Comparison of the HER2/neu gene amplification assesment by differential PCR and immunohistochemistry in breast cancer patients in Isfahan

Background and aim: Amplification of the oncogene HER-2/neu influences the breast cancer progression, prognosis and therapy. Accurate assessment of HER-2/neu status of the tumor is important for management of the disease and correct selection of patients who are able to respond to trastuzumab neu treatment. This study designed to evaluate the HER-2/neu gene status in breast cancer specimens by differential PCR and to show the concordance between these evaluations and IHC test in some specimens. Methods: In this case-control study the quantitative accuracy of differential PCR was evaluated and then 67 fresh and 16 formalin fixed paraffin embedded breast cancer tissues were analysed using this method. IHC reports were available only for 27 of these samples. Data were analysed using McNemar and kappa test. Results: HER-2/neu gene amplification was estimated (35%) in 29 cases out of 83 specimens (35%), as shown by differential PCR. IHC reports showed that 12 out of the 27 specimens contain overexpressed HER-2/neu. So, there was 70% agreement between these two methods (19 out off 27). Among the 8 discordant samples, 6 cases were negative by IHC but positive by differential PCR and 2 cases with HER-2/neu amplification had normal HER-2/neu expression by IHC. Conclusion: Differential PCR is a simple, rapid and cheap method to determine the gene dosage in samples with small amount of tumor tissue but it does not seem to be so accurate, as it compares the end point products of the PCR. Thus, this method has low accuracy and the observation of 6 cases with HER-2/neu amplification in the absence of HER-2/neu over expression may have come from this reality

Detection of FimH Gene in Uropathogenic Escherichia coli Strains Isolated From Patients With Urinary Tract Infection

Background: Urinary tract infections (UTIs) are one of main health problems caused by many microorganisms, including uropathogenic Escherichia coli (UPEC). UPEC strains are the most frequent pathogens responsible for 85% and 50% of community and hospital acquired UTIs, respectively. UPEC strains have special virulence factors, including type 1 fimbriae, which can result in worsening of UTIs. Objectives: This study was performed to detect type 1 fimbriae (the FimH gene) among UPEC strains by molecular method. Materials and Methods: A total of 140 isolated E. coli strains from patients with UTI were identified using biochemical tests and then evaluated for the FimH gene by polymerase chain reaction (PCR) analysis. Results: The UPEC isolates were identified using biochemical tests and were screened by PCR. The fimH gene was amplified using specific primers and showed a band about 164 bp. The FimH gene was found in 130 isolates (92.8%) of the UPEC strains. Of 130 isolates positive for the FimH gene, 62 (47.7%) and 68 (52.3%) belonged to hospitalized patients and outpatients, respectively. Conclusions: The results of this study indicated that more than 90% of E. coli isolates harbored the FimH gene. The high binding ability of FimH could result in the increased pathogenicity of E. coli; thus, FimH could be used as a possible diagnostic marker and/or vaccine candidate

Molecular analysis of the clavulanic acid regulatory gene isolated from an Iranian strain of Streptomyces clavuligerus, PTCC 1709

Objective: The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). Materials and Methods: In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. Results: The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. Conclusion: The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assa

Evaluation of HER2 gene amplification status in invasive breast cancer patients by Fluorescence in Situ Hybridization analysis and its correlation with clinical features

Precise assessment of HER2 gene status as an important biomarker plays a significant role in identifying the eligible patients for Trastuzumab therapy and determining their clinical outcomes. In this study, the researchers assigned HER2 amplification status in invasive breast cancer specimens by Fluorescence in Situ Hybridization (FISH) and determined its association with other clinical features. Formalin-fixed paraffin embedded tumor tissue specimens of 46 patients with invasive breast cancer were collected from November 2011 till May 2012. HER2status was evaluated by FISH. The Zytolight SPEC HER2/CEN17 dual color probe kit was applied for assessment of HER2status. HER2 gene amplification was defined as HER2/CEP17 ratio>2.2.The association between HER2status and clinical features like tumor grade, tumor type, tumor size, axillary lymph node involvement and age of patient was done using Chi squared test at the 0.05 level of significance (p value). Amplification of HER2 gene was detected in twelve cases (26%). On statistical analysis HER2status showed correlation with tumor grade (p=0.02).There was no correlation between HER2status and tumor type, tumor size, lymph node status and age of patients. The results of this study are consonant with the findings of other studies about the presence of HER2 gene amplification in invasive breast cancer. Statistical analysis showed patients with HER2 amplified gene have tumors with higher grade. In these patients the probability of increased proliferation and metastasis is high therefore evaluation of HER2 gene amplification status in breast cancer patients specially in high grade tumor with an accurate method such as FISH is essentia

Expression Analysis of Interferon Beta Level in HEK293T Cells Using Real-Time PCR and Protein Tests

Background: Interferons are some kind of natural cytokines which express in response to a variety of antigens including viral RNA, bacterial products, and tumor proteins. Interferon beta is used in the treatment of autoimmune diseases such as multiple sclerosis. Moreover, this drug inhibits cellular proliferation as well as angiogenesis and as a result, helps to cure cancer. In this research, in addition to cloning the interferon-beta gene along with conserved kozak sequence after the strong eEf1a promoter in the pBud.CE4.1 vector, the expression of this recombinant gene was compared to basal expression in HEK293T cell line using real-time PCR, SDS PAGE and western blot tests. Materials and Methods: In the beginning, the interferon-beta gene was amplified from the pSVM dhfr vector containing the gene, using primers including BglII and KpnI restriction sites as well as conserved kozak sequence. Then the duplicated gene was digested and inserted in the linear pBud.CE4.1 vector. After ensuring entry of the gene using RFLP, colony PCR and sequencing, the recombinant vector was transfected into E. coli TOP10 competent bacteria. After that, amplified recombinant vector was extracted and transfected into HEK293 cell line. Results: The expression of interferon-beta cloned in pBud.CE4.1 vector showed a 79.9-fold increase, in comparison with the basal expression in HEK293T cell line. Moreover, non-recombinant vector transfection has increased the expression of interferon-beta up to 2.87 times in the cell line that is probably due to the existence of the viral promoter in the vector. Conclusion: Real-time PCR and protein test results showed that recombinant beta interferon gene had been successfully expressed in HEK293T cell line. In order to produce more of this protein, optimization of various conditions are required for the HEK293T cell line

Differential Expression of MiR-18b in PBMC from T1D Patients in Isfahan population

Background: Type 1 diabetes (T1D) is caused by cell-mediated autoimmune attack on pancreatic beta-cells. Previous studies highlight the role of microRNAs (miRNAs) in the pathogenesis of T1D. MiRNAs are small non-coding RNAs involved in the regulation of gene expression post-transcriptionally. In this work, miR-18b was chosen and the differential expression of it was measured between T1D patients and healthy controls from Isfahan population. Materials and Methods: MiR-18b was selected using Bioinformatics studies by miRWalk software. 22 T1D patients and 18 healthy controls from Isfahan population were enrolled in this study. Total RNA of the peripheral blood mononuclear cells (PBMCs) samples were extracted. After cDNA synthesis, the expression profile of miR-18b quantified by means of qPCR method in patients and controls. Finally the results were statistically analyzed. Results: In this study despite our hypothesis, the expression levels of miR-18b didn’t show any significant difference between T1D patients and healthy controls (p value: 0.145). Conclusion: Due to the results of our experimental analysis, it seems that miR-18b doesn’t have any association with T1D disease in Isfahan population

Evaluation of VEGF-111b Anticancer Peptide Production in Transfected Human Cells Using Real time- PCR Analysis

Background and purpose: Endothelial growth factor type b with anti-angiogenic activity and inhibition of tumor growth are considered as new anticancer drugs. The aim of this research was to study the expression of vegf111b in HEK293 human cells. Materials and methods: In this experimental study, HEK293 cells were transfected by pBUD.VEGF111b vector containing the VEGF111B gene through lipofectamine method. The mRNA of transfected cells and control cells were extracted and cDNA was built over it. Then, the expression levels of vegf111b were measured using Real time- PCR. Results: Transfection of HEK293 cells was successfully done and 48 hours after transfection of HEK293 cells, ct of the vegf111b expression in transfected cells was 23.17 and ct of the GAPDH control gene expression in these cells was 21.11. In the control (untransfected) cells the ct of GAPDH was 21.09 and there was no expression of vegf111b in these cells. Conclusion: Expression of Vegf111b recombinant protein in HEK293 cells is the first step for further research on this protein. Current study has provided the possibility of using this product for future research on angiogenesis and cancer treatments

Construction of eukaryotic integrative vectors by insertion of the integration region attB in pSVM

زمینه و هدف: سالیان زیادی است که انواع مختلفی از سلول‌های تخمدان هامستر چینی (CHO) غالباً برای تولید اکثر محصولات دارویی استفاده می‌شود. تولید دائمی در این سلول‌های میزبان، یک چالش بزرگ است. هدف از این مطالعه، همسانه سازی ناحیه ی توالی اتصال در ژنوم باکتری (attB) در وکتور pSVM با استفاده از سیستم نوترکیبی phiC31 برای بقای طولانی وکتور در سلول‌های C‏HO بوده است. روش بررسی: در این مطالعه تجربی آزمایشگاهی، یک جایگاه برش آنزیم محدود کننده‌ی PvuII به عنوان جایگاه کلونینگ توسط نرم افزار CLC در وکتور pSVM انتخاب که برش با این آنزیم منجر به ایجاد دو انتهای صاف در وکتور شد. دو پرایمر اختصاصی برای جداسازی و تکثیر ناحیه‌ی attB از وکتور pDrBB2 توسط نرم افزار 5 Oligo®طراحی شد. ژل الکتروفورز و آنالیز PCR بر روی قطعه تکثیرشده در جهت تایید ساختار آن انجام شد. یافته ها: ناحیه‌ی attB به طور موفق در داخل وکتور pSVM الحاق شد و توسط ژل الکتروفورز نشان داده شد. در جهت تایید ورود ژن attB در وکتور pSVMواکنش colony PCR از کلنی‌های نوترکیب انجام شد و صحت کلونینگ با مشاهده‌ی باند مربوط به ترادف attB انجام گرفت. نتیجه گیری: در این تحقیق، جایگاه attB در پلاسمیدpSVM همسانه سازی گردید. با بهره گیری از این اطلاعات و وجود جایگاه psedo-attP در کروموزوم سلول‌های CHO، می توان بعد از الحاق ژن اینترفرون انسانی در این پلاسمید، و ورود این پلاسمید و پلاسمید حاوی آنزیم اینتگراز به سلول‌های CHO برای اولین بار زمینه‌ی پایداری بیان این پروتئین را در سلول‌های CHO فراهم کرد

Optimization of Real Time PCR for Precise Measurement of HER2 Overexpression in Breast Cancer Specimens

Background: Breast cancer is one of the most prevalent malignancies among women in various countries. HER2 overexpression, which is due to different reasons, occurs in 20-30% of breast cancers. HER2 gene encodes an 185kDa transmembrane glycoprotein with 1255 amino acids. This active product triggers downstream intracellular signaling pathways inducing cell proliferation and cell survival. These activities can be done in an uncontrolled manner in the cases which HER2 expression undergoes up-regulation. The aim of this study was optimization of Real Time PCR condition. Materials and Methods: RNA purification, cDNA synthesis and then optimization of Real Time PCR method performed respectively. In this study, total RNA was extracted from fresh tissue samples, first strand of total cDNA was synthesized and in the following steps, Real Time PCR was performed to be optimized. Results: Although altering the protocol, annealing temperature and concentration of MgCl2 did not make any improvement and beneficial effects on reactions, changing the concentration of primers to 0.24 pm/&mul was influential to eliminate primer dimers of Real Time PCR reactions. It demonstrated that the copy number of GAPDH transcripts is more than HER2 transcripts in normal breast tissues. Therefore, deviation in 2.5 differences between the Ct value of HER2 and GAPDH indicated that the copy number of HER2 transcripts was increased therefore, HER2 underwent overexpression in these cases. Conclusion: Under these optimized conditions, this technique can be applied as a powerful method in clinical laboratories

Recombinant Streptomyces clavuligerus strain including cas2 gene production and analysis its antibiotic overproduction by bioassay

Background: Streptomyces clavuligerus is one of the most important strain that produce clavulanic acid that wildly used in combination of strong but sensitive to β-lactamase antibiotics in clinics. The cas2 is one of the important genes in the biosynthesis pathway of clavulanic acid. Materials and Methods: The recombinant construct pMTcas2 which contain cas2 gene is obtained from Isfahan University. Recombinant plasmid extracts from streptomyces lividans and confirm by enzyme digestion. The streptomyces clavuligerus protoplast was prepared and transformation was done by using polyethylene glycol. Transformation was confirmed by plasmid extraction and PCR using cas2 specific primers. Finally, bioassay method was used to survey the effect of extra copy of cas2 on clavulanic acid production. Result: Plasmid extraction was initially carried out and the structure of plasmid was confirmed by digestion. The typical white colony was seen on protoplast recovery culture containing thiostrepton antibiotic and gray spores were detected after one week. Plasmid extraction was done from transformed strain and transformation was confirmed by PCR. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Conclusion: The bioassay was done and the diameters of zone of inhibition in control and sample were compared. The results of the bioassay show that amplification of the cas2 gene in multicopy plasmids resulted in a 4.1 fold increase in clavulanic acid production. Overproduction of clavulanic acid decreases the cost of its dependent drug production