Dysregulated Expression of Long Non-Coding RNA MINCR and EZH2 in Colorectal Cancer

Background: As critical regulators, lncRNAs have attracted attention from researchers for diagnostic, prognostic, and therapeutic purposes in human carcinogenesis via interfering with mRNAs such as EZH2. Nevertheless, the potent roles and molecular mechanisms of these RNAs in CRC are not clearly known. Methods: In this study, the tissue expressions of lncRNA MINCR and EZH2 mRNA between colorectal tumors and polyps were compared with the adjacent normal tissues collected from 114 Iranian patients, using real-time PCR method. Furthermore, the correlation of the expression levels of MINCR and EZH2 with other clinical parameters was evaluated. Results: The significant overexpression of MINCR and EZH2 were observed in the CRC tissues compared to control tissues (p < 0.0001). This observation confirmed the association of these expression enhancements with the pathological stage of CRC patients. Conclusion: Our findings revealed that the expression of MINCR significantly alters during CRC development, and it can be identified as a potential biomarker for the detection of CRC.

Combination of human papillomaviruses L1 and L2 multiepitope constructs protects mice against tumor cells.

Different types of cancer including cervical (>90%), anal (~88%), vaginal (~40%), and penile (~40%) cancers are associated with human papillomaviruse (HPV) infections. Three prophylactic vaccines (Cervarix, Gardasil, and Gardasil-9) were approved to provide immuno-protection against certain types of HPVs. Currently, next-generation HPV vaccines such as L1/L2-based vaccines are being developed to provide broad-type HPV protection. In this study, we introduced a comprehensive framework for design of L1/L2 polyepitope-based HPV vaccine candidate. This framework started with protein sequence retrieval and followed by conservancy analysis between high-risk HPVs, MHC-I and MHC-II epitope mapping, and B-cell and T-cell epitope mapping. Subsequently, we performed Tap transport and proteasomal cleavage, population coverage, antigenicity, allergenicity and cross-reactivity. After that, peptide-MHCI/II flexible docking and comprehensive conservancy analysis against all HPV types were carried out. The next steps were prediction of interferon-gamma and interleukin-10 inducing epitopes, epitope selection and construct design, tertiary structure prediction, refinement and validation, discontinuous B-cell epitope prediction, vaccine-TLR4 molecular docking, and codon optimization. Our data showed that two designed vaccine constructs harboring 8 L1 peptides or 7 L2 peptides, individually were highly conserved between all well-known HPV types. In addition, the combination of in silico/in vivo approaches indicated the potential ability of L1 and L2 polyepitope constructs for development of next generation prophylactic/therapeutic HPV vaccine.

Association of GRIA1 polymorphisms with ovarian response to human menopausal gonadotropin in Iranian women.

OBJECTIVE: Glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) is a subunit of a ligand-gated ion channel that regulates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by controlling the release of gonadotropin-releasing hormone. Few studies have investigated the association between the GRIA1 gene and human infertility. This study evaluated the association of the GRIA1 rs548294 C > T and rs2195450 G > A polymorphisms with the ovarian response to human menopausal gonadotropin (HMG) in Iranian women. METHODS: One hundred women with histories of at least 1 year of infertility were included. On the second day of menstruation, patients were injected with HMG; on the third day, blood samples were collected. After hormonal analysis, the GRIA1 rs548294 C > T and rs2195450 G > A genotypes of samples were identified via the restriction fragment length polymorphism method, and on day 9, the number of follicles was assessed via ultrasound. RESULTS: For the GRIA1 rs548294 C > T and rs2195450 G > A single nucleotide polymorphisms, the subjects with CT and GG genotypes, respectively, displayed the highest mean FSH level, LH level, and number of follicles on day 9 of the menstrual cycle (p< 0.05). Significant positive correlations were observed between LH and FSH (p< 0.01), LH and follicle count (p< 0.01), FSH and age (p< 0.05), follicle count and age (p= 0.048), and FSH and follicle count (p< 0.01). CONCLUSION: This study showed a significant relationship between GRIA1 polymorphisms and ovarian response to the induction of ovulation. Therefore, determining patients' GRIA1 genotype may be useful for improving treatment and prescribing suitable doses of ovulation-stimulating drugs.

Evaluation of beta-thalassemia in the fetus through cffDNA with multiple polymorphisms as a haplotype in the beta-globin gene.

OBJECTIVE: Invasive biopsy during the pregnancy is associated with an abortion risk of approximately 1% for the fetus. Free fetal DNA in maternal plasma is an excellent source of genetic material for prenatal molecular diagnoses. This study was conducted to investigate beta-thalassemia mutation in the fetus through maternal blood with multiple polymorphisms as haplotypes in the beta-globin gene. METHODS: In this study, a total of 33 beta-thalassemia carrier (minor) couples were genotyped by ARMS-PCR for IVSII-IG>A mutation. During pregnancy, 10mL of blood was collected from pregnant women, and DNA was extracted by the magnetic bead-based extraction, and fetal DNA was enriched with AMPure XP kit. Five polymorphisms in 4 haplotype groups were evaluated by the Sanger Sequencing method. Finally, results were compared with those of the invasion method. RESULTS: Participants in study were 33 couples, mean age of the men was 26±5 years, and mean age of women was 23±4 years, and mean MCV, MCH, HbA2 blood parameters were 62.4±5.3, 19.6±3.1, 4.2±2.1 respectively. A total of 33 fetuses were genotyped for IVSII-IG>A mutation. Nine fetuses were affected, 10 fetuses were normal and 14 fetuses were carrier of beta-thalassemia. Sensitivity and specificity of Sanger Sequencing were equal to 88.8% and 91.6% respectively. Positive and negative predictive values were obtained as 80% and 95.6%, respectively. CONCLUSION: Mutational status of the fetus can be assessed by determining inheritance of paternally-derived alleles based on detection of haplotype-associated SNP in maternal plasma. Magnetic-based DNA extraction and fetal DNA enrichment are very simple and easy to perform and have satisfactory accuracy.

Development of multiepitope therapeutic vaccines against the most prevalent high-risk human papillomaviruses.

Aim: Our goal was the development of DNA- or peptide-based multiepitope vaccines targeting HPV E7, E6 and E5 oncoproteins in tumor mouse model. Materials & methods: After designing the multiepitope E7, E6 and E5 constructs from four types of high risk HPVs (16, 18, 31 & 45) using bioinformatics tools, mice vaccination was performed by different homologous and heterologous modalities in a prophylactic setting. Then, anti-tumor effects of the best prophylactic strategies were studied in a therapeutic setting. Results: In both prophylactic and therapeutic experiments, groups receiving homologous E7+E6+E5 polypeptide, and heterologous E7+E6+E5 DNA prime/polypeptide boost were successful in complete rejection of tumors. Conclusion: The designed multiepitope constructs can be considered as promising candidates to develop effective therapeutic HPV vaccines.

Evaluation of Myc Gene Expression as a Preventive Marker for Increasing the Implantation Success in the Infertile Women.

BACKGROUND: There are numerous couples worldwide currently suffering from infertility. Several factors, including genetic abnormalities are involved in infertility. In this study, we investigated the expression of myc gene in uterine tissue of infertile women. The protein encoded by this gene is one of the important transcription factors involved in the expression of many genes in the embryonic growth, and development pathways. METHODS: There are about 45 samples of uterine tissue from women with primary and secondary infertility were involved in this study. After extracting RNA and synthesizing cDNA, using specific primers for the myc gene and the beta-actin gene (as an internal control), gene expression was evaluated by Real-time RT-PCR method. RESULTS: The results of myc gene expression analysis showed no significant pattern between the affected and healthy women, however decreasing of its expression should not be rejected. CONCLUSIONS: This study is the first report about myc gene expression and its relation with the primary and secondary infertility. Myc gene expression study at different times of sexual period of infertile woman is suggested. Also, we proposed here, as a preventive strategy, improvement of the expression level of myc gene by some methods, such as hormone therapy, can increase the implantation success in the infertile women.

Association of Candidate Single Nucleotide Polymorphisms Related to Candidate Genes in Patients With Schizophrenia.

INTRODUCTION: Schizophrenia is a chronic heterogenic neurodevelopment disorder. Many genes interfere in the development of SCZ. All four genes, NrCAM, PRODH, ANK3, and ANKK1, which were evaluated in this study, were previously reported to be associated with Schizophrenia. The NrCAM contributes to creating cognitive deficiencies through the CAM's signaling pathway. PRODH plays a vital role in creating SCZ negative symptoms through the signaling pathway of glutamatergic and NMDA receptors. ANK3 affects ion channel and molecular adhesion in Ranvier and initial segments of axons, leading to mental retardation, sleep disorder, and SCZ. ANKK1 encodes a protein kinase and was reported to be associated with alcohol addiction, Attention Deficit Hyperactivity Disorder (ADHD), and SCZ. METHODS: The subjects were selected from Schizophrenic patients referring to the Psychiatric Ward of Imam-Hussein Hospital and Schizophrenic Patients Support Institution (AHEBBA). 95 (30 Schizoaffective patients, 57 Paranoid patients, and 8 disorganized) patients were recruited as the subjects in the present case-control association study. 120 healthy subjects were recruited from the Tehran Medical Genetics Laboratory staff and a group of students from the Islamic Azad University of Science and Research in Tehran. The genotypes were determined with molecular genotyping techniques of PCR-RFLP, ARMS-PCR, and Cycle sequencing. Results were analyzed by the Chi-Square test using SPSS V. 24 and R, SNP STATE Package to investigate significant differences between cases and controls. RESULTS: The incidence of schizophrenia was 68% and 32% among men and women, respectively. The evaluation of the allelic association between schizophrenia and all the candidate SNPs showed a significant association between NrCAM's SNP rs10235968 and SCZ (P=0.001). Haplotype T, T, C in rs10235968, rs6967368, rs3763463, respectively, within the NrCAM gene, showed significant association with schizophrenia disorder (P=0.0001). CONCLUSION: No association was found between other candidate SNPs and SCZ among the subjects.

LNA Inhibitor in microRNA miR-23b as a Potential Anti-proliferative Option in Human Hepatocellular Carcinoma.

PURPOSE: Dysregulation of microRNAs (miRNAs) has been shown to be involved in the pathogenesis and progression of many malignancies. Human hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the third cause of cancer-related deaths. Recent data suggest that microRNA-23b (miR-23b) is significantly high in different types of cancer, specifically human hepatocellular carcinoma. Locked nucleic acid (LNA)-modified oligonucleotides have recently been suggested as a novel approach for targeting miRNAs as antisense-based gene silencing. The aim of this study was to explore the functional role of LNA-anti-miR-23b in a HepG2 (hepatocarcinoma) cell line. METHODS: HepG2 cells were transfected with LNA-anti-miR-23b for 24, 48, and 72 h. Quantitative real-time reverse transcriptase-PCR (qRT-PCR) was performed to assess miR-23b expression by LNA-anti-miR-23b. The viability of the cells was evaluated by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. RESULTS: LNA-anti-miR-23b was successfully transfected into human HepG2 cells and suppressed the miR-23b. LNA-anti-miR-23b reduced the invasive behaviors of HepG2 cells after 24 h, compared to untreated cells and scrambled LNA-transfected cells, and this effect was more pronounced after 72 h. CONCLUSIONS: Our findings suggest that inhibition of miR-23b could be used as a novel approach in inhibition of HCC proliferation.

Correlation of HER2, MDM2, c-MYC, c-MET, and TP53 Copy Number Alterations in Circulating Tumor Cells with Tissue in Gastric Cancer Patients: A Pilot Study

Background: The analysis of the gene copy number alterations in tumor samples are increasingly used for diagnostic and prognostic purposes in patients with gastric cancer (GC). However, these procedures are not always applicable due to their invasive nature. In this study, we have analyzed the copy number alterations of five genes (HER2, MDM2, c-MYC, c-MET, and TP53) with a fixed relevance for GC in the circulating tumor cells (CTCs) of GC patients, and, accordingly, as a potential approach, evaluated their usage to complete primary tumor biopsy. Methods: We analyzed the status of the copy number alterations of the selected genes in CTCs and matched biopsy tissues from 37 GC patients using fluorescence in situ hybridization. Results: HER2 amplification was observed in 2 (5.41%) samples. HER2 gene status in CTCs showed a strong agreement with its status in 36 out of 37 patients' matched tissue samples (correlation: 97.29%; Kappa: 0.65; p < 0.001). MDM2 amplification was found only in 1 (2.70%) sample; however, the amplification of this gene was not detectable in the CTCs isolated from this patient. c-MYC amplification was observed in 3 (8.11%) samples, and the status of its amplification in the CTCs indicated a complete agreement with its status in the matched tissue samples (correlation: 100%; Kappa: 1.0). Conclusion: Our work suggests that the amplification of HER2 and c-MYC is in concordance with the CTCs and achieved biopsies, and, consequently, CTCs may act as a non-invasive alternative for recording the amplification of these genes among GC patients.

In silico/In vivo analysis of high-risk papillomavirus L1 and L2 conserved sequences for development of cross-subtype prophylactic vaccine.

Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. Nowadays, the virus-like particles (VLPs) based on L1 proteins have been considered as the best candidate for vaccine development against HPV infections. Two commercial HPV (Gardasil and Cervarix) are available. These HPV VLP vaccines induce genotype-limited protection. The major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. Thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk HPV. In this study, we developed DNA constructs (based on L1 and L2 genes) that were potentially immunogenic and highly conserved among the high-risk HPV types. The framework of analysis include (1) B-cell epitope mapping, (2) T-cell epitope mapping (i.e., CD4+ and CD8+ T cells), (3) allergenicity assessment, (4) tap transport and proteasomal cleavage, (5) population coverage, (6) global and template-based docking, and (7) data collection, analysis, and design of the L1 and L2 DNA constructs. Our data indicated the 8-epitope candidates for helper T-cell and CTL in L1 and L2 sequences. For the L1 and L2 constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of 95.55% and 96.33%, respectively. In vitro studies showed high expression rates of multiepitope L1 (~57.86%) and L2 (~68.42%) DNA constructs in HEK-293T cells. Moreover, in vivo studies indicated that the combination of L1 and L2 DNA constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against C3 tumor cells (the percentage of tumor-free mice: ~66.67%). Thus, the designed L1 and L2 DNA constructs would represent promising applications for HPV vaccine development.

A comprehensive in silico analysis for identification of therapeutic epitopes in HPV16, 18, 31 and 45 oncoproteins.

Human papillomaviruses (HPVs) are a group of circular double-stranded DNA viruses, showing severe tropism to mucosal tissues. A subset of HPVs, especially HPV16 and 18, are the primary etiological cause for several epithelial cell malignancies, causing about 5.2% of all cancers worldwide. Due to the high prevalence and mortality, HPV-associated cancers have remained as a significant health problem in human society, making an urgent need to develop an effective therapeutic vaccine against them. Achieving this goal is primarily dependent on the identification of efficient tumor-associated epitopes, inducing a robust cell-mediated immune response. Previous information has shown that E5, E6, and E7 early proteins are responsible for the induction and maintenance of HPV-associated cancers. Therefore, the prediction of major histocompatibility complex (MHC) class I T cell epitopes of HPV16, 18, 31 and 45 oncoproteins was targeted in this study. For this purpose, a two-step plan was designed to identify the most probable CD8+ T cell epitopes. In the first step, MHC-I and II binding, MHC-I processing, MHC-I population coverage and MHC-I immunogenicity prediction analyses, and in the second step, MHC-I and II protein-peptide docking, epitope conservation, and cross-reactivity with host antigens' analyses were carried out successively by different tools. Finally, we introduced five probable CD8+ T cell epitopes for each oncoprotein of the HPV genotypes (60 epitopes in total), which obtained better scores by an integrated approach. These predicted epitopes are valuable candidates for in vitro or in vivo therapeutic vaccine studies against the HPV-associated cancers. Additionally, this two-step plan that each step includes several analyses to find appropriate epitopes provides a rational basis for DNA- or peptide-based vaccine development.

The rs4846049 polymorphism in the 3'UTR region of the MTHFR gene increases the migraine susceptibility in an Iranian population.

INTRODUCTION: Migraine is a painful complex neurovascular disease characterized by recurrent moderate-to-severe headaches. Increased level of homocysteine is related to dilation of cerebral vessels and endothelial injury that could trigger migraine attacks. Functional polymorphisms in the MTHFR gene affect homocysteine metabolism and, therefore, play an important role in the etiology of the disease. OBJECTIVES: We aimed to investigate the possible association between MTHFR gene rs4846049, C677T, and A1298C polymorphisms and the risk of migraine in Iranian population. METHODS: In this genetic association study, 498 individuals were enrolled, including 223 migraine patients and 275 healthy controls. Genotyping was performed using tetra-primer ARMS-PCR for rs4846049 and PCR-restriction fragment length polymorphism for C677T and A1298C polymorphisms. RESULTS: The association between rs4846049 and C677T polymorphisms and migraine was observed. For the rs4846049 polymorphism, the association was detected under a dominant model (P=0.007; odds ratio [OR] =0.60; 95% confidence interval [CI], 0.41-0.87), and for the C677T polymorphism, the TT genotype frequency was significantly different in the studied groups (P=0.009; OR =2.48; 95% CI, 1.25-4.92). No significant differences in the genotype or allele frequencies were found for the A1298C polymorphism between the migraineurs and controls. CONCLUSION: Present data provide evidence for the association of rs4846049 and C677T polymorphisms in the MTHFR gene and migraine. Further studies are required to validate the significance of the studied genetic variations in diverse ethnic populations.

Bias in detection of Mycobacterium tuberculosis polyclonal infection: Use clinical samples or cultures?

The application of MIRU-VNTR has unveiled that infection by Mycobacterium tuberculosis can be polyclonal. Our comparative study demonstrated that based on the studied samples (clinical specimen or culture) detection of polyclonal M. tuberculosis infection can be significantly different.

Evaluation of the impact of polyclonal infection and heteroresistance on treatment of tuberculosis patients.

Mixed strain infections of Mycobacterium tuberculosis make diagnosis, treatment, and control of tuberculosis (TB) more difficult. This study was aimed to evaluate the relationship between mixed infections, antibiotic resistance patterns and treatment of TB patients. In this study, among 2850 suspected TB clinical samples, a total of ninety-six clinical samples from 66 TB confirmed patients were subjected to the 24-locus variable-number tandem repeat method to evaluate the prevalence of mixed infections. For all studied strains, 288 colonies (three individual clones for each sample) were isolated from different colonies and separately analyzed by the Drug Susceptibility Test (DST). For all patients, follow up was done after 6 months of treatment. Based on direct 24 loci MIRU-VNTR, in the 66 TB patients, 53% (35/66) showed mixed infection. In the mixed samples, 45.71% (16/35) showed different antibiotic resistant patterns. Among the mixed infection patients, eight (22.9%; 8/35) showed treatment failure after six- month therapy. Six of these non-treated patients (75%; 6/8) had different antibiotic resistant patterns. We conclude that mixed infections, have a negative impact on treatment of TB patients especially when co-infecting M. tuberculosis strains display heteroresistance.

Cloning, Expression, and Cost Effective Purification of Authentic Human Epidermal Growth Factor With High Activity.

BACKGROUND: Epidermal growth factor (EGF) plays a fundamental role in the healing of wounds relating to skin damage, the cornea, and the gastrointestinal tract. OBJECTIVES: The aim of this study is the cloning, expression, and purification of recombinant human EGF (rhEGF), and an assessment of its activity. MATERIALS AND METHODS: In the present experimental study, a synthetic pET28a (+) -hEGF construct was prepared. In order to ligate hEGF into pET24a (+), the PCR technique was performed, using special primers that possess restriction enzyme sites, which are also located in appropriate sites in pET24a (+). After transferring this construct into E. coli cells, protein expression was performed under standard conditions. Protein solubilization was done by urea. hEGF purification and refolding were carried out using gradient dialysis against the urea. We used RP-HPLC to compare between rhEGF and commercial rhEGF as a control. Finally, an MTT assay was performed to assess the viability of the NIH 3T3 cells treated with various concentrations of rhEGF. RESULTS: Dialysis after urea solubilization caused precipitation of unwanted proteins, resulting in achievement of purified EGF with > 90% purity, without the need for expensive and time-consuming process. The MTT assay results showed that our rhEGF activate significantly higher proliferation of NIH 3T3 cells in comparison to the control (P-values were < 0.0001), in total concentrations and times evaluated CONCLUSIONS: Via our purification protocol, a sufficient amount of bioactive recombinant human epidermal growth factor was obtained in just a few affordable steps, with superlative purity.

Comparing the Marginal Adaptation of Cold Ceramic and Mineral Trioxide Aggregate by Means of Scanning Electron Microscope: An In vitro Study.

BACKGROUND: Long-term success of endodontic surgeries is often influenced by the type of root-end filling material (RFM). The aim of present study was to compare the marginal adaptation of two different RFM, cold ceramic (CC) and mineral trioxide aggregate (MTA), using scanning electron microscope (SEM). MATERIALS AND METHODS: About 20 extracted human single-rooted teeth were collected and stored into sodium hypochlorite 5.25%. The teeth were decronated from the cemento-enamel junction to prepare 16 mm roots. The working length was measured, and 1/3 coronal of the canal was prepared by Gates-Glidden drills. Apical flaring was followed by K file size # 40-70 based on step back technique. After filling of the canals, 3 mm above the apex was cut at 90° to the long axis. Furthermore, 3 mm of the filling was removed from the apical part using the ultrasonic device. All of the prepared specimens were divided into two groups and were retro filled by MTA and CC. The roots were cut horizontally from 1 mm above the apical part, and dentin-filling material interface was observed by SEM. Finally, the collected data were analyzed by Mann-Whitney test and using SPSS software version 18 at a significant level of 0.05. RESULTS: The mean interfacial adaptation was higher in CC group. However, no significant differences were observed by statistical test (P = 0.35). CONCLUSION: Both CC and MTA had similar marginal adaptation as RFM however in vivo studies are recommended for better determination.

Expression of COLLAGEN 1 and ELASTIN Genes in Mitral Valvular Interstitial Cells within Microfiber Reinforced Hydrogel.

OBJECTIVE: The incidence of heart valve disease is increasing worldwide and the number of heart valve replacements is expected to increase in the future. By mimicking the main tissue structures and properties of heart valve, tissue engineering offers new options for the replacements. Applying an appropriate scaffold in fabricating tissue-engineered heart valves (TEHVs) is of importance since it affects the secretion of the main extracellular matrix (ECM) components, collagen 1 and elastin, which are crucial in providing the proper mechanical properties of TEHVs. MATERIALS AND METHODS: Using real-time polymerase chain reaction (PCR) in this experi- mental study, the relative expression levels of COLLAGEN 1 and ELASTIN were obtained for three samples of each examined sheep mitral valvular interstitial cells (MVICs)-seeded onto electrospun poly (glycerol sebacate) (PGS)-poly (ε-caprolactone) (PCL) microfibrous, gelatin and hyaluronic acid based hydrogel-only and composite (PGS-PCL/hydrogel) scaffolds. This composite has been shown to create a synthetic three-dimensional (3D) microenvironment with appropriate mechanical and biological properties for MVICs. RESULTS: Cell viability and metabolic activity were similar among all scaffold types. Our results showed that the level of relative expression of COLLAGEN 1 and ELASTIN genes was higher in the encapsulated composite scaffolds compared to PGS-PCL-only and hydrogel-only scaffolds with the difference being statistically significant (P<0.05). CONCLUSION: The encapsulated composite scaffolds are more conducive to ECM secretion over the PGS-PCL-only and hydrogel-only scaffolds. This composite scaffold can serve as a model scaffold for heart valve tissue engineering.

Sequence analysis of tyrosinase gene in ocular and oculocutaneous albinism patients: introducing three novel mutations.

PURPOSE: Albinism is a heterogeneous genetic disorder of melanin synthesis that results in hypopigmented eyes (in patients with ocular albinism) or hair, skin, and eyes (in individuals with oculocutaneous albinism). It is associated with decreased visual acuity, nystagmus, strabismus, and photophobia. The tyrosinase gene is known to be involved in both oculocutaneous albinism and autosomal recessive ocular albinism. In this study, we aimed to screen the mutations in the TYR gene in the nonsyndromic OCA and autosomal recessive ocular albinism patients from Iran. METHODS: The tyrosinase gene was examined in 23 unrelated patients with autosomal recessive ocular albinism or nonsyndromic OCA using DNA sequencing and bioinformatics analysis. RESULTS: TYR gene mutations were identified in 14 (app. 60%) albinism patients. CONCLUSIONS: We found 10 mutations, 3 of which were novel. No mutation was found in our ocular albinism patients, but one of them was heterozygous for the p.R402Q polymorphism.

Expression of bax and bcl2 Genes in MDMA-induced Hepatotoxicity on Rat Liver Using Quantitative Real-Time PCR Method through Triggering Programmed Cell Death.

BACKGROUND: 3-4methylenedioxymethamphetamine (MDMA) is a synthetic and psychoactive drug, which is known popularly as Ecstasy and has toxic effects on human organs. OBJECTIVES: Considering the potential toxic interaction, this study was performed to quantify the expression of bax and bcl2 genes in MDMA-induced hepatotoxicity on rat liver. Subsequently, we evaluated pentoxifylline as a possible protective drug on hepatotoxicity. MATERIALS AND METHODS: Adult male Wistar rats weighting 250 - 300 grams were used in the study. The rats were equally distributed into four experimental groups (5 rat/group). MDMA was dissolved in PBS and injected intraperitoneally (IP) including untreated control, MDMA (MDMA dissolved in PBS), treated-1 (MDMA followed by PTX) and treated-2 (PTX followed by MDMA). All animals given MDMA received 3 doses of 7.5mg/kg with two hours gap between doses. Liver tissue was removed after anaesthetizing. Subsequently, RNA isolation, cDNA synthesis and Real-Time PCR were performed. Finally, data analyzed statistically to determine significantly differences between the groups (P value < 0.05). RESULTS: Using Real-Time quantitative PCR results, the gene expression ratio of bcl2 were calculated 93.80±20.64, 340.45 ± 36.60 and 47.13 ± 5.84 fold in MDMA, treated-1 and treated-2 groups, respectively. Furthermore, this ratio for bax gene obtained 2.13±0.33 fold in MDMA, 1.55 ± 0.26 fold in treated-1 and 10.44 ± 1.56 fold in treated-2 groups. CONCLUSIONS: The present study focused on molecular mechanism of MDMA in programmed cell death using gene expression quantification of a pro-apoptotic and anti-apoptoic gene in MDMA-induced hepatotoxocity. The results showed that MDMA prompted apoptosis in liver and pentoxifylline protected against hepatotoxicity before and after taking MDMA.

Gene Expression Profiles of BAD and Bcl-xL in the CA1 Region of the Hippocampus Following Global Ischemic/Reperfusion and FK-506 Administration.

BACKGROUND: The hippocampus is a tiny nub in the mammalian brain that is involved in forming, organizing, and storing memories. Global cerebral ischemia (GCI) and reperfusion induced apoptosis lead to cell injury and death. FK-506 is a strong immunosuppressant drug that has neuroprotective effects on the hypoxic-ischemic effects of brain damage. BAD and Bcl-xL are pro-apoptotic and anti-apoptotic genes, respectively. These genes belong to The B-cell lymphoma-2 (Bcl-2) family. OBJECTIVES: In this study, we assessed the neurotrophic properties of FK-506 on expression of the BAD and Bcl-xL genes in the hippocampus following global ischemia and reperfusion. MATERIALS AND METHODS: In the present experimental study, adult male Wistar rats were obtained and housed under standard conditions in the Tehran University of Medical Science in Iran. Rats were equally distributed in groups of three among the following groups: normal control, treated-1 (ischemia/reperfusion), and treated-2 (ischemia/reperfusion followed by FK-506). Global ischemia was induced for animals in the treated-1 and treated-2 groups. In treated-2, two doses of FK-506 were injected: one dose as an IV injection immediately after reperfusion and another as an intra-peritoneal (IP) injection after 48 hours. Then, the hippocampus tissue was removed after anaesthetizing the rats. RNA was isolated, cDNA was synthesized, and real-time PCR was performed. Finally, the obtained data were analyzed statistically (P value ˂ 0.05). RESULTS: The quantitative results of real-time PCR show that the mRNA expression ratio of Bcl-xL down-regulated was 0.75 ± 0.06 in the ischemia/reperfusion group versus 1.57 ± 0.09 in the control group (P value < 0.001), whereas Bcl-xL gene expression was greater in the ischemia/reperfusion +FK506 group (1.93 ± 0.15) than in the ischemia/reperfusion group. Moreover, the mRNA expression ratio of BAD up-regulated in the ischemia/reperfusion + FK506 group was 3.65 ± 0.49 compared to Normal control (1.39 ± 0.09) and Ischemia/reperfusion + FK506 was 1.09 ± 0.20 (P value < 0.001). CONCLUSIONS: The analysis of the pro-apoptotic gene to anti-apoptotic gene expression ratio (BAD /Bcl-xL) confirmed that expression of the pro-apoptotic gene significantly decreased (P value ˂ 0.001) under the ischemia/reperfusion condition. In contrast, the expression of the anti-apoptotic gene increased after administration of FK-506 (P value ˂ 0.001).