The integrative multi-omics approach identifies the novel competing endogenous RNA (ceRNA) network in colorectal cancer.

Circular RNAs (circRNA) are known to function as competing endogenous RNA (ceRNA) in various cancers by regulating microRNAs (miRNA). However, in colorectal cancer (CRC), the precise pathological role of circ000240/miRNA/mRNA remains indeterminate. The expression level of hsa_circ_000240 was evaluated using qRT-PCR in matching pairs of CRC tumor and adjacent normal tissue samples in our laboratory. Then, to determine whether hsa_circ_000240 acted as a ceRNA in CRC, the linked miRNAs and gene targets were retrieved. Topological analysis of candidate genes using a network approach identified the most critical hub genes and subnetworks related to CRC disease. Microarray and bulk RNA sequencing analyses were utilized to comprehensively evaluate the expression levels of both miRNA and mRNA in CRC. Single-cell RNA-seq analysis was also used to evaluate the significant overall survival (OS) genes at the cellular level. ATAC-seq data provided insights into candidate genes' accessible chromatin regions. The research uncovered a considerable upregulation of hsa_circ_000240 in CRC tissues. Three miRNAs interacted with the target circRNA. One thousand six hundred eighty intersected genes regulated by three miRNAs were further identified, and the relevant functionality of identified neighbor genes highlighted their relevance to cancer. The topological analysis of the constructed network has identified 33 hub genes with notably high expression in CRC. Among these genes, eight, including CHEK1, CDC6, FANCI, GINS2, MAD2L1, ORC1, RACGAP1, and SMC4, have demonstrated a significant impact on overall survival. The utilization of single-cell RNA sequencing unequivocally corroborated the augmented expression levels of CDC6 and ORC1 in individuals with CRC, alongside their noteworthy connection with the infiltration of immune cells. ATAC-seq analyses revealed altered accessibility regions in Chr2, 4, and 12 for CDC6 and ORC1 high-expression. Correlation analysis of CDC6 and ORC1 further highlighted the association of candidate gene expression with exhaustion markers such as CTLA4, CD247, TIGIT, and CD244. The candidate genes exhibit a positive correlation with chromatin remodeling and histone acetylation. These epigenetic modifications play a significant role in influencing the cancer progression following expression of CDC6 and ORC1 in CRC. Additionally, results showed that the methylation rate of the promoter region of CDC6 was elevated in CRC disease, confirming the functional importance of CDC6 and their interaction with hsa_circ_000240 and associated ceRNA in CRC. In conclusion, this study highlights hsa_circ_000240's role as a ceRNA in CRC. It opens new avenues for further dissection of CDC6, ORC1, and underlying novel epigenetics and immunotherapy targets for CRC therapy.

Following the Trace of HVS II Mitochondrial Region Within the Nine Iranian Ethnic Groups Based on Genetic Population Analysis.

The Iranian gene pool is seen as an important human genetic resource for investigating the region connecting Mesopotamia and the Iranian plateau. The main objective of this study was to explore gene flow in nine Iranian ethnic/subpopulation groups (402 samples) by examining mtDNA HVS2 sequence variations. This then allowed us to detect mtDNA HVS2 sequence mutations in two independent thalassemia and cystic fibrosis patient sample groups. The patient groups did not explicitly belong to any of the aforementioned nine subpopulations. Across all subpopulations, the haplogroups B4a1c3a, H2a2a1, N10b, H2a2a2, and J1 were seen to be predominant. High haplogroup diversities along with admixture of the exotic groups were observed in this study. The Arab subpopulation was shown to be independent from the others. It was revealed that there is a far distant relationship between Arab and Azeri groups. The thalassemia patient group, represented an almost random sample of most Iranian ethnic groups, and revealed few significant differences (P < 0.05) in their HVS2 sequence. It turned out that the IVS II-I (G → A) mutation in the thalassemia ß-globin gene was highly significant. Since the thalassemia patients in the present study represent many unique haplotypes, we can begin to comprehend the importance of mtDNA with this disease and the necessity for more studies in this context.

Integrative and in-vitro analysis reveal hsa_circ_001787 can act as a diagnostic biomarker for colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) are a new kind of non-coding RNA(ncRNA). Throughout research, we see an increase in the number of studies demonstrating that circRNAs occupy a pivotal role in the growth and advancement of human tumors. Nevertheless, hsa_circ_001787's role in the evolution of colorectal cancer (CRC) remains unclear. This current study ascertained the expression level of circRNA001787 in CRC specimens and neighboring healthy tissues, and investigated the miRNAs associate with hsa_circ_001787, as well as the relationship between hsa_circ_001787 and pathological factors. METHOD: First, the expression level of hsa_circ_001787 was measured in 43 matched Tissues from CRC and normal tissues through using real-time quantitative reverse transcription PCR (qRT-PCR). Second, based on circular RNA-microRNA and microRNA-mRNA pairs, a circRNA-miRNA-mRNA network was created. The survival rate of mRNAs was investigated through the GEPIA in the network. Regarding the elucidated function analysis of hsa_circ_001787, The biological, molecular, cellular function (GO) and pathway (KEGG) enrichment was obtained. RESULT: We detected that hsa_circ_001787 expression level was significantly down expressed in CRC tissue versus paired CRC histological normal tissue. The area under the curve (AUC) was 0.83. The expression level of hsa_circ_001787 was significantly associated with pathological factors such as tumor grade and the primary site of the tumor. Based on the hsa_circ_001787, a novel circRNA/miRNA/mRNA network has been built up, four miRNAs, and 24 mRNA. The pathway of mRNAs analyzed in the pathogenesis of CRC. Four genes distinguished via the GEPIA database were positively linked to the overall survival of CRC patients. CONCLUSION: Our study suggested that hsa_circ_001787 was significantly down-regulated in CRC. We might be able to use this as a new biomarker in the screening of CRC. Furthermore, our finding achieves a broader understanding of the regulatory mechanisms by which hsa_circ_001787 acts as ceRNA in colorectal cancer.

Graphene Oxide Negatively Regulates Cell Cycle in Embryonic Fibroblast Cells.

BACKGROUND: Unique properties of graphene and its derivatives make them attractive in the field of nanomedicine. However, the mass application of graphene might lead to side effects, which has not been properly addressed in previous studies, especially with regard to its effect on the cell cycle. METHODS: The effect of two concentrations (100 and 200 µg/mL) of nano- and microsized graphene oxide (nGO and mGO) on apoptosis, cell cycle, and ROS generation was studied. The effect of both sizes on viability and genotoxicity of the embryonic fibroblast cell cycle was evaluated. MTT and flow cytometry were applied to evaluate the effects of graphene oxide (GO) nanosheets on viability of cells. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The results of this study showed that GO disturbed the cell cycle and nGO impaired cell viability by inducing cell apoptosis. Interestingly, both nGO and mGO blocked the cell cycle in the S phase, which is a critical phase of the cell cycle. Upregulation of TP53-gene transcripts was also detected in both nGO- and mGO-treated cells compared to the control, especially at 200 µg/mL. DNA content of the treated cells increased; however, because of DNA degradation, its quality was decreased. CONCLUSION: In conclusion, graphene oxide at both nano- and micro-scale damages cell physiology and increases cell population in the S phase of the cell cycle.

Quantification of mitochondrial DNA damage and copy number in circulating blood of patients with systemic sclerosis by a qPCR-based assay

Abstract Background: Although not fully understood, oxidative stress has been implicated in the pathogenesis of different autoimmune diseases such as systemic sclerosis. Accumulating evidence indicates that oxidative stress can induce mitochondrial DNA (mtDNA) damage and variations in mtDNA copy number (mtDNAcn). Objective: The aim of this study was to explore mtDNAcn and oxidative DNA damage byproducts in peripheral blood of patients with systemic sclerosis and healthy controls. Methods: Forty six patients with systemic sclerosis and forty nine healthy subjects were studied. Quantitative real-time PCR used to measure the relative mtDNAcn and the oxidative damage (oxidized purines) of each sample. Results: The mean mtDNAcn was lower in patients with systemic sclerosis than in healthy controls whereas the degree of mtDNA damage was significantly higher in cases as compared to controls. Moreover, there was a negative correlation between mtDNAcn and oxidative DNA damage. Study limitations: The lack of simultaneous analysis and quantification of DNA oxidative damage markers in serum or urine of patients with systemic sclerosis and healthy controls. Conclusion: These data suggest that alteration in mtDNAcn and increased oxidative DNA damage may be involved in the pathogenesis of systemic sclerosis.

Gene Panel Testing in Hereditary Breast Cancer.

BACKGROUND: Breast cancer (BC) is a highly complex, heterogeneous and multifactorial disease and is the most commonly diagnosed cancer and the leading cause of cancer-related mortality in women worldwide. Family history and genetic mutations are important risk factors for BC. While studies in twins have estimated that about 10%-30% of BC are due to hereditary factors, only 4%-5% of them are due to mutations in BRCA1 or BRCA2 genes. Our aim was to investigate the role of other BC genes in familial BC among the Iranian population. METHODS: We selected 61 BC patients who were wild-type for BRCA1 and BRCA2 mutations but who met the criteria for hereditary BC based on the American College of Medical Genetics and Genomics (ACMG) and the National Comprehensive Cancer Network (NCCN) guidelines. We performed targeted sequencing covering the exons of 130 known cancer susceptibility genes based on the Cancer Gene Census list. RESULTS: We found seven mutations in seven known BC susceptibility genes (RAD50, PTEN, TP53, POLH, DKC1, WRN and CHEK2) in seven patients including two pathogenic frameshift variants in RAD50 and WRN genes, four pathogenic missense variants in TP53, PTEN, POLH, and DKC1 genes and a pathogenic splice donor variant in the CHEK2 gene. The presence of all these variants was confirmed by Sanger sequencing and Gap reverse transcription-polymerase chain reaction (RT-PCR) for the splice variant. In silico analysis of all of these variants predicted them to be pathogenic. CONCLUSION: Panel testing of BC patients who met the established criteria for hereditary BC but who were negative for BRCA1/2 mutations provided additional relevant clinical information for approximately 11.5% of the families. Our findings indicate that next generation sequencing (NGS) is a powerful tool to investigative putative mutagenic variants among patients who meet the criteria for hereditary BC, but with negative results on BRCA1/2 testing.

Association of MicroRNA Polymorphisms With Hepatocellular Carcinoma in an Iranian Population.

BACKGROUND: Single nucleotide polymorphisms (SNPs) can modulate various biological processes by influencing microRNA (miRNA) biogenesis and altering target selection. Common SNPs may alter the processing of miRNA and may be associated with hepatocellular carcinoma (HCC). We investigated the relationship between miR-499A>G, miR-149C>T, miR-196a2T>C, and miR-146aG>C and HCC susceptibility, examining the interaction of the miRNAs with hepatitis B virus (HBV). METHODS: We evaluated the associations of miR-499A>G (rs3746444), miR-149C>T (rs2292832), miR-196a2T>C (rs11614913), and miR-146aG>C (rs2910164) with HCC susceptibility in 100 HCC patients (70 males and 30 females) and 120 healthy controls (70 males and 50 females), using the PCR-restriction fragment length polymorphism method. RESULTS: For miR-499A>G, the frequencies of the AG genotype and G allele were higher in female HCC patients than in female controls (P=0.02 and 0.045, respectively). The frequency of the A allele was higher in HBV-positive HCC patients than in controls (P=0.019). For miR-149C>T, the frequency of the CC genotype was higher in female HCC patients than in female controls (P=0.009). For miR-196a2T>C, the frequencies of the CT and CC genotypes and the C allele were higher in HBV-positive HCC patients than in controls (P<0.001, P=0.009, and P<0.001, respectively). The frequencies of miR-146aG>C polymorphisms did not differ between HCC patients and controls. CONCLUSIONS: miR-499A>G, miR-149C>T, and miR-196a2T>C were associated with the development of HCC in women and/or that of HBV-related HCC. They can be considered genetic risk factors for the development of HCC among Iranians.

Targeting histone demethylases KDM5A and KDM5B in AML cancer cells: A comparative view.

Epigenetic modifications play an important role in initiation and progression of cancers including acute myeloid leukemia. Among different epigenetic modifiers, lysine specific demethylases have been noticed as potential therapeutic targets. KDM5 family of histone demethylases which removes methyl marks from lysine residues of H3, are frequently found in the promoter region of transcriptionally active genes resulting in repression of expression. Here we have compared the effects of KDM5A and KDM5B downregulation on HL-60 cell line behavior. KDM5A/5B knockdown resulted in lower viability of HL-60 cells in addition to modified cell cycle distribution and sub-G1 accumulation. Induction of apoptosis was observed in both knockdown cells. But in spite of similarity in their role, downregulation of KDM5A showed more efficient anti-leukemic effects in comparison to KDM5B. Cells showed higher accumulation in sub-G1 and apoptosis occurred significantly higher and also earlier after KDM5A reduction. Expression analysis confirmed almost 5 and 4 fold increased expression for bax and caspase-3 after downregulation of KDM5A in comparison to KDM5B. Due to the present study we propose KDM5A as a potential target for therapeutic aspects of acute myeloid leukemia although further investigations are needed.

In Vitro Generation of Glucose-Responsive Insulin-Secreting Cells from PDX1-Overexpressing Human-Induced Pluripotent Stem Cell Derived from Diabetic Patient.

Pancreatic and duodenal homeobox 1 (PDX1), a member of the homeodomain-containing transcription factor family, is a key transcription factor for pancreas development and mature ß-cell function. In this study, induced overexpression of PDX1 resulted in producing susceptible cells for pancreatic differentiation and was well beneficial to enhance ß-cell production, maturation, function, and survival. Induced PDX1 overexpression in harmony with a set of signaling molecules involves in guiding the signaling pathways toward pancreas development, leaded to high-efficient in vitro generation of ectopic insulin-producing cells (IPCs) with the effectively reduced number of polyhormonal cells and increased number of insulin (INS) single-positive cells. This strategy yielded 85.61% glucose-responsive insulin-positive cells in vitro, which was seven times higher than the basal level, and electron microscopy images revealed the presence of mature ß-cell secretory granules. The generation of glucose-responsive insulin-secreting ß-like cells from human-induced pluripotent stem cells (hiPSCs) in vitro would provide a promising approach to produce an unprecedented cell source for cell transplantation therapy in diabetes without the ethical obstacle of embryonic stem cells and would bypass immune rejection. These cells are an invaluable source for disease modeling, drug discovery, and pharmacogenomics studies as well.

Pancreatic Endoderm-Derived From Diabetic Patient-Specific Induced Pluripotent Stem Cell Generates Glucose-Responsive Insulin-Secreting Cells.

Human-induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient- and disease-specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin-producing pancreatic ß-cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five-step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose-responsive insulin-producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut-tube endoderm, posterior foregut, pancreatic endoderm, and endocrine precursor. Each stage of differentiation were characterized by stage-specific markers. The produced cells exhibited many properties of functional ß-cells, including expression of critical ß-cells transcription factors, the potency to secrete C-peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin-secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs-derived glucose-responsive insulin-secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. J. Cell. Physiol. 232: 2616-2625, 2017. © 2016 Wiley Periodicals, Inc.

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

STUDY QUESTION: Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? SUMMARY ANSWER: WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. WHAT IS KNOWN ALREADY: Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. STUDY DESIGN, SIZE, DURATION: This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. PARTICIPANTS/MATERIALS, SETTING, METHODS: WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. MAIN RESULTS AND THE ROLE OF CHANCE: WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G > A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G > A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. WIDER IMPLICATIONS OF THE FINDINGS: Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. STUDY FUNDING/COMPETING INTERESTS: This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

HLA-G allele and haplotype frequencies in a healthy population of Iran.

The human leukocyte antigen (HLA)-G molecule is expressed in cytotrophoblast cells, adult thymic epithelial cells, erythroblasts, pancreatic islets and mesenchymal stem cells. Although, HLA-G expression in allotransplanted patients is correlated with a better allograft acceptance, it is associated with an advanced grade of the tumor in cancer. In addition to the role on the immune system, HLA-G is also involved in successful pregnancy through the embryo implantation, fetal survival and the initial steps of hematopoiesis and angiogenesis. The aim of this study was determination of HLA-G allele frequencies in a healthy population of Iran. In this research, we selected 100 samples from healthy Iranian individuals and henceforth, we used polymerase chain reaction (PCR) followed by sequencing technique for exon 2, 3, 4 and intron 2 of the gene for evaluating the HLA-G alleles frequencies. Investigation of intronic (intron 2) variation is the novelty of our study. The obtained results indicated thirteen alleles of HLA-G in Iranian individuals including G*01:01:01:01, G*01:06, G*01:01:01:06, G*01:01:02, G*01:01:03, G*01:01:05, G*01:01:06, G*01:01:07, G*01:01:08, G*01:03, G*01:04:01, G*01:04:03, and G*01:04:04. According to this study, the most prevalent alleles in the Iranian population were G*01:01:01:01 (52.5%), G*01:01:02 (16%) and G*01:04:03 (14.5%) and also the lowest alleles regarding the frequency were G*01:01:01:06 (0.5%) and G*01:03 (0.5%). The results of G*01:01:01:01 and G*01:04:01 frequencies showed some similarities with the polish population. Our results were similar to the north Indian population for the frequencies of G*01:06 and G*01:01:02.

Expression and prognostic significance of bcl-2 and bax in the progression and clinical outcome of transitional bladder cell carcinoma.

OBJECTIVE: To evaluate the mRNA expression ratio of Bcl-2/Bax both in normal and tumoral bladder tissues of patients with transitional cell carcinoma (TCC) of bladder and investigate potential correlation between this expression ratio and clinical outcome. MATERIALS AND METHODS: In this experimental study, we used real time-PCR to investigate the expression of Bcl-2 and Bax both in normal and tumoral bladder tissues. The Bcl-2/ Bax expression ratio was determined in tumoral bladder tissues of patients with transitional cell carcinoma of the bladder (n=40) and correlation between expression ratios and the emergence of early relapses in a follow-up of 14-30 months was examined. RESULTS: Relapse-free time in 14/31 patients (45.16%) with Bcl-2/Bax>1 was shorter than 9 months (range of 2-9 months) with 5.7 months average median while 17/31 patients (54.84%) with Bcl-2/Bax<1 are currently relapse-free (14-30 months). Bcl-2 and Bax expression levels were not solely correlated with clinical outcome and progression of carcinogenesis. CONCLUSION: The mRNA expression ratio of Bcl-2/Bax in tumoral bladder tissues may serve as a significant prognostic indicator in predicting the clinical outcome in low grade non-invasive bladder cancer.

The mitochondrial C16069T polymorphism, not mitochondrial D310 (D-loop) mononucleotide sequence variations, is associated with bladder cancer.

BACKGROUND: Bladder cancer is a relatively common and potentially life-threatening neoplasm that ranks ninth in terms of worldwide cancer incidence. The aim of this study was to determine deletions and sequence variations in the mitochondrial displacement loop (D-loop) region from the blood specimens and tumoral tissues of patients with bladder cancer, compared to adjacent non-tumoral tissues. METHODS: The DNA from blood, tumoral tissues and adjacent non-tumoral tissues of twenty-six patients with bladder cancer and DNA from blood of 504 healthy controls from different ethnicities were investigated to determine sequence variation in the mitochondrial D-loop region using multiplex polymerase chain reaction (PCR), DNA sequencing and southern blotting analysis. RESULTS: From a total of 110 variations, 48 were reported as new mutations. No deletions were detected in tumoral tissues, adjacent non-tumoral tissues and blood samples from patients. Although the polymorphisms at loci 16189, 16261 and 16311 were not significantly correlated with bladder cancer, the C16069T variation was significantly present in patient samples compared to control samples (p < 0.05). Interestingly, there was no significant difference (p > 0.05) of C variations, including C7TC6, C8TC6, C9TC6 and C10TC6, in D310 mitochondrial DNA between patients and control samples. CONCLUSION: Our study suggests that 16069 mitochondrial DNA D-Loop mutations may play a significant role in the etiology of bladder cancer and facilitate the definition of carcinogenesis-related mutations in human cancer.

Proteome analysis of post-transplantation recovery mechanisms of an EAE model of multiple sclerosis treated with embryonic stem cell-derived neural precursors.

Multiple sclerosis (MS) is a chronic inflammatory and progressive disorder of the central nervous system (CNS), which ultimately causes demyelination and subsequent axonal injury. Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model to study the etiology and pathogenesis of MS. This model can also be used to investigate various therapeutic approaches for MS. Herein; we have treated a score 3 EAE mouse model with an embryonic stem cell-derived neural precursor. Clinical analysis showed recovery of the EAE model of MS following transplantation. We analyzed the proteome of spinal cords of healthy and EAE samples before and after transplantation. Proteome analysis revealed that expressions of 86 spinal cord protein spots changed in the EAE or transplanted mouse compared to controls. Mass spectrometry resulted in identification of 72 proteins. Of these, the amounts of 27 differentially expressed proteins in EAE samples returned to sham levels after transplantation, suggesting a possible correlation between changes at the proteome level and clinical signs of EAE in transplanted mice. The recovered proteins belonged to various functional groups that included disturbances in ionic and neurotransmitter release, apoptosis, iron hemostasis, and signal transduction. Our results provided a proteomic view of the molecular mechanisms of EAE recovery after stem cell transplantation. BIOLOGICAL SIGNIFICANCE: In this study, we applied proteomics to analyze the changes in proteome pattern of EAE mouse model after embryonic stem cell-derived neural precursor transplantation. Our proteome results clearly showed that the expression levels of several differentially expressed proteins in EAE samples returned to sham levels after transplantation, which suggested a possible correlation between changes at the proteome level and decreased clinical signs of EAE in transplanted mice. These results will serve as a basis to address new questions and design new experiments to elucidate the biology of EAE and recovery after transplantation. A thorough understanding of stem cell-mediated therapeutic mechanisms might result in the development of more efficacious therapies for MS than are currently available.

BAK, BAX, and NBK/BIK proapoptotic gene alterations in Iranian patients with ataxia telangiectasia.

INTRODUCTION: Ataxia telangiectasia (AT) is an autosomal recessive multisystem disorder characterized by variable immunodeficiency, progressive neurodegeneration, occulocutaneous telangiectasia, and an increased susceptibility to malignancies. This study was designed to study the role of proapoptotic BAK, BAX, and NBK/BIK genes in a group of patients with AT to elucidate the possible role of these genes in progression of malignancies in this disease. MATERIALS AND METHODS: Fifty Iranian patients with AT were investigated in this study. The entire coding regions of the BAK gene (exons 2-6), NBK/BIK gene (exons 2-5), and BAX gene (exons 1-7) were amplified using polymerase chain reaction (PCR). The PCR products were separated by 2% agarose gel electrophoresis, and all positive samples were verified by direct sequencing of PCR products using the same primers used for PCR amplification, BigDye chemistry, and Avent 3100 Genetic Analyzer following the manufacturer's instructions (Applied Biosystems). RESULTS: Eight of fifty Iranian AT patients (16%) exhibited a C > T transition in exon 2 (c342C > T) of the BAK gene, while none of the healthy controls had such alteration (P = 0.0001). Higher frequency of another nucleotide substitution in the noncoding region of exon 7 in BAX gene (6855G > A) was also identified in 68% of the patient group versus 24% in the controls (P < 0.0001). Sequence alteration in intronic region of the NBK/BIK gene IVS4-12delTC was observed in 52% of AT patients, which was significantly higher than 20% in the control group (P = 0.0023). Another variant IVS1146C > T in the intronic region of the BAX gene was found in 78% of patients, which was significantly higher than 10% in the controls (P < 0.0001). Frequency of alteration in intronic region of exon 3 of the BAX gene (IVS3 + 14A > G) was also significantly higher in the AT patients (P < 0.0001). DISCUSSION: Several alterations in the proapoptotic genes BAK, NBK/BIK, and BAX were found in our study, which could elucidate involvement of the mitochondrial pathway mediated apoptosis in accelerating and developing of cancers and in immunopathogenesis of AT. Such altered apoptosis in AT could play some roles in developing cancers in this group of patients.

The influence of uric acid treatments on liver glutathione system prevent oxidative damages in experimental autoimmune encephalomyelitis mice.

Recently we reported that antioxidant system in brain and spinal cord in experimental autoimmune encephalomyelitis (EAE) mice is mainly affected at early stages of the disease [M. Zargari, A. Allameh, M.H. Sanati, T. Tiraihi, S.H. Lavasani, O. Emadyan, Relationship between the clinical scoring and demyelination in central nervous system with total antioxidant capacity of plasma during experimental autoimmune encephalomyelitis development in mice, Neurosci. Lett. 412 (2007), 24-28]. The aim of the present study was to investigate the role of uric acid (UA) on antioxidant system in liver and plasma of EAE mice. EAE was induced in C57/BL6 mice (n=60), followed by i.p. administration of UA (10mg/kg BW) in 30 mice at three distinct clinical stages (A: prior to onset, B: after onset, C: after development of EAE). Livers were removed and processed for measurement of lipid peroxidation products, reduced glutathione (GSH), and glutathione S-transferase (GST) and total antioxidant capacity of plasma (FRAP). The results showed that lipid peroxidation products in liver of EAE mice was increased significantly ( approximately 85%) as compared to normal. UA administration to EAE mice caused a significant suppression of liver lipid peroxidation products ( approximately 45%) at early stages (A and B). There was an inverse relationship between lipid peroxidation and cellular GSH in liver. GSH was significantly depleted in mice liver during the EAE progression, but it was recovered ( approximately 29%) when UA was injected before the onset of the disease (groups A and B). Plasma total antioxidant capacity was significantly decreased during the development of EAE, however it was subsided in mice treated with UA as compared to the corresponding controls (21%) in groups A and B. Elevated liver GST as a result of EAE induction was reversed in mice treated with UA particularly in groups A and B. These results indicate that hepatic glutathione system, particularly GST plays a major role in modulation of oxidative damages to central nervous system (CNS) during EAE induction. The positive response of antioxidant system to UA administration in EAE mice was corroborated with improvement of clinical manifestation of the animals.

Use of D11S2179 and D11S1343 as markers for prenatal diagnosis of ataxia telangiectasia in Iranian patients.

Ataxia telangiectasia (AT) is an autosomal recessive disorder with an estimated prevalence of 1/40,000 to 1/100,000 in reported populations. There is a 25% possibility for having an affected child when parents are carriers for the ATM gene mutation. There is no cure available for this disease and prenatal testing is strongly recommended for prevention of this disease. Although the preferred method is the direct mutation analysis of the ATM gene, the large size of the ATM gene with 63 exons and the large number of possible mutations in patients considerably limit efficiency of mutation analysis as a diagnostic choice. Indirect method is a better tool when parents are not carriers of founder mutation and pass different mutations to their children. Indirect molecular diagnosis using ATM-related molecular markers facilitates prenatal diagnosis of AT children. In this study, four molecular markers: D11S2179, D11S1787, D11S535, D11S1343 are genotyped in 19 unrelated families from different regions of Iran. Those markers are amplified using extracted sequence primers from the Gene Bank with their described PCR conditions. Amplified products were separated using denaturing PAGE gels, and data were analyzed to detect their pattern of inheritance in each family. In all families, segregation of alleles was according to Mendelian inheritance, and affected chromosomes were distinguishable from unaffected ones. All carriers and affected patients were diagnosed accurately. Thus, this method is effectively useful in prenatal diagnosis of AT.

Gene dosage is not responsible for the upregulation of MRP1 gene expression in adult leukemia patients.

BACKGROUND: Upregulation of multidrug resistance-associated protein (MRP1) gene has been detected in many in vitro systems and could be the basis of the drug resistance phenotype in vivo. Increase in gene dosage and overexpression are two major mechanisms for increasing MRP1 expression level. In many drug resistant cell lines, MRP1 gene amplification has been detected. However, it is not yet known whether gene amplification plays a role in inducing the multidrug resistance phenotype clinically. METHODS: To establish whether MRP1 gene copy number is a common feature of the upregulation of MRP1 expression in cancer patients, we studied the MRP1 gene copy number in leukemia patients by fluorescent in situ hybridization (FISH) and real-time PCR. This involved determination of the MRP1 gene copy number and mRNA level in the peripheral blood of 52 adult leukemic patients and ten healthy volunteers. The leukemic CCRF-CEM cell line (drug sensitive) and its drug-resistant subline CCRF-E1000, which has MRP1 overexpression, were used as controls. RESULTS: The MRP1 gene copy number in CCRF-CEM was normal but increased significantly in CCRF-E1000 cell line. However, in the presence or absence of MRP1 overexpression, increase in gene dosage was not detected in patients. CONCLUSIONS: Our data suggest that the increase in MRP1 gene dosage observed in resistant cell lines is not responsible for the upregulation of MRP1 expression in leukemic patients.

ATM Gene Mutations Detection in Iranian Ataxia-Telangiectasia Patients.

Ataxia-Telangiectasia (AT) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, radiation sensitivity and cancer predisposition. The ATM gene on human chromosome 11q22.3 has recently been identified as the gene responsible for ataxia-telangiectasia (AT). The gene mutated in AT, which has been designated as the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. More than 100 mutations are broadly distributed throughout the ATM gene. The large size of the ATM gene (66 exons spanning ~150kb of genomic DNA) together with the diversity and broad distribution of mutations in AT patients, greatly limits the utility of direct mutation screening as a diagnostic tool. In this study, 20 families with at least one affected child clinically suspected to have ataxia-telangiectasia were examined and their DNA was extracted and amplified with standard methods. Sequencing methods were used to detect the new point mutation. Four exons which were hot spots for point mutations in ATM gene were detected by PCR-SSCP or PCR-RFLP.