International Journal of Molecular and Cellular Medicine (IJMCM)

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One of the applications of gold nanoparticles (GNPs) in medicine is radiation dose-enhancing effect. Although there are many simulations, in vitro and in vivo evidence that GNPs can enhance significantly the radiation dose effect of orthovoltage beams. These beams compared with megavoltage (MV) beams, have limited applications in radiotherapy. In order to evaluate GNPs radiosensitization performance with MV beams in-vivo, we used two most clinically used X-ray beams (6 and 18 MV) with the dose of 20 Gy for each mouse. Intratumoral injection of 50 nm GNPs with the concentration of 5 mg ml-1 was applied to melanoma tumor growing in the left leg of 7 to 8 mice in 4 control and treatment groups of C57BL/6 mice. Albeit, using 10 cm plexiglass jig phantom in the beam path improved the radiation - treatments, the statistical differences between groups were not significant. According to the results, it is concluded that mice can be treated with smaller tumors and higher concentrations of GNPs in MV radiation beams.

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Drug resistance (especially multiple drug resistance) in Mycobacterium tuberculosis makes global concerns in treatment and control of tuberculosis. Rapid diagnosis of drug resistant strains of the bacteria has vital importance in the prognosis of the disease. The aim of this study was to identify the mutations responsible for drug resistance in Mycobacterium tuberculosis strains derived from patients with tuberculosis using line probe assay (LPA) method which rapidly detect drug resistant strains and respective mutations. Sputum samples from tuberculosis patients were collected and cultured on Lowenstein– Jensen medium, and then the colonies of Mycobacterium tuberculosis from cultures of 54 bacterial positive cases were randomly chosen for DNA extraction. Bacterial DNA was extracted using standard Cetyl Trimethyl Ammonium Bromide (CTAB) method. In order to identify drug resistant strains and related mutations, LPA method was applied. Three subjects out of 54 investigated cases were resistant to quinolone (5.5%), and resistance to kanamycin/ amikacin, streptomycin, rifampin, and isoniazid were observed in 3 (5.5%), 4 (7.4%), 3 (5.5%), and 2 (3.7%) of the Mycobacterium tuberculosis strains, respectively. In the present study, 4 cases (7.4%) were detected to be resistant to more than one drug. Since LPA is a rapid method that simultaneously detects mutations involved in drug resistance, applying this method in the prediction of drug resistance and selecting appropriate treatment in tuberculosis patients is recommended.

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Bladder cancer (BC) ranks the second most common genitourinary tract malignant tumor with high mortality and 70% recurrence rate worldwide. MiRNAs expression has noticeable role in bladder tumorigenesis. The purpose of this study was to assess miR-200c, miR-30b and miR-141 in tissue samples of patients with BC and healthy adjacent tissue samples and their association with muscle invasion, grade and the size of the tumor. Transurethral resection tissue samples were collected from thirty- five newly diagnosed untreated patients with BC from 2013 to 2014. The control group consisted of adjacent normal urothelium. All samples, observed by two pathologists, were diagnosed transitional cell carcinomas (TCC) with the proportion of tumor cells greater than 80%. Total RNA including miRNAs was extracted from about 50 mg tissue samples by applying TRIzol reagent. 2(-ΔΔ CT) method was used to calculate relative quantification of miRNA expression. Two of 35 patients were females and the other 33 were males. Invasion to bladder muscle was observed in 23 (67%) cases. MiR-141, miR-200-c and miR30-b were up-regulated in 91%, 79% and 64% of malignant tissues, respectively. Down-regulation of miR-141 had a strong association with muscle invasion (P= 0.017). Significant inverse correlation between grading and miRNA-141 level was observed (P= 0.043).

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Cryptosporidium is one of the most common causes of childhood diarrhea in developing countries. The aim of this randomized pilot study was to detect and characterize infective species and determine the genotypes of Cryptosporidium parasites in pediatric patients suffering from diarrhea in North West of Iran. A total of 113 fecal samples were collected from diarrheic children hospitalized in Tabriz Pediatric Hospital. The amplification of small subunit ribosomal RNA gene was performed using a nested polymerase chain reaction protocol and its products were digested using two restriction enzymes for Cryptosporidium species and genotype differentiation. Cryptosporidium oocysts were found in 2 (1.76%) children with diarrhea and restriction pattern revealed the presence of C.parvum bovine genotype in both positive fecal samples. The findings indicate that Cryptosporidium parvum is responsible for cryptosporidiosis in children in the study region and probably zoonotic transmission is the predominant route of parasite transmission.

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Memory formation is the most important aspect of a vaccine which can guarantee long-lasting immunity and protection. The main aim of the present study was to evaluate the memory immune responses after immunization with a mini vaccine. Mice were immunized with human immunodeficiency virus-1 P24-Nef fusion peptide and then cellular and humoral immune responses were evaluated. In order to determine long-lived memory, immune responses were monitored for 20 weeks after final immunization. The results showed that the candidate vaccine induced proliferation and cytotoxic T lymphocyte responses and shifted cytokine patterns to T helper-1 profile. Evaluation of humoral immune responses also showed an increase in total peptide specific-IgG titer and a shift to IgG2a humoral response. Monitoring of immune responses at weeks 4, 12 and 20 after last immunization showed that immunologic parameters have been sustained for 20 weeks. Our findings support the notion that long-lived memory responses were achieved using a mini vaccine immunization.

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Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.

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Torque Teno virus (TTV) is a transfusion transmitted virus that seems to be involved in several complications such as acute respiratory diseases, liver diseases, AIDS, cancer, and autoimmune reactions.  In the present study the frequency of TTV was investigated among β- thalassemia (BT) and haemodialysis (HD) patients (high risk patients for TTV) in Mazandaran province, Iran. DNA was extracted from the serum of 82 BT and 100 HD patients, and nested PCR method was applied to detect TTV DNA. The aspartate transaminase (AST) and alanine transaminase (ALT) enzyme levels in BT patients were measured using photometric assay.  The mean ages of BT and HD patients were 23.4± 5.4 and 48.8 ± 8.2 years, respectively.  21% of HD, and 26.8% of BT patients were infected with TTV, respectively. The frequency of TTV was not significantly different between two groups of patients and there was no significant correlation between sex and TTV infection.   The mean AST and ALT levels in TTV positive BT patients was not significantly higher than TTV negative cases. The present study showed that TTV prevalence in BT patients with recurrent blood transfusion was not significantly higher than HD patients. The investigation of TTV prevalence in healthy individuals is recommended to identify if transfusion or dialysis is associated with higher TTV infection. Besides, although TTV infection did not change AST and ALT enzymes in BT patients, the liver involvement may still exist in these patients.

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The type IV pili (T4P) is a major virulence factor of Pseudomonas aeruginosa (P. aeruginosa) that is associated with primary adhesion, biofilm formation and twitching motility. This study focuses on the introduction of a novel biologically active subunit vaccine derived from the disulfide loop (DSL) of P. aeruginosa pilin. We investigated the expression of the novel PilA in-frame with pET26a vector, which contains three domains, that each domain contains three tandem repeats. The flexible (GGGGS) and (GGGGS)3 linkers were linked between the three tandem repeats and each pilA domain, respectively.  The recombinant construct (pET26b/pilA) was transformed and expressed in Escherichia coli BL21 (DE3). The reactivity of specific antiserum against PilA was assessed by ELISA method. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis and twitching inhibition assays. The pET26b/pilA plasmid was confirmed by enzymatic digestion. The purified PilA protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen was 1:8. The results of opsonophagocytosis assay revealed that the antibodies raised against PilA promoted phagocytosis of the PAO1 and 6266E strains, to some extent (17.5% and 16.3%, respectively), so that the twitching inhibition test confirmed this result.  Taken together, these are preliminary results based on a first chimerical structure failure in order to induce antibodies that promote the opsonization and eradication of the pathogen. Therefore, the biological activity of the PilA protein showed that it should be introduced with other proteins or target antigens against P. aeruginosa in the future studies.

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Bladder cancer is the second most common cancer in the genitourinary tract, showing often recurrence and progresse into invasive states. Epigenetic changes, such as microRNA alteration are involved in bladder cancer tumorigenesis through a variety of signaling pathways. The epigenetic state depends on geographic and lifestyle conditions. The aim of this study was to investigate the expression level of microRNA-99a and microRNA-205 in bladder cancer in Iranian populations and to determine the relationship between their expressions with clinicophatological features. 36 patients with bladder cancer were included in the study. The control group was the healthy adjacent tissue of the same patients. Total RNA was extracted from approximately 50 mg tissue using TRIzol reagent. cDNA was synthesized and Real-Time PCR was carried out using specific primers. The Unisp6 rRNA was used as a reference gene. A significant decrease was found in the expression level of miR-99a in tumor samples, compared to healthy adjacent tissues (P<0.001). The increased expression level of miR-99a was significantly associated with muscle invasion (P=0.02). The receiver operating characteristic (ROC) analysis for miR-99a showed AUC value equal to 0.944, with specificity of 97%, sensitivity of 91%, and cut off value of 8.31 (P<0.001). A significant association was found between smoking and miR-99a (P=0.04) and miR-205 (P= 0.01) expression levels. Dramatic down-regulation of miR-99a in bladder cancer tissues confirmed the tumor suppressor role of miR-99a in bladder cancer. A higher amount of miR-99a expression was associated with invasive bladder cancer. According to ROC analysis, miR-99a could be considered as a valuable diagnostic biomarker.

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Colorectal cancer (CRC) is one of the most prevalent diagnosed cancers and a common cause of cancer-related mortality. Despite effective clinical responses, a large proportion of patients undergo resistance to radiation therapy. Therefore, the identification of efficient targeted therapy strategies would be beneficial to overcome cancer radioresistance. Doublecortin-like kinase 1 (DCLK1) is an intestinal and pancreatic stem cell marker that showed overexpression in a variety of cancers. The transfection of DCLK1 siRNA to ‎normal HCT-116 cells was performed, and then cells were irradiated with X-rays. The effects of DCLK1 inhibition on cell survival, apoptosis, cell cycle, DNA damage response (ATM and γH2AX proteins), epithelial- mesenchymal transition (EMT) related genes (vimentin, N‐cadherin, and E-cadherin), cancer stem cells markers (CD44, CD133, ALDH1, and BMI1), and β‐catenin signaling pathway (β‐catenin) were evaluated. DCLK1siRNA downregulated DCLK1 expression in HCT-116 cells at both mRNA and protein levels (P < 0.01). Colony formation assay showed a significantly reduced cell survival in the DCLK1 siRNA transfected group in comparison with the control group following exposure to 4 and 6 Gy doses of irradiation (P < 0.01). Moreover, the expression of cancer stem cells markers (P < 0.01), EMT related genes (P < 0.01), and DNA repair proteins including pATM (P < 0.01) and γH2AX (P < 0.001) were significantly decreased in the transfected cells in comparison with the nontransfected group after radiation. Finally, the cell apoptosis rate (P < 0.01) and the number of cells in the G0/G1 phase in the silencing DCLK1 group was increased (P < 0.01). These findings suggest that DCLK1 can be considered a promising therapeutic target for the treatment of radioresistant human CRC.

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MicroRNAs (miRNAs) have emerged as essential gene expression regulators associated with human diseases such as colorectal cancer (CRC). The purpose of this study was to evaluate the expression of miR-330-3p and its target gene BMI1 in tissue samples of patients with CRC, polyp, and healthy adjacent tissue samples and their association with clinicopathological and demographic factors such as age, tumor stage, grade, and lymph node invasion of the tumor. Following the extraction of total RNA from approximately 50 mg of colon and rectum tissue of 82 patients with CRC, 13 polypoid lesions, and 26 marginal healthy tissues using RiboEx reagent, cDNA synthesis was performed, and then quantitative real-time PCR was used to detect the expression levels of miR-330-3p and BMI1. Alterations in the gene expression were assessed using the 2(-∆∆ CT) method. The expression of miR-330-3p in all of the CRC samples was significantly lower than in adjacent healthy tissues and polyp (P<0.001). BMI1 was up-regulated in 97.9% of CRC tissue compared to healthy adjacent tissues and polyps (P<0.001). A negative reverse correlation between the miR-330-3p and BMI1 gene was observed in the CRC samples (r= -0.882, P<0.001). Down-regulation of miR-330-3p and BMI1 overexpression strongly correlates with higher tumor stage and lymph node invasion. The AUC for miR-330-3p and BMI1expression was 0.982 (sensitivity, 98.5%; specificity, 78.8%), and 0.971 (sensitivity, 97.6%; specificity, 84.6%) (P<0.001), respectively. Our results indicated that miR-330-3p and BMI1 expression probably could be considered potential diagnostic or prognostic biomarkers for CRC patient.

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Polycystic ovary syndrome (PCOS) is the most prevalent endocrine disorder of women in reproductive age with significant effects on reproductive and metabolic functions. Many molecular players may be involved in PCOS pathology; however, miRNAs possess great ability in gene expression control in normal ovarian function and folliculogenesis. We appraised the relative expression of miR-146a, miR-222, miR-9, and miR-224 in serum and follicular fluid (FF) of PCOS patients compared to control subjects. PCOS (n = 35) and control (n = 30) subjects were recruited in the study during their enrolment in IVF cycles. Serum and FF of human subjects were collected and stored. Total RNA was isolated from samples and cDNA was synthesized using miRNA-specific stem-loop RT primers. Quantitative real-time PCR was used to evaluate the expression of miRNAs relative to U6 expression. The predictive value of miRNAs’ expression for discrimination of PCOS patients from control subjects was evaluated by receiver-operating characteristic (ROC) curve analysis. miR-224 was not detected in serum and FF samples. Significantly, higher levels of miR-146a and miR-9 in serum of PCOS group were detected. In contrast, relative expression of miR-146a and miR-9 significantly decreased in FF. In PCOS group, relative expression of all detected miRNAs was elevated in serum in comparison to FF, whereas in control group no change was noticed. Combination of FF miRNAs showed improved predictive value with area under the ROC curve (AUC) of 0.84, 93.8% sensitivity, and 83.3% specificity. Contradicting alternations of miRNAs in serum and FF are indicative of different sources of miRNAs in body fluids. Presumptive target genes of studied miRNAs in signaling pathways may show the potential role of these miRNA in folliculogenesis.