Background: Despite the dramatic efficacy of erlotinib, an EGFR tyrosine kinase inhibitor (TKI), most of nonsmall cell lung cancer (NSCLC) patients ultimately acquire resistance to this agent. Different studies indicated that miRNA-125a-5p is down-regulated in human lung cancer cells and may function as a tumor suppressor by targeting EGFR. However, the biological function of miRNA-125a-5p in NSCLC resistance to EGFR-TKIs is not fully understood. In this study the effect of miRNA-125a-5p on cell proliferation, apoptosis and sensitivity of the A549 lung cancer cells to erlotinib was investigated. Methods: After miRNA-125a-5p transfection, the expression levels of EGFR mRNA were measured by QRT-PCR. Trypan blue assays were performed to evaluate the proliferation of the A549 lung cancer cells. The cytotoxic effects of miRNA-125a-5p and erlotinib, alone and in combination, were determined using MTT assay. Combination index study was performed using the method of Chou-Talalay. Apoptosis was assessed using an ELISA cell death assay kit. Results: MiRNA-125a-5p clearly reduced the expression of EGFR mRNA in a time dependent manner, causing marked cell proliferation inhibition and spontaneous apoptosis (p<0.05, relative to control). Pretreatment with miRNA-125a-5p synergistically increased the cytotoxic effect of erlotinib and decreased its IC 50. Furthermore, miRNA-125a-5p significantly enhanced the apoptotic effect of erlotinib. Negative control miRNA had no significant effect on biological parameter of the tumor cells. Conclusions: Our data suggest that suppression of EGFR by miRNA-125a-5p can effectively trigger apoptosis and overcome EGFR-TKs resistance of lung cancer cells. Therefore, miRNA-125a-5p may be a potential therapeutic adjuvant in patients with lung cancer.

Background: Despite effective activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as erlotinib, all non-small cell lung cancer (NSCLC) patients eventually acquire resistance to these agents. Studies have demonstrated that down-regulation of miRNA-145 leads to enhancement of EGFR expression, cell proliferation and metastasis. The aim of this study was to investigate the effect of miRNA-145 on sensitivity of the A549 NSCLC cells to erlotinib. Methods: Quantitative real-time PCR was used to examine the effect of miRNA-145 on EGFR expression. The effect of miRNA-145 on cell growth and sensitivity the lung cancer cells to erlotinib was examined by trypan blue and MTT assays, respectively. The combination index was calculated using the non-constant method of Chou-Talalay. Apoptosis was determined by ELISA cell death assay. Results: We found that miRNA-145 was markedly suppressed the expression of EGFR and inhibited the cancer cell growth, relative to blank control and negative control miRNA (p<0.05). Pretreatment with miRNA-145 synergistically enhanced the sensitivity of the lung cancer cells to erlotinib. Results of apoptosis assay revealed that miRNA-145 can induce apoptosis and increase the erlotinib-mediated apoptosis. Conclusions: Our data demonstrate that miRNA-145 play a critical role in the lung cancer cell growth, survival and EGFR-TKIs resistance possibly by regulation of EGFR. Therefore, miRNA-145 replacement therapy can become a new therapeutic strategy in lung cancer.

BackgroundClinical reasoning plays a major role in the ability of doctors to make a diagnosis and reach treatment decisions. This paper describes the use of four clinical reasoning tests in the second National Medical Science Olympiad in Iran: key features (KF), script concordance (SCT), clinical reasoning problems (CRP) and comprehensive integrative puzzles (CIP). The purpose of the study was to design a multi instrument for multiple roles approach in clinical reasoning field based on the theoretical framework, KF was used to measure data gathering, CRP was used to measure hypothesis formation, SCT and CIP were used to measure hypothesis evaluation and investigating the combined use of these tests in the Olympiad. A bank of clinical reasoning test items was developed for emergency medicine by a scientific expert committee representing all the medical schools in the country. These items were pretested by a reference group and the results were analyzed to select items that could be omitted. Then 135 top-ranked medical students from 45 medical universities in Iran participated in the clinical domain of the Olympiad. The reliability of each test was calculated by Cronbach's alpha. Item difficulty and the correlation between each item and the total score were measured. The correlation between the students' final grade and each of the clinical reasoning tests was calculated, as was the correlation between final grades and another measure of knowledge, i.e., the students' grade point average.ResultsThe combined reliability for all four clinical reasoning tests was 0.91. Of the four clinical reasoning tests we compared, reliability was highest for CIP (0.91). The reliability was 0.83 for KF, 0.78 for SCT and 0.71 for CRP. Most of the tests had an acceptable item difficulty level between 0.2 and 0.8. The correlation between the score for each item and the total test score for each of the four tests was positive. The correlations between scores for each test and total score were highest for KF and CIP. The correlation between scores for each test and grade point average was low to intermediate for all four of the tests.ConclusionThe combination of these four clinical reasoning tests is a reliable evaluation tool that can be implemented to assess clinical reasoning skills in talented undergraduate medical students, however these data may not generalizable to whole medical students population. The CIP and KF tests showed the greatest potential to measure clinical reasoning skills. Grade point averages did not necessarily predict performance in the clinical domain of the national competitive examination for medical school students.

BackgroundPolymerase chain reaction (PCR) is preferred to other methods for detecting Escherichia coli (E. coli) in water in terms of speed, accuracy and efficiency. False positive result is considered as the major disadvantages of PCR. For this reason, reverse transcriptase-polymerase chain reaction (RT-PCR) can be used to solve this problem. The aim of present study was to determine the efficiency of RT-PCR for rapid detection of viable Escherichia coli in drinking water samples and enhance its sensitivity through application of different filter membranes.Materials and methodsSpecific primers were designed for 16S rRNA and elongation Factor II genes. Different concentrations of bacteria were passed through FHLP and HAWP filters. Then, RT-PCR was performed using 16srRNA and EF –Tu primers. Contamination of 10 wells was determined by RT-PCR in Arak city. To evaluate RT-PCR efficiency, the results were compared with most probable number (MPN) method.ResultsRT-PCR is able to detect bacteria in different concentrations. Application of EF II primers reduced false positive results compared to 16S rRNA primers. The FHLP hydrophobic filters have higher ability to absorb bacteria compared with HAWB hydrophilic filters. So the use of hydrophobic filters will increase the sensitivity of RT-PCR.ConclusionRT-PCR shows a higher sensitivity compared to conventional water contamination detection method. Unlike PCR, RT-PCR does not lead to false positive results. The use of EF-Tu primers can reduce the incidence of false positive results. Furthermore, hydrophobic filters have a higher ability to absorb bacteria compared to hydrophilic filters.

Vibrio cholerae is the causative agent of cholera and annually leads to death of thousands of people around the globe. Two factors in the pathogenesis of this bacterium are its pili and flagella. The main subunits of pili TcpA, TcpB, and FlaA are the constituent subunit of flagella. In this study, we studied the ability of pili and flagella subunits to stimulate immune responses in mice. After amplification of TcpA, TcpB, and FlaA genes using PCR, they were cloned in expression plasmids. After production of the above-mentioned proteins by using IPTG, the proteins were purified and then approved using immunoblot method. After injection of the purified proteins to a mice model, immune response stimulation was evaluated by measuring the levels of IgG1 and IgG2a antibody titers, IL5 and IFN-γ. Immune response stimulation against pili and flagella antigens was adequate. Given the high levels of IL5 titer and IgG1 antibody, the stimulated immune response was toward Th1. Humoral immune response stimulation is of key importance in prevention of cholera. Our immunological analysis shows the appropriate immune response in mice model after vaccination with recombinant proteins. The high level of IL5 and low level of IFN-γ show the activation of Th2 cell response.