Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma: Supplementary Issue: Biomarkers for Colon Cancer

Colorectal cancer (CRC) is the third most common malignancy and the second most common cause of cancer death worldwide. Early detection of CRC can improve patient survival rates; thus, the identification of noninvasive diagnostic markers is urgently needed. MicroRNAs (miRNAs) have extensive potential to diagnose several diseases, including cancer. In this study, we compared the expression pattern of miRNAs from plasma and stool samples of patients with early stages of CRC (I, II) with that of healthy subjects. We performed miRNA profiling using microarrays on plasma and stool samples of eight patients with CRC and four healthy subjects. Seven miRNAs were found to be underexpressed in both plasma and stool samples of patients with CRC versus healthy subjects. Then, we aimed to verify two out of these seven differentially expressed miRNAs (let-7a-5p and let-7f-5p) by quantitative reverse transcriptase polymerase chain reaction on a larger set of plasma and stool samples of 51 patients with CRC and 26 healthy subjects. We confirmed the results of microarray analysis since their expression was significantly lower in stool and plasma samples of patients with CRC. Moreover, receiver operating characteristic curve analysis demonstrated that fecal let-7f expression levels have significant sensitivity and specificity to distinguish between patients with CRC and healthy subjects. In conclusion, if the results are confirmed in larger series of patients, underexpressed let-7a-5p and let-7f-5p miRNAs in both plasma and stool samples of patients with CRC may serve potentially as noninvasive molecular biomarkers for the early detection of CRC.Peer reviewe

A Rapid and Cost-Effective Method for the Purification of Streptococcus pneumoniae Antiserum Using Tangential Flow Filtration Method: Rapid method for purification of S. Pneumoniae antiserum

Antibodies are important agents in the laboratory diagnosis of various microorganisms such as Streptococcus pneumoniae. In this study, we prepared and purified IgG antibody against Streptococcus pneumoniae using novel methods to be used in ELISA and agglutination diagnostic kits. First, Streptococcus pneumoniae was cultured, harvested, and inactivated. Then, bacteria were injected into four mature New Zealand white rabbits, and antisera were developed. Afterward, immunoglobulins were purified using ammonium sulfate precipitation, diafilteration using Tangential Flow Filtration, and ion-exchange chromatography. The purified antibody was then biotinylated and used in ELISA. Gel electrophoresis results showed that the antibody was highly pure. Agglutination test on the primary antigen and clinical samples was four-plus. ELISA results showed that the sensitivity and specificity of the test were 95% and 100%, respectively. Results indicated that our fast method was suitable for anti-Streptococcus pneumoniae IgG purification. Repetitive qualitative and quantitative experiments confirmed high purity of the immunoglobulin. Thus, it could be a suitable candidate to be used in laboratory diagnostic kits. HIGHLIGHTS A rapid and cost-effective method for the purification of Streptococcus pneumoniae antiserum. IgG antibody was prepared and purified by using tangential flow filtration method. The purified antibody can be used in ELISA and agglutination diagnostic kits

Investigating the Relation between miR-31 and RhoA Expressions in Breast Cancer Clinical Samples and Cell Lines: A Controversial Matter

Breast cancer is the most prevalent diagnosed cancer and the second cause of cancer death among women worldwide. There are different mechanisms that play crucial roles in the onset and progression of breast cancer including microRNAs. microRNAs are small noncoding RNAs that regulate gene expression by repressing translation post-transcriptionally. miR-31 is an integrin modulator implicated in different cellular processes such as apoptosis, cell cycle control, and DNA repair. According to the literature, RhoA is one of the genes regulated by miR-31. It has an important role in actin-myosin contraction and subsequently in cell motility and migration in metastasis cascade. Breast cancer cell lines, MCF-7 and MDA-MB-231, as well as normal breast cells, MCF-10A, were cultured. RNA extraction, cDNA synthesis, and SYBR Green I quantitative real-time PCR were used to investigate the expression of miR-31 and RhoA. In addition, 10 metastatic breast cancer clinical samples were analyzed to assess miR-31 and RhoA expression, and normal cells from the same patients were used as controls. Pearson’s correlation co-efficient was applied to find out any probable relation between miR-31 and RhoA expression. Gene expression analyses in MCF-7 cell line showed downregulation of miR-31 while RhoA was upregulated in the cell line (inverse correlation). miR-31 and RhoA were both upregulated in metastatic MDA-MB-231 cell line and downregulated in 90% of clinical samples. Pearson’s correlation co-efficient showed complete positive correlation between miR-31 and RhoA expression. The expression of miR-31 and RhoA is positively correlated, and it is declined in metastatic breast that cancer clinical samples save MDA-MB-231 cells. Unlike previous reports, we found that miR-31 is not the main silencer of RhoA expression. Therefore, more investigation on genes and miRNAs affecting metastasis process can elucidate new biomarkers and therapeutic targets for metastatic breast cancer.Highlights miR-31 is an important miRNA implicated in different cellular processes as well as cancer.The protein product of RhoA gene plays a role in actin-myosin contraction and cell motility in cancer metastasis.We approved bioinformatically and experimentally that RhoA is one of the genes regulated by miR-3

Production of Brucella abortus Antiserum in Goats and its Comparison with Conventional Rabbit Antiserum: Production of Brucella abortus antiserum in goats

Brucellosis, caused by Brucella species, is common among humans and animals, and is one of the most common infectious diseases in Iran. Several assays are available to detect brucellosis, but serological tests may be the only method used in many laboratories. In Iran, different kits are produced in the Pasteur Institute based on agglutination, such as Rose Bengal and 2-mercaptoethanol (2-ME). The positive antiserum control used in these kits is produced in rabbits. The purposeof this study was to produce the antiserum in goats and to compare the titer, quality, and quantity with the antiserum produced in rabbits. The goat immunization was performed by intramuscular injection. Seven days after the last injection, sera were collected. The produced antibody was used in slide and tube agglutination tests with different antigens. The results indicated that the antiserum, produced by the goats had a high quality and quantity. Slide agglutination test showed positive results at 1/6400 dilution with goat antiserum (4+) and 1/1600 dilution with rabbit antiserum (1+). The application of the goats is a better and more appropriate choice, in terms of both cost and quantity, when a high concentration of serum is required. In addition, one goat can provide a higher amount of antiserum compared to several rabbits. HIGHLIGHTS This study presents a robust and time-saving method for the production of an efficient antiserum. Goat is a better and more appropriate host when higher amount of serum is required. The amount of anti-serum produced in goat is approximately 8 times greater than that of rabbit

Determining buffer conditions for downstream processing of VLP-based recombinant hepatitis B surface antigen using multimodal resins in bind-elute and flow-through purification modes

Abstract The difficulties in purification of VLP-based recombinant hepatitis B surface antigen (rHBsAg) are mainly emerged from inefficient semi-purification step plus proteins physicochemical properties and these issues make the downstream processing (DSP) very lengthy and expensive. In this study, optimization of rHBsAg (recombinantly-expressed in Pichia pastoris) DSP was performed using selection of buffering conditions in the semi-purification step. In the semi-purification optimization step, up to 73% of the protein impurities were eliminated and the utmost increase in rHBsAg purity (ca. 3.6-fold) was achieved using 20 mM sodium acetate, pH 4.5. By using rHBsAg binding and nonbinding situations obtained from the response surface plot in design of experiments (DOE), additional bind-elute and flow-through purification mode experiments were conducted and rHBsAg with high purity (near 100%) and recovery (> 83%) was achieved. Following assessment of critical quality attributes (i.e., purity, particle size distribution, host cell DNA, host cell protein, secondary structures, specific activity and relative potency), it was indicated that the characteristics of rHBsAg purified by the new DSP were similar or superior to the ones obtained from conventional DSP. The purification performance of the resin was constantly retained (97–100%) and no significant resin damage took place after 10 adsorption–elution–cleaning cycles. The new DSP developed for production of rHBsAg in this study can substitute the conventional one with granting satisfactory target protein quality, long-lasting resin efficacy, shorter and less expensive process. This process may be also employable for purification of both non-VLP- and VLP- based target proteins expressed in the yeast

Microsatellite instability detection using BAT-25 and BAT-26 by Real Time PCR and HPLC in colorectal cancer

Background: Colorectal Cancer (CRC) is the third common cancer in the world. One of the pathways in colorectal tumor genesis is Microsatellite Instability (MSI+). MSI is detected in about 15% of all colorectal cancers. Colorectal tumors with MSI have dis-tinctive features compared with Microsatellite Stable (MSS) tumors. Due to the high percentage of MSI+ in patients with CRC in Iran, screening of this type of CRC is im-perative. In current study, two markers (BAT-26 and BAT-25) were used to determine an appropriate screening technique with high sensitivity and specificity to diagnose MSI status in patients with CRC. Methods: Allelic variation in two markers (BAT-26 and BAT-25) was analyzed in tis-sues and sera of 44 normal volunteers and tumor and matched normal mucosal tissues as well as sera of 44 patients with sporadic colorectal cancer by Real Time PCR (Hy-bridization probe) and High-Performance Liquid Chromatography (HPLC) techniques. The sensitivity and specificity of Real Time PCR and HPLC compared with sequencing as gold standard. The data were statistically analyzed using Student’s t-test and 2 or fisher exact test, where applicable with (P<0.05). Receiver-operating-characteristic (ROC) curves were used to evaluate the sensitivity and specificity. Results: The sensitivity and specificity of BAT-26 with Real Time PCR method (Hy-bridization probe) were 100% in comparison with gold standard method. Whereas the sensitivity and specificity of BAT-26 and BAT-25 with HPLC were 83%, 100% and 50%, 97%, respectively. Neither HPLC nor Real time PCR could detect circulating DNA with MSI property in sera. Conclusion: The sensitivity and specificity of real time PCR in MSI detection is the same as sequencing method and more than HPLC. BAT-26 marker is more sensitive than BAT-25 and MSI detection with Real time PCR could be considered as an accu-rate method to diagnose MSI in CRC tissues not sera

Over-expression of NOTCH1 as a biomarker for invasive breast ductal carcinoma

Breast cancer is the leading cause of cancer-related death in women worldwide. Invasive ductal carcinoma (IDC) is the most frequent invasive form of breast cancer followed by metastasis. There is no accepted marker for distinguishing this form from other less aggressive forms of breast cancer. Therefore, finding new markers especially molecularly detectable ones are noteworthy. It has been shown that NOTCH1 has been overexpressed in the patients with breast cancer, but no study has investigated the expression of NOTCH1 and its correlation with other molecular and hormonal markers of breast cancer so far. In the current study, 20 breast cancer tissues and 20 matched adjacent normal breast tissue from breast cancer patients were obtained and categorized in two groups: patients with IDC and patient with other types of breast cancer. Gene expression analysis using real-time PCR showed that the NOTCH1 gene was significantly overexpressed in patients with IDC. We also found a slight correlation between NOTCH1 overexpression and p53 accumulation in the cancerous cells confirmed by Immunohistochemistry (IHC). This results showed that it is possible to introduce NOTCH1 expression as a novel biomarker of IDC, alone or preferably accompanied by IHC of p53. We also can design new therapeutic agents targeting NOTCH1 expression for inhibition of metastasis in ductal breast carcinoma

Simultaneous Underexpression of let-7a-5p and let-7f-5p microRNAs in Plasma and Stool Samples from Early Stage Colorectal Carcinoma

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