis of N, E, and RDRP. The threshold cycle (Ct) values for N, RDRP, and E targets of 100 clinical samples for identifying SARS-CoV-2 were then evaluated using quantitative real-time PCR (qRT-PCR). This result suggests N gene as a potential target for the detection of the SARS-CoV‐2, since it was observed to be highly expressed in the nasopharyngeal or oropharynges of COVID-19 patients (P

Background and purpose: Coronavirus disease 2019 (COVID-19) is a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an indirect ELISA method was designed to measure the human IgM and IgG antibodies against SARS-CoV-2. Materials and methods: Protein sequence of nucleocapsid antigen from SARS-CoV-2 was expressed in E. coli BL21 and then was purified by chromatography. The purified protein was confirmed by SDS-PAGE and Western blotting. An indirect ELISA method was designed to measure the specific IgG and IgM antibodies against SARS-CoV-2 using recombinant N protein. The optimized ELISA method was then applied to measure the IgG and IgM antibodies in 61 infected or recovered COVID-19 patients and in 31 healthy controls. Finally, data obtained from the designed ELISA method were compared with those of a commercially approved ELISA kit. Results: The recombinant nucleocapsid protein was successfully expressed and purified which was confirmed by SDS-PAGE and Western blotting. The amount of optical densities obtained from the designed ELISA method was similar to those of the commercial kit in 61 patients and 31 controls. The sensitivity and specificity of the designed ELISA method for IgG were 100% compared with the commercial ELISA kit, while the sensitivity and specificity for IgM were 96.72 and 96.77, respectively. Conclusion: Serological tests alone are not suitable for diagnosis; however, their combination with molecular tests increases the accuracy and sensitivity of the COVID-19 diagnosis. These tests are also vauable for epidemiological studies.

PurposeRobust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness.MethodsWe conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 (IFI27) in COVID-19 patients.ResultsWe show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27-like genes were highly upregulated in the blood samples of severely infected patients.ConclusionThese data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus.

One of the most common and most critical mortality causes in modern society is gastrointestinal (GI) cancer. Helicobacter pylori is one of the most potent risk factors of gastric cancer. This bacterium can suppress the immune system by various pathogenic factors and mechanisms, lead to chronic inflammatory responses, and play a critical role in the induction of cancer. The first part of this article begins with a description of H. pylori carcinogenicity and in the next part, the roles of innate immunity pattern recognition receptors (PRRs), Toll-like receptors (TLRs), cytokines, and inflammatory regulatory mechanisms in H. pylori infection are discussed. According to our current knowledge, screening and on-time diagnosis of the disease, timely treatment, and immediate eradication of H. pylori are the most critical ways to barricade gastric cancer progression. In the end, it is suggested that, considering the expensive costs of gastric cancer treatment, H. pylori diagnostic screening tests be done at least annually in middle-aged and at-risk people.

Objective(s): Frizzled-7, the most common receptor of the Wnt signaling pathway, was significantly over-expressed in gastric (GC) and colorectal (CRC) cancers and stimulated tumorigenesis. The extracellular domain of Fzd7 (sFzd7) as a decoy receptor, could competitively bound with ligands and antagonize the interaction between Fzd7 receptors and Wnt ligands. Materials and Methods: We expressed and purified the extracellular region of Fzd7 including cysteine-rich domain (33 aa–185 aa) from Escherichia coli by chromatography. The effect of sFzd7 was evaluated on AGS gastric and SW480 colon cancer cell lines expressing high levels of Fzd7 receptor. Accordingly, cell viability and apoptosis were measured using MTT and flow cytometry assays, respectively. Real-Time PCR determined the relative expression of the β-catenin and cyclin-D1 genes. Results: After three days of treatment with sFzd7, the viability of AGS and SW480 cell lines was decreased in a dose-dependent manner. In addition, sFzd7 at concentrations of 10 and 20 ug/ml increased the rate of apoptosis. Especially at the concentration of 20 ug/ml, the apoptosis rate was remarkably high in AGS (P-value= 0.003) and SW480 cells (P-value= 0.0007). Finally, the expressions of β-catenin (P-value= 0.01) and cyclin-D1 (P-value= 0.02) were obviously decreased in SW480 cells. The same results were obtained in AGS cells, although not statistically significant. Conclusion: sFzd7 decoy receptor inhibits tumor cell progression by attenuating the Wnt pathway through inhibiting Fzd7 receptors and Wnt ligand interaction. Hence, sFzd7 can be proposed as a candidate therapy for GC and CRC cells with high levels of Fzd7 expression.

Background: Antimalarial drug resistance is one of the important challenges for governments in the fight against malaria. Molecular surveillance of antimalarial drug resistance supports early detection of how the recommended treatments work. This allows immediate action to reduce any threat and prevent it from spreading. Therefore, the aim of this study was to evaluate the frequency of dihydrofolate reductase (dhfr) mutants in Plasmodium falciparum resistance to pyrimethamine in Iranian malaria patients. Materials and Methods: In 2020, 27 patients (21 males and 5 females) with imported P. falciparum cases were studied. The nested-PCR technique first confirmed the species in all samples and then amplification was done by the semi-nested-PCR method in order to detect single nucleotide polymorphisms (SNPs) in dhfr gene related to pyrimethamine resistance. Results: All samples in the 18S rRNA gene had species-specific bands for P. falciparum strains. In the sequence analysis of pfdhfr gene amplification after comparison with the standard strain (wild type), 21 patients had a double mutation (C59R+S108N) and six patients had a triple mutation (N51I+C59R+S108N) of pyrimethamine resistance. Conclusion: The results of this study showed that the susceptibility of P. falciparum to pyrimethamine in the treatment of malaria is significantly reducing. These findings can raise concerns about pyrimethamine resistance in P. falciparum. Due to the high emergence of double and triple mutants related to pyrimethamine resistance, the malaria surveillance and treatment systems in Iran, the use of pyrimethamine should be considered.

Expression and location of nucleolin are often abnormal in malignancies, which may result in the production of autoantibodies. Despite this, the identification of such autoantibodies may be essential for the early diagnosis and prognosis of cancers. In this investigation, the recombinant nucleolin protein was generated using an Escherichia coli expression system and was used an indirect enzyme-linked immunosorbent assay to detect anti-nucleolin autoantibodies in cancer patients' sera. Lung cancer patients' autoantibodies displayed the highest seroreactivity with the recombinant protein, with area under the curve of 0.948 and sensitivity and specificity of 85% and 96.67%, respectively (accuracy=92%). Anti-nucleolin autoantibodies were linked with lung tumor size (r=0.793), tumor, node, metastasis staging (r=0.643), and proliferation (r=0.744). These autoantibodies distinguished patients with early-stage lung cancer from healthy controls. Since anti-nucleolin autoantibodies are strongly linked to tumor size, clinical staging, and growth, they can be used to measure how well a treatment is working.

In the article published in volume 32, issue 215, 2022,http://jmums.mazums.ac.ir/article-1-17695-fa.html the affilifations of Naghi Shahabi Majd, Hosein Ranjbaran, and Shabanali Khodashenas were published incorrectly, which are now corrected.

Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been a major concern since it first emerged. Prevention of Coronavirus 2019 disease (COVID-19) and slowing the epidemic and prevalence of the disease are the main goals of health institutions all over the world. it is very difficult to control the disease and predict its prevalence due to the continuous changes in the form of single nucleotide polymorphism (SNP) and mutations in this virus. In fact, some changes and mutations in Corona 2019 virus directly affect the pathogenicity and its transmission. The most important mutations that occur in the spike or RBD part of the SARS-CoV-2 virus, change the function of the virus, its severity of pathogenesis, and creates new variants. Mutations in these variants change the ability of the virus in binding to human cells, the rate of transmission, and make it easier to escape the immune system which plays a role in the effectiveness or ineffectiveness of vaccines. This study reviews eight major mutations in the spike or RBD region, their presence in different variants of Corona 2019 virus, and their relationship with response rate in infected people. Search keywords included COVID-19, mutation, variant, coronavirus, and respiratory infection in Google Scholar, PubMed, Springer, ScienceDirect, and Scopus databases. The main variations made from SARS-CoV-2 are Alfa (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) that cause the infection to spread faster and the adequacy of vaccination on these variations have not been conclusively analyzed at this point.

Background and purpose: Human papillomavirus (HPV) is one of the infectious agents that causes genital and non-genital warts and skin cancers in humans. The E7 protein of this virus is one of the small oncoproteins that may be the main target in therapeutic vaccines. E7 protein with HIV-1 Tat peptide (49-59), plays a protective role that cause immune Th1 and CTLs response. The aim of this study was to design a recombinant HIV-1 Tat 49-59 / HPV16, 18, 6, 11 E7 protein in vitro and the function of this antigen for MHC-1 expression. Materials and methods: In this study, macrophage cell line J774 was used to evaluate the expression of MHC-1 and the function of Tat peptide in delivery of recombinant protein to the surface of MHC-1. J744 mouse macrophage cell line was treated by10, 50, and 100 μg of each of the proteins with and without Tat peptide. After 24 hours, the cells were collected and then analyzed by flow cytometry. Results: In this study, J774 cells were treated and analyzed by E7-Tat and E7 proteins at different concentrations. The study showed that 10 μg of E7-Tat protein increased the expression of MHC-1, while at higher concentrations of this protein, the expression of MHC-1 molecule decreased considerably compared to the group without Tat peptide. Conclusion: E7-Tat protein at lower concentrations can act as a stimulant for increasing the MHC-1 expression and could be used in therapeutic vaccines.

Background and purpose: Given the role of cancer stem cells in cancer, the aim of this study was to determine the expression of LGR5 marker in gastric cancer and its association with cagA genotype of Helicobacter pylori infection. Materials and methods: A case-control study was performed in gastric biopsy specimens from antrum and body during endoscopic examination of patients attending Sari Touba Clinic, 2017-2018. After reviewing the patient records, the samples from those aged 50 and higher were studied. Case group included gastric cancer specimens with H. pylori infection (n=30) and control group included non-cancerous samples with H. pylori infection (n=30). LGR5 expression and presence of cagA were evaluated by IHC and PCR methods, respectively. Results: The mean ages of gastric cancer and control group were 69.5±10.1 and 62.3±7.8, respectively (P= 0.003). Twenty three patients (76.7%) in cancer group and 24 patients (80%) in control group were positive for cagA genotype. Overexpression of LGR5 was observed in 15 patients (51.7%) with gastric cancer and 11 patients (39.3%) in control group (P= 0.429). LGR5 was also overexpressed in 18 cases (40.9%) with cagA positive genotype and 8 cases (61.5%) with negative cagA genotype (P=0.22). Conclusion: High expression of LGR5 was observed in half of patients with gastric cancer but it was not significantly associated with cagA H.pylori genotype.

BackgroundIvermectin which was widely considered as a potential treatment for COVID-19, showed uncertain clinical benefit in many clinical trials. Performing large-scale clinical trials to evaluate the effectiveness of this drug in the midst of the pandemic, while difficult, has been urgently needed.MethodsWe performed two large multicenter randomized, double-blind, placebo-controlled clinical trials evaluating the effectiveness of ivermectin in treating inpatients and outpatients with COVID-19 infection. The intervention group received ivermectin, 0.4mg/kg of body weight per day for 3 days. In the control group, placebo tablets were used for 3 days.ResultsData for 609 inpatients and 549 outpatients were analyzed. In hospitalized patients, complete recovery was significantly higher in the ivermectin group (37%) compared to placebo group (28%; RR, 1.32 [95% CI, 1.04–1.66]; p-value = 0.02). On the other hand, the length of hospital stay was significantly longer in the ivermectin group with a mean of 7.98 ± 4.4 days compared to the placebo receiving group with a mean of 7.16 ± 3.2 days (RR, 0.80 [95% CI, 0.15–1.45]; p-value = 0.02). In outpatients, the mean duration of fever was significantly shorter (2.02 ± 0.11 days) in the ivermectin group versus (2.41 ± 0.13 days) placebo group with p value = 0.020. On the day seventh of treatment, fever (p-value = 0.040), cough (p-value = 0.019), and weakness (p-value = 0.002) were significantly higher in the placebo group compared to the ivermectin group. Among all outpatients, 7% in ivermectin group and 5% in placebo group needed to be hospitalized (RR, 1.36 [95% CI, 0.65–2.84]; p-value = 0.41). Also, the result of RT-PCR on day five after treatment was negative for 26% of patients in the ivermectin group versus 32% in the placebo group (RR, 0.81 [95% CI, 0.60–1.09]; p-value = 0.16).ConclusionOur data showed, ivermectin, compared with placebo, did not have a significant potential effect on clinical improvement, reduced admission in ICU, need for invasive ventilation, and death in hospitalized patients; likewise, no evidence was found to support the prescription of ivermectin on recovery, reduced hospitalization and increased negative RT-PCR assay for SARS-CoV-2 5 days after treatment in outpatients. Our findings do not support the use of ivermectin to treat mild to severe forms of COVID-19.Clinical Trial Registrationwww.irct.ir IRCT20111224008507N5 and IRCT20111224008507N4.

Background: Trametes species possess remarkable immunomodulatory and anticancer effects which are mainly related to the activation of innate immune receptors by their polysaccharide constituents. In this study, we investigate the effect of Trametes gibbosa (Pers.) Fr. polysaccharide fraction (TGP) on activation of TLR-4 receptor and subsequent release of IL-8 in HEK-Blue™ hTLR4 cells. Materials and Methods: The polysaccharide fraction was purified using ethanol precipitation and dialysis methods. The total sugar content and monosaccharide composition were analyzed by phenol–sulfuric acid and chromatographic methods. FT-IR spectroscopy was also performed for structure characterization of the polysaccharide. The activation of TLR4 was determined by measuring the secreted embryonic alkaline phosphatase in the culture media. Results: The results indicated that the total sugar content of TGP was about 90%, which glucose was the major constituents. FT-IR analysis showed the characteristic bands of polysaccharides. TGP was able to activate the TLR-4 signaling pathway in a dose-dependent manner. Moreover, the significant increase of IL-8 was observed in cells treating with TGP. The HEK-Blue Null2™ reporter cells lacking TLR4, did not respond to LPS and TGP. Conclusion: The results suggest that TLR4 signaling cascade serve as targets for immunomodulatory activity of T. gibbosa which could address the anticancer properties of Trametes species.

Background: The Methicillin-resistant Staphylococcus aureus (MRSA) strains cause wide spectrum infections in hospitals and are responsible for the majority of the community-acquired infections. The emergence of community-acquired MRSA in patients without risk factors represents a high risk for public health. Objectives: In this study, we aimed to molecular typing the nasal carriage MRSA strains harbored mecA gene isolated from healthy children living in the North of Iran and also to determine the pattern of vancomycin susceptibility in these isolates. Methods: The MRSA strains were identified using standard microbiological and molecular procedures. The antibiotic susceptibility test was performed according to the clinical and laboratory standards institute. The MRSA strains were characterized by spa typing. Results: In total, 14 different patterns of spa genes were obtained from MRSA isolates in this study, which included t790 (23.07%), t2962 (15.38%), t267 (15.38%), t159 (7.69%) and t701, t094, t1816, t11332, t937, t2135, t180, t160 [1 (3.84%) isolate per each type]. The rate of resistant isolates to cefoxitin, , cefalotin, clindamycin, cefazolin, co-amoxiclav, co-trimoxazole, and cephalexin antibiotics were 69.23%, 100%, 46.15%, 96.15%, 100%, 3.48%, and 100%, respectively. In total, six (23.07%) revealed vancomycin-intermediate resistant phenotype and five (19.23%) isolates revealed vancomycin-resistant S. aureus phenotype. Conclusions: This study showed the different variants of MRSA strains based on molecular typing, among healthy children. Continuous surveillance studies to monitor MRSA should be encouraged to gain a better understanding of the circulating MRSA strains and prevent community spread of this pathogen.

The role of immune checkpoint receptors in T-cell exhaustion has been demonstrated in several cancers. We investigated the co-expression of TIGIT/PD-1 and LAG-3/PD-1 cells in patients with chronic lymphocytic leukemia (CLL). The frequencies of TIGIT+PD-1+CD8+and LAG-3+PD-1+CD8+cells and relative mRNA expression of LSECtin and CD155 were examined in PBMCs from 33 CLL patients and 20 controls. The percentage of TIGIT+PD-1+CD8+cells was significantly higher in CLL patients than in control subjects, with the preference in advanced stage patients. However, LAG-3+PD-1+CD8+cell percentage was significantly lower in CLL patients than in the control subjects and no significant difference were found between the early and advanced stages of the disease. An increase in the mRNA expression level of LSECtin, but not that of CD155, was observed in CLL patients compared to the control subjects. Collectively, a higher co-expression of PD-1 and TIGIT on CD8+ T-cells in CLL compared to control subjects suggests an important role of TIGIT in T-cell exhaustion in CLL patients especially those with advanced disease.

The antifungal resistance in non-fumigatus Aspergillus spp., as well as Aspergillus fumigatus, poses a major therapeutic challenge which affects the entire healthcare community. Mutation occurrence of cyp51 gene paralogs is the major cause of azole resistance in Aspergillus spp. To obtain a full map of genomic changes, an accurate scan of the entire length of the Aspergillus genome is necessary. In this study, using whole genome sequencing (WGS) technique, we evaluated the mutation in cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA genes in different clinical isolates of Aspergillus fumigatus, Aspergillus niger, Aspergillus tubingensis, Aspergillus welwitschiae and Aspergillus terreus which responded to minimum inhibitory concentrations of itraconazole above 16 µg mL−1. We found different nonsynonymous mutations in the cyp51A, cyp51B, Cdr1B, AtrR, Hmg1, HapE and FfmA gene loci. According to our findings, Aspergillus species isolated from different parts of the world may represent different pattern of resistance mechanisms which may be revealed by WGS.

Abstract 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.

Background and purpose: Activation-induced cytidine deaminase (AID) is a B-cell specific enzyme responsible for somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes within the B-cell follicle of peripheral lymphoid organs. Ectopic overexpression of the enzyme leads to mutations in non-B cells and Escherichia coli (E.coli) genes. However, induction of mutations in E.coli by expression of AID requires highly regulated conditions. Therefore, the aim of this study was to construct a plasmid expressing AID under the control of tightly regulated temperature-sensitive promoter of bacteriophage lambda.. Materials and methods: The AID gene was isolated from PGEMT recombinant plasmid, containing AID and cloned into PTG19-T vector. Then, AID was ligated to an expression plasmid under the control of left promoter of bacteriophage lambda and mutant thermolabile cI857 repressor. Finally, the resistance marker of the engineered plasmid was replaced by tetracycline resistance gene. Results: The whole open reading frame of AID was ligated into a plasmid containing a cI857-sensitive receptor lambda bacteriophage. The sequence and reading frame accuracy of the cloning was confirmed by electrophoresis of PCR products on agarose gel, restriction enzyme digestion, and nucleotide sequencing methods. Conclusion: In current study, the AID was successfully cloned into an expression plasmid containing thermo-sensitive promoter of bacteriophage lambda. The recombinant plasmid could be used in different strains of E.coli to produce mutator strains.

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 (n = 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 themocusa 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.

Background and purpose: Increasing resistance to Quinolones in Escherichia coli and Klebsiella pneumonia in Sari, has caused many problems in treatment. Mutation in gyrA gene lead to changes in amino acids and resistance against Fluoroquinolones in E. coli and K. pneumonia. This study aimed at identifying remarkable mutations in E. coli and K. pneumonia isolates using PCR-SSCP analysis. Materials and methods: Antibiotic sensitivity test (ciprofloxacin, nalidixic acid) was performed using Agar Disk Diffusion method. Resistance to fluoroquinolones was confirmed by E-test. (MIC experiment). We used PCR-SSCP method to detect mutation in gyrA (ser83 – asp 87) genes. Then, the PCR products were randomly sequenced. Results: From 103 isolates, 65 (63.2 %) were E. coli and 38 (36.8%) were K. pneumoniae. In all E.coli isolates resistant to Ciprofloxacin, at least one mutation was observed. Also, in all K. pneumoniae samples resistant to Ciprofloxacin, at least one mutation was seen and in 14 samples two mutation points were observed, but in 5 samples that were sensitive to Ciprofloxacin no mutation was observed. Conclusion: This study showed that the mutation in gyrA genes is closely related to quinolones resistance. High prevalence of quinolones resistance in E. coli and K. pneumoniae isolates requires more appropriate infection control programs.

Background : Allergic asthma is a chronic airway inflammatory disease often determined with degrees of inflammation, hypersensitivity, bronchial constriction, and airway changes. Th1, Th2, and Th17 cells are the main cells involved in asthma pathophysiology. To evaluate Th1, Th2, and Th17 functions by assessing INF-g, IL-4, and IL-17 gene and protein levels in asthma patients and healthy controls. Materials and methods: In total, 44 individuals of Iranian ethnicity including 24 patients with allergic asthma and 20 healthy controls were enrolled. Peripheral blood mononuclear cells of all participants were isolated and cDNA was synthesized following RNA extraction. Gene expression and protein levels of INF-g, IL-4, and IL-17 were evaluated by real-time polymerase chain reaction and sandwich ELISA, respectively. Results: The results of this study showed that the gene expression of IL-4 and IL-17 in patients was increased significantly compared to the control group (p = 0.046 and 0.03, respectively) whereas that of IFN-g was significantly decreased in the group of patients (p = 0.021). Compared to the healthy controls, serum levels of IL-17 and IL-4 were significantly increased in asthma patients (p = 0.015 and 0.03, respectively). Conclusion: Higher IL-17 and IL-4 mRNA expression and serum levels in asthma patients than healthy controls highlight the role of Th2 and Th17 cells in asthma pathogenesis and their potential as therapeutic targets.

Aspergillus flavus is one of the most important agents of invasive and non-invasive aspergillosis, especially in tropical and subtropical regions of the world, including Iran. Aspergillus oryzae is closely related to A. flavus, and it is known for its economic importance in traditional fermentation industries. Reports of infection due to A. oryzae are scarce. Several studies reported that differentiating these two species in clinical laboratories is not possible using MALDI-TOF or by targeting fungal barcode genes, such as Internal Transcribed Spacer (ITS) and β-tubulin (benA). The species-level identification of causative agents and the determination of antifungal susceptibility patterns can play significant roles in the outcome of aspergillosis. Here, we aimed to investigate the discriminatory potential of cyp51A PCR-sequencing versus that of the ITS, benA and calmodulin (CaM) genes for the differentiation of A. flavus from A. oryzae. In a prospective study investigating the molecular epidemiology of A. flavus in Iran between 2008 and 2018, out of 200 clinical isolates of A. flavus, 10 isolates showed >99% similarity to both A. flavus and A. oryzae. Overall, the ITS, β-tubulin and CaM genes did not fulfil the criteria for differentiating these 10 isolates. However, the cyp51A gene showed promising results, which warrants further studies using a larger set of isolates from more diverse epidemiological regions of the world.

Background: HIV infected persons are also at risk for Infection with other pathogens, like Hepatitis B virus (HBV) and Hepatitis C virus (HCV). One of the main steps in the planning of treatment and prevention is the study of the HIV positive population who have HBV and/or HCV infection. The objective of this study was to determine the prevalence of HIV and Hepatitis B and C dual infection in north of Iran Materials and methods: Blood samples were collected into EDTA containing tubes by sterile syringes and needles from 83 HIV positive patients which were previously confirmed by Real-time Polymerase chain reaction in the HIV center of the north of IRAN. A structured questionnaire was used to obtain socio-demographic data from participants. Samples were screened for Hepatitis B surface antigen (HBsAg) and anti-HCV antibody. All non-reactive samples were recorded as negative. Results: Out of the 83 patients, 50(60%) were male and 33 (40%) were female. Positive hepatitis C antibody was found in 28 (33%) and positive hepatitis B surface antigen was present in 15 (18%) of patients. The frequency of all these three viruses co-infection was 7 (8%). Conclusion: Seroprevalence of HCV and HIV co-infection was high and it was strongly related to mutual ways of acquisition.

Background and purpose: Allergic asthma is a chronic inflammatory disorder with increased inflammation and bronchial over react to stimulants. Th1 and Th2 cells are the main cells involved in the pathophysiology of asthma. The function of these cells is under the influence of T-bet and GATA3 transcription factors. This study aimed to investigate the impairment of immune responses in patients with allergic asthma compared with controls. Materials and methods: In a case-control study, 24 patients with allergic asthma and 10 healthy individuals were recruited. The Mononuclear cells (PBMCs) were isolated and cDNA was synthesized after RNA extraction. Gene’s expressions of T-bet and GATA3 were evaluated by Real-time Polymerase chain reaction and their relationships with risk factors for asthma were analyzed using statistical tests. Results: In our study, the GATA3 gene expression in patients increased 29 times more than that in controls (P= 0.002) but the expression of T-bet declined (0.43, P =0.32). Evaluation of T-bet / GATA3 showed that this ratio was significantly lower in patients compared with that in the controls (P

Background and Purpose: Hyphal wall protein 1 (HWP1) is an important adhesin which usually is expressed on the germ tube and hyphal surface produced by different Candida species. The hyphal wall protein-coding gene (HWP1) was evaluated as a novel identification and phylogenetic marker in Candida tropicalis, C. orthopsilosis, C. parapsilosis and C. glabrata. Materials and Methods: Initially, four specific primer pairs were designed, and the target was amplified and finally sequenced. A total of 77 Candida isolates from four different species were included in the study. Consensus sequences were used for the evaluation of phylogenetic tree using the CLC Genome Workbench, GENEIOUS, and MEGA softwares and the levels of nucleotide and amino acid polymorphism were assessed. Results: According to the results, the specific amplified fragments of HWP1 gene were useful for the differentiation of four species. Intra-species variation was observed only in C. tropicalis with two DNA types. The phylogenetic tree of Candida species based on the HWP1 gene showed consistency in topology with those inferred from other gene sequences. Conclusion: We found that HWP1 gene was an excellent marker for the identification of non-albicans Candida species as well as the phylogenetic analysis of the most clinically significant Candida species.

ABO blood type antigens are polymorphic, inherited structures presented on the surface of red blood cells. Although ABO blood group antigens is the most important antigens in transfusion medicine, its main role is not clearly clarified. Correlation between ABO blood group and susceptibility to certain infectious and non-infectious diseases is a controversial issue. Since, lack of blood type antigens has not been related to significant diseases or health risks. There are many research studies that suffice to prove that blood antigens are biologically active, carbohydrate structures involving to the “cell physiology and human pathology”. This review summarizes the available data concerning the blood group antigens correlation with different aspects of health. We therefore investigated that certain blood type antigens can affect susceptibility to diseases or make a phenotype resistant.

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.

Background: Prevalence of community acquired Methicillin-Resistant Staphylococcus Aureus (MRSA) infection and colonization has been increased in recent years. The aim of this study was to determine the nasal colonization with MRSA agent and its antibiotic resistance pattern in children. Methods: Participants were 277 elementary school students. Sampling was performed for 6 months from February to August 2017. The sample size was determined by the Cochran formula. MRSA strains were identified by using conventional lab techniques including catalase, coagulase and DNase tests. The oxacillin and cefoxitin discs diffusion method was used for detection of MRSA strain, and the determination of the strain resistance pattern was performed by Kirby-Bauer disk diffusion test by using 10 antibiotics. Results: MRSA strains were isolated from the nose of 81 of 277 children (29.24%). Of 81 MRSA isolates, the highest rate of resistance was belonged to cefixime and cephalexin with 100% resistance followed by amoxicillin-clavulanic acid (97%) and cephalothin (95%), while the lowest rate of resistance was related to co-trimoxazole (12.5%). The resistance rate was 87% for cefazolin, 60% for clindamycin, 65% for cefoxitin, and 45% for vancomycin. Furthermore, no significant association was found between demographic variables (application of ointments, history of hospitalization, history of antibiotic consumption, mucosal infection, etc.) and MRSA colonization. Conclusions: The prevalence of MRSA infection is high. Hence, there is a need for rational use of empiric antibiotic therapy to reduce the mortality and morbidity rate caused by this infection. The emergence of vancomycin resistant staphylococcus aureus infection is expected in the future.

Background: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4) proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein. Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain) and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 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 ROM4 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 ROM4 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 ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.

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.

Background: Breast cancer is the most common female malignancy and the leading cause of cancer mortality in women worldwide. The human epidermal growth factor receptor2 (HER2) is a transmembrane tyrosine kinase receptor that is usually overexpressed in human breast cancers. Stable cell lines heterogeneously overexpressing HER2 are highly required as in vitro models for breast cancer research. The aim of this study was to establish a stable cell line overexpressing HER2. Materials and Methods: CHO Cells were transfected with linearized pCVN/HER2 plasmid and selected for the recombinant cells with G418 antibiotic. Expression of HER2 in the transfected cells was analyzed using western blotting and immunofluorescence. Results: We found that the recombinant cells stably expressed high levels of HER2 proteins that were mostly concentrated on the cell membrane. Conclusions: The cell line established here provides a useful in vitro model for breast cancer research and any HER-related studies.

Background: Understanding of cellular and molecular mechanisms involved in cell/tumor growth and progression has led to molecular-targeted therapy. HER1 and HER2 are two main oncogenic components of HER-receptor family considered as targets for cancer research and therapy. Reliable results of HER- related research and targeted therapies dependent upon cell line with known HER expression status. But there are no published studies focusing on determination of HER1 and HER2 receptors expression in CHO and HEK-293 cell lines. Materials and Methods: Absolute expressions of HER1 and HER2 in CHO, HEK-293, MDA-MB-468 and SKBR3 cells were evaluated using quantitative real-time PCR. Absolute expression quantification was carried out by serial dilution plasmids harboring HER1 and HER2 to extrapolate standard curve. Results: Real-time PCR amplification was successfully optimized by HER1 and HER2 plasmids with high efficiency and their copy numbers were calculated. The results showed that HER1 was not expressed in CHO and HEK-293 cells but these cells express HER2 up to 9.4 × 102 and 1.1 × 105 copy/µg, respectively. MDA-MB-468 and SKBR3 cells express HER1 up to 8.4 × 105 and 9.7 × 104, respectively. In addition, the expression of HER2 is higher on the surface of SKBR3 cell (6.5 × 106 copy/µg). Conclusion: Our study provides a proven insight to HER1 and HER2 expression status in CHO, HEK-293, MDA-MB-468 and SKBR3 cells. Heterologous expression of HER1 and HER2 in cell lines is the first step in structural and functional studies. Since HER1 is not expressed in CHO and HEK-293 endogenously, these cells are ideal for HER receptor related studies.

Background: Taq DNA polymerase is a very important enzyme for molecular biological studies such as DNA amplification and DNA sequencing by the PCR. It is a standard enzyme that is used in 90% of molecular biology labs today. The aim of this study was to produce Taq DNA polymerase enzyme in E. coli by a reliable, practical, simple and low cost method.Materials and Methods: In this study, the Taq gene was amplified from the genomic DNA of Thermus aquaticus and cloned into pTrc99A vector. Recombinant plasmid is expressed in E. coli strain TOP10. Product protein is extracted and purified. Expression of gene was analyzed by SDS-PAGE and gene amplification.Results: SDS-PAGE showed an approximately 94 KDa protein. The density of protein bands in agarose gel electrophoresis indicated that the purified enzyme is more active than the non purified one.Conclusion: The protocols described in this paper lead to the production of pure and active enzyme that can be applied in both teaching and research laboratories.

Production and approval of trastuzumab (Herceptin®) for the treatment of metastatic breast cancer (MBC) was a millstone in antibody-based targeted therapy in the cancer treatment. However, despite the early success in the clinical trials, trastuzumab failed to appreciate the initial attraction due to development of resistance to the drug. The majority of patients who benefit from the drug acquire resistance to it and experience tumor recurrence within 1 year. Several molecular and cellular mechanisms underlying the resistance to trastuzumab have been proposed. In this review, first, we provide a brief history leading to production of trastuzumab. Also we consider the cellular and molecular antitumor effects of trastuzumab and then, we discuss the mechanisms underlying trastuzumab resistance in four levels.

Cutaneous leishmaniasis is a neglected tropical disease and a major public health in the most countries. Leishmania major is the most common cause of cutaneous leishmaniasis. In the Leishmania 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 L. major 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 (p-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, L. major 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.