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Sample records for methylation independent pcr

  1. Methylation-Specific PCR Unraveled

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    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  2. Culture independent PCR: an alternative enzyme discovery strategy

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glyco...... the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology....

  3. Transient neonatal diabetes mellitus with macroglossia diagnosed by methylation specific PCR (MS-PCR

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    Hye Young Jin

    2010-03-01

    Full Text Available Transient neonatal diabetes mellitus (TNDM has been associated with paternal uniparental isodisomy of chromosome 6, paternally inherited duplication of 6q24, or a methylation defect at a CpG island of the ZAC or HYMAI gene. We experienced a case of TNDM in which the patient presented with hyperglycemia, macroglossia, and intrauterine growth retardation, caused by a paternally derived HYMAI. An 18-day-old female infant was admitted to the hospital because of macroglossia and recurrent hyperglycemia. In addition to the macroglossia, she also presented with large fontanelles, micrognathia, and prominent eyes. Serum glucose levels were 200–300 mg/dL and they improved spontaneously 2 days after admission. To identify the presence of a maternal methylated allele, bisulfite-treated genomic DNA from peripheral blood was prepared and digested with BssHII after polymerase chain reaction (PCR amplification with methylation-specific HYMAI primers. PCR and restriction fragment length polymorphism analysis showed that the patient had only the paternal origin of the HYMA1 gene. TNDM is associated with a methylation defect in chromosome 6, suggesting that an imprinted gene on chromosome 6 is responsible for this phenotype.

  4. Application of multiplex nested methylated specific PCR in early diagnosis of epithelial ovarian cancer.

    Science.gov (United States)

    Wang, Bi; Yu, Lei; Yang, Guo-Zhen; Luo, Xin; Huang, Lin

    2015-01-01

    To explore the application of multiplex nested methylated specific polymerase chain reaction (PCR) in the early diagnosis of epithelial ovarian carcinoma (EOC). Serum and fresh tissue samples were collected from 114 EOC patients. RUNX3, TFPI2 and OPCML served as target genes. Methylation levels of tissues were assessed by multiplex nested methylated specific PCR, the results being compared with those for carcinoma antigen 125 (CA125). The serum free deoxyribose nucleic acid (DNA) methylation spectrum of EOC patients was completely contained in the DNA spectrum of cancer tissues, providing an accurate reflection of tumor DNA methylation conditions. Serum levels of CA125 and free DNA methylation in the EOC group were evidently higher than those in benign lesion and control groups (p0.05). The sensitivity, specificity and positive predicative value (PPV) of multiplex nested methylated specific PCR were significantly higher for detection of all patients and those with early EOC than those for CA125 (pnested methylated specific PCR (p>0.05), but there was no significant difference in sensitivity (p>0.05). Serum free DNA methylation can be used as a biological marker for EOC and multiplex nested methylated specific PCR should be considered for early diagnosis since it can accurately determine tumor methylation conditions.

  5. Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

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    Ito Takashi

    2011-06-01

    Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

  6. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    Science.gov (United States)

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. A robust internal control for high-precision DNA methylation analyses by droplet digital PCR.

    Science.gov (United States)

    Pharo, Heidi D; Andresen, Kim; Berg, Kaja C G; Lothe, Ragnhild A; Jeanmougin, Marine; Lind, Guro E

    2018-01-01

    Droplet digital PCR (ddPCR) allows absolute quantification of nucleic acids and has potential for improved non-invasive detection of DNA methylation. For increased precision of the methylation analysis, we aimed to develop a robust internal control for use in methylation-specific ddPCR. Two control design approaches were tested: (a) targeting a genomic region shared across members of a gene family and (b) combining multiple assays targeting different pericentromeric loci on different chromosomes. Through analyses of 34 colorectal cancer cell lines, the performance of the control assay candidates was optimized and evaluated, both individually and in various combinations, using the QX200™ droplet digital PCR platform (Bio-Rad). The best-performing control was tested in combination with assays targeting methylated CDO1 , SEPT9 , and VIM . A 4Plex panel consisting of EPHA3 , KBTBD4 , PLEKHF1 , and SYT10 was identified as the best-performing control. The use of the 4Plex for normalization reduced the variability in methylation values, corrected for differences in template amount, and diminished the effect of chromosomal aberrations. Positive Droplet Calling (PoDCall), an R-based algorithm for standardized threshold determination, was developed, ensuring consistency of the ddPCR results. Implementation of a robust internal control, i.e., the 4Plex, and an algorithm for automated threshold determination, PoDCall, in methylation-specific ddPCR increase the precision of DNA methylation analysis.

  8. Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

    Science.gov (United States)

    Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

    2016-01-01

    Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

  9. Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) technique among various cell types of bulls

    OpenAIRE

    Phutikanit, Nawapen; Suwimonteerabutr, Junpen; Harrison, Dion; D'Occhio, Michael; Carroll, Bernie; Techakumphu, Mongkol

    2010-01-01

    Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR) assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR) to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII). The native genomic ...

  10. Absolute quantification of DNA methylation using microfluidic chip-based digital PCR.

    Science.gov (United States)

    Wu, Zhenhua; Bai, Yanan; Cheng, Zule; Liu, Fangming; Wang, Ping; Yang, Dawei; Li, Gang; Jin, Qinghui; Mao, Hongju; Zhao, Jianlong

    2017-10-15

    Hypermethylation of CpG islands in the promoter region of many tumor suppressor genes downregulates their expression and in a result promotes tumorigenesis. Therefore, detection of DNA methylation status is a convenient diagnostic tool for cancer detection. Here, we reported a novel method for the integrative detection of methylation by the microfluidic chip-based digital PCR. This method relies on methylation-sensitive restriction enzyme HpaII, which cleaves the unmethylated DNA strands while keeping the methylated ones intact. After HpaII treatment, the DNA methylation level is determined quantitatively by the microfluidic chip-based digital PCR with the lower limit of detection equal to 0.52%. To validate the applicability of this method, promoter methylation of two tumor suppressor genes (PCDHGB6 and HOXA9) was tested in 10 samples of early stage lung adenocarcinoma and their adjacent non-tumorous tissues. The consistency was observed in the analysis of these samples using our method and a conventional bisulfite pyrosequencing. Combining high sensitivity and low cost, the microfluidic chip-based digital PCR method might provide a promising alternative for the detection of DNA methylation and early diagnosis of epigenetics-related diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Detection of methylated CDO1 in plasma of colorectal cancer; a PCR study.

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    Keishi Yamashita

    Full Text Available BACKGROUND: Cysteine biology is important for the chemosensitivity of cancer cells. Our research has focused on the epigenetic silencing of cysteine dioxygenase type 1 (CDO1 in colorectal cancer (CRC. In this study, we describe detection of CDO1 methylation in the plasma of CRC patients using methylation specific PCR (Q-MSP and extensive analysis of the PCR reaction. METHODS: DNA was extracted from plasma, and analysed for methylation of the CDO1 gene using Q-MSP. The detection rate of CDO1 gene methylation was calculated and compared with that of diluted DNA extracted from "positive control" DLD1 cells. CDO1 gene methylation in the plasma of 40 CRC patients that were clinicopathologically analysed was then determined. RESULTS: (1 The cloned sequence analysis detected 93.3% methylation of the promoter CpG islands of the CDO1 gene of positive control DLD1 cells and 4.7% methylation of the negative control HepG2 CDO1 gene. (2 DLD1 CDO1 DNA could not be detected in this assay if the extracted DNA was diluted ∼1000 fold. The more DNA that was used for the PCR reaction, the more effectively it was amplified in Q-MSP. (3 By increasing the amount of DNA used, methylated CDO1 could be clearly detected in the plasma of 8 (20% of the CRC patients. However, the percentage of CRC patients detected by methylated CDO1 in plasma was lower than that detected by CEA (35.9% or CA19-9 (23.1% in preoperative serum. Combination of CEA/CA19-9 plus plasma methylated CDO1 could increase the rate of detection of curable CRC patients (39.3% as compared to CEA/CA19-9 (25%. CONCLUSION: We have described detection of CDO1 methylation in the plasma of CRC patients. Although CDO1 methylation was not detected as frequently as conventional tumor markers, analysis of plasma CDO1 methylation in combination with CEA/CA19-9 levels increases the detection rate of curable CRC patients.

  12. A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

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    Tierling Sascha

    2010-06-01

    Full Text Available Abstract Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2 and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

  13. Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR technique among various cell types of bulls

    Directory of Open Access Journals (Sweden)

    Carroll Bernie

    2010-03-01

    Full Text Available Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII. The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Results Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90% were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8% and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05. Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05. Conclusions The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic

  14. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

    Science.gov (United States)

    Barault, L; Amatu, A; Bleeker, F E; Moutinho, C; Falcomatà, C; Fiano, V; Cassingena, A; Siravegna, G; Milione, M; Cassoni, P; De Braud, F; Rudà, R; Soffietti, R; Venesio, T; Bardelli, A; Wesseling, P; de Witt Hamer, P; Pietrantonio, F; Siena, S; Esteller, M; Sartore-Bianchi, A; Di Nicolantonio, F

    2015-09-01

    O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P alkylating agents. © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  15. Tissue-specific methylation of human insulin gene and PCR assay for monitoring beta cell death.

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    Mohamed I Husseiny

    Full Text Available The onset of metabolic dysregulation in type 1 diabetes (T1D occurs after autoimmune destruction of the majority of pancreatic insulin-producing beta cells. We previously demonstrated that the DNA encoding the insulin gene is uniquely unmethylated in these cells and then developed a methylation-specific PCR (MSP assay to identify circulating beta cell DNA in streptozotocin-treated mice prior to the rise in blood glucose. The current study extends to autoimmune non-obese diabetic (NOD mice and humans, showing in NOD mice that beta cell death occurs six weeks before the rise in blood sugar and coincides with the onset of islet infiltration by immune cells, demonstrating the utility of MSP for monitoring T1D. We previously reported unique patterns of methylation of the human insulin gene, and now extend this to other human tissues. The methylation patterns of the human insulin promoter, intron 1, exon 2, and intron 2 were determined in several normal human tissues. Similar to our previous report, the human insulin promoter was unmethylated in beta cells, but methylated in all other tissues tested. In contrast, intron 1, exon 2 and intron 2 did not exhibit any tissue-specific DNA methylation pattern. Subsequently, a human MSP assay was developed based on the methylation pattern of the insulin promoter and human islet DNA was successfully detected in circulation of T1D patients after islet transplantation therapy. Signal levels of normal controls and pre-transplant samples were shown to be similar, but increased dramatically after islet transplantation. In plasma the signal declines with time but in whole blood remains elevated for at least two weeks, indicating that association of beta cell DNA with blood cells prolongs the signal. This assay provides an effective method to monitor beta cell destruction in early T1D and in islet transplantation therapy.

  16. High Specificity of Quantitative Methylation-Specific PCR Analysis for MGMT Promoter Hypermethylation Detection in Gliomas

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    Paola Parrella

    2009-01-01

    Full Text Available Normal brain tissue from 28 individuals and 50 glioma samples were analyzed by real-time Quantitative Methylation-Specific PCR (QMSP. Data from this analysis were compared with results obtained on the same samples by MSP. QMSP analysis demonstrated a statistically significant difference in both methylation level (P=.000009 Mann Whitney Test and frequencies (P=.0000007, Z-test in tumour samples as compared with normal brain tissues. Although QMSP and MSP showed similar sensitivity, the specificity of QMSP analysis was significantly higher (93%; CI95%: 84%–100% as compared with MSP (64%; 95%CI: 46%–82%. Our results suggest that QMSP analysis may represent a powerful tool to identify glioma patients that will benefit from alkylating agents chemotherapy.

  17. Enrichment of methylated molecules using enhanced-ice-co-amplification at lower denaturation temperature-PCR (E-ice-COLD-PCR) for the sensitive detection of disease-related hypermethylation.

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    Mauger, Florence; Kernaleguen, Magali; Lallemand, Céline; Kristensen, Vessela N; Deleuze, Jean-François; Tost, Jörg

    2018-05-01

    The detection of specific DNA methylation patterns bears great promise as biomarker for personalized management of cancer patients. Co-amplification at lower denaturation temperature-PCR (COLD-PCR) assays are sensitive methods, but have previously only been able to analyze loss of DNA methylation. Enhanced (E)-ice-COLD-PCR reactions starting from 2 ng of bisulfite-converted DNA were developed to analyze methylation patterns in two promoters with locked nucleic acid (LNA) probes blocking amplification of unmethylated CpGs. The enrichment of methylated molecules was compared to quantitative (q)PCR and quantified using serial dilutions. E-ice-COLD-PCR allowed the multiplexed enrichment and quantification of methylated DNA. Assays were validated in primary breast cancer specimens and circulating cell-free DNA from cancer patients. E-ice-COLD-PCR could prove a useful tool in the context of DNA methylation analysis for personalized medicine.

  18. PCR

    African Journals Online (AJOL)

    Elham

    2013-07-03

    Jul 3, 2013 ... was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on .... products' differentiation was easy.

  19. Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

    International Nuclear Information System (INIS)

    Shah, Jinesh N; Shao, Genze; Hei, Tom K; Zhao, Yongliang

    2008-01-01

    Hypermethylation of the TGFBI promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the TGFBI promoter in human lung and prostate cancer specimens. Methylation-specific primers were designed based on the methylation profiles of the TGFBI promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP. Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the TGFBI promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the TGFBI promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (p < 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the TGFBI promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (p < 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the TGFBI gene in human lung and prostate tumor cell lines. We successfully optimized a MSP method for the precise and efficient screening of TGFBI promoter methylation status. Dense methylation of the TGFBI promoter correlated with the extent of TGFBI gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, TGFBI promoter methylation can be used as a potential

  20. Digital quantification of gene methylation in stool DNA by emulsion-PCR coupled with hydrogel immobilized bead-array.

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    Liu, Yunlong; Wu, Haiping; Zhou, Qiang; Song, Qinxin; Rui, Jianzhong; Zou, Bingjie; Zhou, Guohua

    2017-06-15

    Aberrations of gene methylation in stool DNA (sDNA) is an effective biomarker for non-invasive colorectal cancer diagnosis. However, it is challenging to accurately quantitate the gene methylation levels in sDNA due to the low abundance and degradation of sDNA. In this study, a digital quantification strategy was proposed by combining emulsion PCR (emPCR) with hydrogel immobilized bead-array. The assay includes following steps: bisulfite conversion of sDNA, pre-amplification by PCR with specific primers containing 5' universal sequences, emPCR of pre-amplicons with beaded primers to achieve single-molecular amplification and identification of hydrogel embedding beads coated with amplicons. The sensitivity and the specificity of the method are high enough to pick up 0.05% methylated targets from unmethylated DNA background. The successful detection of hypermethylated vimentin gene in clinical stool samples suggests that the proposed method should be a potential tool for non-invasive colorectal cancer screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

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    Sérgio D.J. Pena

    1999-06-01

    Full Text Available We have developed a non-isotopic technique based on methylation-specific PCR (MSP of the FMR1 promoter for the identification of fragile X full mutations among men. DNA samples were first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We designed two primer pairs: one produced a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other generated an 84-bp product from the treated non-methylated promoter. In normal males only the 84-bp fragment was seen, while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with absence or weak visualization of the 84-bp band. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15. Using the methylation-specific PCR we identified with 100% sensitivity and accuracy eight previously diagnosed FRAXA male patients mixed with 42 normal controls. Because of its simplicity and high efficiency, methylation-specific PCR may become the method of choice for the diagnosis of the fragile X syndrome in mentally retarded males.Nós desenvolvemos uma técnica não-isotópica baseada na PCR para a identificação de mutações completas da síndrome do X-frágil em homens. O método é baseado na PCR específica para metilação do promotor do gene FMR1. Amostras de DNA são tratadas com bissulfito de sódio para converter citosinas não-metiladas para uracilo, seguindo-se amplificação por PCR com oligonucleotídeos iniciadores específicos para DNA metilado versus não-metilado. Desenhamos dois iniciadores: um produz um fragmento de 142 pb da ilha CpG metilada tratada com bissulfito de sódio, enquanto o outro gera um produto de 84 pb do

  2. Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women's Cancers.

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    Thomas E Bartlett

    Full Text Available We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221, which validates in two independent data sets from Mayo Clinic (n = 198 and TCGA (n = 358, with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes.

  3. Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women's Cancers.

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    Bartlett, Thomas E; Jones, Allison; Goode, Ellen L; Fridley, Brooke L; Cunningham, Julie M; Berns, Els M J J; Wik, Elisabeth; Salvesen, Helga B; Davidson, Ben; Trope, Claes G; Lambrechts, Sandrina; Vergote, Ignace; Widschwendter, Martin

    2015-01-01

    We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV) based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221), which validates in two independent data sets from Mayo Clinic (n = 198) and TCGA (n = 358), with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF) binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes.

  4. Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species.

    Science.gov (United States)

    Springer, Jan; Goldenberger, Daniel; Schmidt, Friderike; Weisser, Maja; Wehrle-Wieland, Elisabeth; Einsele, Hermann; Frei, Reno; Löffler, Jürgen

    2016-03-01

    PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.

  5. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; Di Nicolantonio, F.

    2015-01-01

    O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA

  6. Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer

    NARCIS (Netherlands)

    Barault, L.; Amatu, A.; Bleeker, F. E.; Moutinho, C.; Falcomatà, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; de Braud, F.; Rudà, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; de Witt Hamer, P.; Pietrantonio, F.; Siena, S.; Esteller, M.; Sartore-Bianchi, A.; di Nicolantonio, Federica

    2015-01-01

    Background: O6-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. Patients and methods: We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and

  7. Development of a multiplex methylation-specific PCR as candidate triage test for women with an HPV-positive cervical scrape

    International Nuclear Information System (INIS)

    Snellenberg, Suzanne; Strooper, Lise MA De; Hesselink, Albertus T; Meijer, Chris JLM; Snijders, Peter JF; Heideman, Daniëlle AM; Steenbergen, Renske DM

    2012-01-01

    Quantitative methylation-specific PCR (qMSP) analysis for determining the methylation status of (candidate) tumor suppressor genes has potential as objective and valuable test to triage high-risk human papillomavirus (hrHPV) positive women in cervical screening. Particularly combined methylation analysis of a panel of genes shows most promising clinical performance, with sensitivity levels that equal or exceed that of cytology. However, the wide application of such methylation marker panels is hampered by the lack of effective multiplex assays allowing simultaneous methylation detection of various targets in a single reaction. Here, we designed and analyzed a multiplex qMSP assay for three genes whose methylation was previously found to be informative for cervical (pre)cancer (i.e. CADM1, MAL and hsa-miR-124-2) as well as a reference gene β-actin. Based on our experience, we discuss the optimization of the parameters that provide a practical approach towards multiplex qMSP design. Primers and PCR reagents were optimized for multiplex qMSP purposes and the resulting assay was analytically validated on serial dilutions of methylated DNA in unmethylated DNA, and compared with singleplex counterparts on hrHPV-positive cervical scrapings. Upon optimization, including primer redesign and primer limiting assays, the multiplex qMSP showed the same analytical performance as the singleplex qMSPs. A strong correlation between the obtained normalized ratios of the singleplex and multiplex qMSPs on cervical scrapes was found for all three markers: CADM1 (R 2 =0.985), MAL (R 2 =0.986) and hsa-miR-124-2 (R 2 =0.944). Multiplex qMSP offers a promising approach for high-throughput diagnostic analysis of the methylation status of multiple genes, which after proper design and validation can be equally specific, sensitive and reproducible as its singleplex versions

  8. FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    Directory of Open Access Journals (Sweden)

    Lu Jia

    2011-10-01

    Full Text Available Abstract Background Although a variety of methods and expensive kits are available, molecular cloning can be a time-consuming and frustrating process. Results Here we report a highly simplified, reliable, and efficient PCR-based cloning technique to insert any DNA fragment into a plasmid vector or into a gene (cDNA in a vector at any desired position. With this method, the vector and insert are PCR amplified separately, with only 18 cycles, using a high fidelity DNA polymerase. The amplified insert has the ends with ~16-base overlapping with the ends of the amplified vector. After DpnI digestion of the mixture of the amplified vector and insert to eliminate the DNA templates used in PCR reactions, the mixture is directly transformed into competent E. coli cells to obtain the desired clones. This technique has many advantages over other cloning methods. First, it does not need gel purification of the PCR product or linearized vector. Second, there is no need of any cloning kit or specialized enzyme for cloning. Furthermore, with reduced number of PCR cycles, it also decreases the chance of random mutations. In addition, this method is highly effective and reproducible. Finally, since this cloning method is also sequence independent, we demonstrated that it can be used for chimera construction, insertion, and multiple mutations spanning a stretch of DNA up to 120 bp. Conclusion Our FastCloning technique provides a very simple, effective, reliable, and versatile tool for molecular cloning, chimera construction, insertion of any DNA sequences of interest and also for multiple mutations in a short stretch of a cDNA.

  9. Intra-Gene DNA Methylation Variability Is a Clinically Independent Prognostic Marker in Women’s Cancers

    Science.gov (United States)

    Bartlett, Thomas E.; Jones, Allison; Goode, Ellen L.; Fridley, Brooke L.; Cunningham, Julie M.; Berns, Els M. J. J.; Wik, Elisabeth; Salvesen, Helga B.; Davidson, Ben; Trope, Claes G.; Lambrechts, Sandrina; Vergote, Ignace; Widschwendter, Martin

    2015-01-01

    We introduce a novel per-gene measure of intra-gene DNA methylation variability (IGV) based on the Illumina Infinium HumanMethylation450 platform, which is prognostic independently of well-known predictors of clinical outcome. Using IGV, we derive a robust gene-panel prognostic signature for ovarian cancer (OC, n = 221), which validates in two independent data sets from Mayo Clinic (n = 198) and TCGA (n = 358), with significance of p = 0.004 in both sets. The OC prognostic signature gene-panel is comprised of four gene groups, which represent distinct biological processes. We show the IGV measurements of these gene groups are most likely a reflection of a mixture of intra-tumour heterogeneity and transcription factor (TF) binding/activity. IGV can be used to predict clinical outcome in patients individually, providing a surrogate read-out of hard-to-measure disease processes. PMID:26629914

  10. Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR

    OpenAIRE

    Hristova, Krassimira R.; Lutenegger, Christian M.; Scow, Kate M.

    2001-01-01

    The fuel oxygenate methyl tert-butyl ether (MTBE), a widely distributed groundwater contaminant, shows potential for treatment by in situ bioremediation. The bacterial strain PM1 rapidly mineralizes and grows on MTBE in laboratory cultures and can degrade the contaminant when inoculated into groundwater or soil microcosms. We applied the TaqMan quantitative PCR method to detect and quantify strain PM1 in laboratory and field samples. Specific primers and probes were designed for the 16S ribos...

  11. Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients.

    Science.gov (United States)

    Sartore-Bianchi, Andrea; Pietrantonio, Filippo; Amatu, Alessio; Milione, Massimo; Cassingena, Andrea; Ghezzi, Silvia; Caporale, Marta; Berenato, Rosa; Falcomatà, Chiara; Pellegrinelli, Alessio; Bardelli, Alberto; Nichelatti, Michele; Tosi, Federica; De Braud, Filippo; Di Nicolantonio, Federica; Barault, Ludovic; Siena, Salvatore

    2017-01-01

    O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment. Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS). One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p alkylating agents. Their combination could enhance patient selection in this setting. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Culture-independent identification and quantification of Gallibacterium anatis (G. anatis) by real-time quantitative PCR

    DEFF Research Database (Denmark)

    Wang, Chong; Robles, Francisco; Ramirez, Saul

    2016-01-01

    Gallibacterium is a genus within the family Pasteurellaceae characterized by a high level of phenotypic and genetic diversity. No diagnostic method has yet been described, which allows species-specific identification of Gallibacterium anatis. The aim of this study was to develop a real...... published conventional PCR method and culture-based identification, respectively. The detection rates were 97%, 78% and 34% for the current qPCR, the conventional PCR and the culture-based identification method, respectively. The qPCR assay was able to detect the gene gyrB in serial dilutions of 10......-time quantitative PCR (qPCR) method allowing species-specific identification and quantification of G. anatis. A G. anatis specific DNA sequence was identified in the gyrase subunit B gene (gyrB) and used to design a TaqMan probe and corresponding primers. The specificity of the assay was tested on 52 bacterial...

  13. Prenatal famine and genetic variation are independently and additively associated with DNA methylation at regulatory loci within IGF2/H19.

    Directory of Open Access Journals (Sweden)

    Elmar W Tobi

    Full Text Available Both the early environment and genetic variation may affect DNA methylation, which is one of the major molecular marks of the epigenome. The combined effect of these factors on a well-defined locus has not been studied to date. We evaluated the association of periconceptional exposure to the Dutch Famine of 1944-45, as an example of an early environmental exposure, and single nucleotide polymorphisms covering the genetic variation (tagging SNPs with DNA methylation at the imprinted IGF2/H19 region, a model for an epigenetically regulated genomic region. DNA methylation was measured at five differentially methylated regions (DMRs that regulate the imprinted status of the IGF2/H19 region. Small but consistent differences in DNA methylation were observed comparing 60 individuals with periconceptional famine exposure with unexposed same-sex siblings at all IGF2 DMRs (P(BH<0.05 after adjustment for multiple testing, but not at the H19 DMR. IGF2 DMR0 methylation was associated with IGF2 SNP rs2239681 (P(BH = 0.027 and INS promoter methylation with INS SNPs, including rs689, which tags the INS VNTR, suggesting a mechanism for the reported effect of the VNTR on INS expression (P(BH = 3.4 × 10(-3. Prenatal famine and genetic variation showed similar associations with IGF2/H19 methylation and their contributions were additive. They were small in absolute terms (<3%, but on average 0.5 standard deviations relative to the variation in the population. Our analyses suggest that environmental and genetic factors could have independent and additive similarly sized effects on DNA methylation at the same regulatory site.

  14. Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

    Science.gov (United States)

    Kristensen, Lasse S; Wojdacz, Tomasz K; Thestrup, Britta B; Wiuf, Carsten; Hager, Henrik; Hansen, Lise Lotte

    2009-12-21

    The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. MS-HRM and SMART-MSP can be successfully used for single locus

  15. Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje

    2011-01-01

    To facilitate quantitative risk assessment in the meat production chain, there is a need for culture-independent quantification methods. The aim of this study was to evaluate the use of flotation, a non-destructive sample preparation method based on traditional buoyant density centrifugation...

  16. Detection of circulating tumor cells with CK20 RT-PCR is an independent negative prognostic marker in colon cancer patients - a prospective study.

    Science.gov (United States)

    Hinz, Sebastian; Hendricks, Alexander; Wittig, Amke; Schafmayer, Clemens; Tepel, Jürgen; Kalthoff, Holger; Becker, Thomas; Röder, Christian

    2017-01-13

    Detection of circulating (CTC) or disseminated tumor cells (DTC) has been associated with negative prognosis and outcome in patients with colorectal cancer, though testing for these cells is not yet part of clinical routine. There are several different methodological approaches to detect tumor cells but standardized detection assays are not implemented so far. In this prospective monocentric study 299 patients with colon cancer were included. CTC and DTC were detected using CK20 RT-PCR as well as immunocytochemistry staining with anti-pan-keratin and anti-EpCAM antibodies. The primary endpoints were: Evaluation of CTC and DTC at the time of surgery and correlation with main tumor characteristics and overall (OS) and disease free survival (DFS). Patients with detectable CTC had a 5-year OS rate of 68% compared to a 5-year OS rate of 85% in patients without detectable CTC in the blood (p = 0.002). Detection of DTC in the bone marrow with CK20 RT-PCR was not associated with a worse OS or DFS. Detection of pan-cytokeratin positive DTC in the bone marrow correlated with a significantly reduced 5-year OS rate (p = 0.048), but detection of DTC in the bone marrow with the anti-EpCAM antibody did not significantly influence the 5-year OS rate (p = 0.958). By multivariate analyses only detection of CTC with CK20 RT-PCR in the blood was revealed to be an independent predictor of worse OS (HR1.94; 95% CI 1.0-3.7; p = 0.04) and DFS (HR 1.94; 95% CI 1.1-3.7; p = 0.044). Detection of CTC with CK20 RT-PCR is a highly specific and independent prognostic marker in colon cancer patients. Detection of DTC in the bone marrow with CK20 RT-PCR or immunohistochemistry with anti-EpCAM antibody is not associated with a negative prognostic influence.

  17. The monofunctional alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine triggers apoptosis through p53-dependent and -independent pathways

    International Nuclear Information System (INIS)

    Kim, W.-J.; Beardsley, Dillon I.; Adamson, Aaron W.; Brown, Kevin D.

    2005-01-01

    One of the cellular responses to DNA damaging events is the activation of programmed cell death, also known as apoptosis. Apoptosis is an important process in limiting tumorigenesis by eliminating cells with damaged DNA. This view is reinforced by the finding that many genes with pro-apoptotic function are absent or altered in cancer cells. The tumor suppressor p53 performs a significant role in apoptotic signaling by controlling expression of a host of genes that have pro-apoptotic or pro-survival function. The S N 1 DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) triggers apoptosis and the upregulation/phosphorylation of p53; however, the mechanism(s) governing MNNG-induced cell death remain unresolved. We observed that the human lymphoblastoid cell line WTK-1, which expresses mutant p53, shows far less sensitivity to the cytotoxic effects of MNNG than the closely related, p53-normal line TK-6. Exposure to 15 μM MNNG (LD50 at 24 h in TK-6) leads to a kinetically slower rate of apoptotic onset in WTK-1 cells compared to TK-6 as judged by viability assays and approaches that directly examine apoptotic onset. Similar results were obtained using an unrelated human lymphoblastoid line B310 expressing reduced levels of p53 due to E6 oncoprotein expression, indicating that MNNG activates both p53-dependent and -independent apoptotic mechanisms and that these two mechanisms are discernable by the rates which they trigger apoptotic onset. We document, during time points corresponding to peak apoptotic response in TK6, WTK-1, B310, and B310-E6, that these cell lines show marked decreases in mitochondrial transmembrane potential and increases in cytochrome c within the cytosolic fraction of MNNG-treated cells. Consistent with these events, we observed that both caspase-9 and -3 are activated in our panel of lymphoblastoid cells after MNNG exposure. We also found, using both broad spectrum and specific inhibitors, that blocking caspase activity in TK-6 and

  18. A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

    Science.gov (United States)

    Kelly, A D; Kroeger, H; Yamazaki, J; Taby, R; Neumann, F; Yu, S; Lee, J T; Patel, B; Li, Y; He, R; Liang, S; Lu, Y; Cesaroni, M; Pierce, S A; Kornblau, S M; Bueso-Ramos, C E; Ravandi, F; Kantarjian, H M; Jelinek, J; Issa, J-Pj

    2017-10-01

    Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP + ). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP + =not reached, CIMP - =1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP + =2.34, A-CIMP - =1.00; P=0.01). Hypermethylation in A-CIMP + favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP + was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP + function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP + AML should be validated prospectively.

  19. The intervening domain from MeCP2 enhances the DNA affinity of the methyl binding domain and provides an independent DNA interaction site.

    Science.gov (United States)

    Claveria-Gimeno, Rafael; Lanuza, Pilar M; Morales-Chueca, Ignacio; Jorge-Torres, Olga C; Vega, Sonia; Abian, Olga; Esteller, Manel; Velazquez-Campoy, Adrian

    2017-01-31

    Methyl-CpG binding protein 2 (MeCP2) preferentially interacts with methylated DNA and it is involved in epigenetic regulation and chromatin remodelling. Mutations in MeCP2 are linked to Rett syndrome, the leading cause of intellectual retardation in girls and causing mental, motor and growth impairment. Unstructured regions in MeCP2 provide the plasticity for establishing interactions with multiple binding partners. We present a biophysical characterization of the methyl binding domain (MBD) from MeCP2 reporting the contribution of flanking domains to its structural stability and dsDNA interaction. The flanking disordered intervening domain (ID) increased the structural stability of MBD, modified its dsDNA binding profile from an entropically-driven moderate-affinity binding to an overwhelmingly enthalpically-driven high-affinity binding. Additionally, ID provided an additional site for simultaneously and autonomously binding an independent dsDNA molecule, which is a key feature linked to the chromatin remodelling and looping activity of MeCP2, as well as its ability to interact with nucleosomes replacing histone H1. The dsDNA interaction is characterized by an unusually large heat capacity linked to a cluster of water molecules trapped within the binding interface. The dynamics of disordered regions together with extrinsic factors are key determinants of MeCP2 global structural properties and functional capabilities.

  20. Direct Detection and Identification of Enteroviruses from Faeces of Healthy Nigerian Children Using a Cell-Culture Independent RT-Seminested PCR Assay

    Directory of Open Access Journals (Sweden)

    Temitope Oluwasegun Cephas Faleye

    2016-01-01

    Full Text Available Recently, a cell-culture independent protocol for detection of enteroviruses from clinical specimen was recommended by the WHO for surveillance alongside the previously established protocols. Here, we investigated whether this new protocol will show the same enterovirus diversity landscape as the established cell-culture dependent protocols. Faecal samples were collected from sixty apparently healthy children in Ibadan, Nigeria. Samples were resuspended in phosphate buffered saline, RNA was extracted, and the VP1 gene was amplified using WHO recommended RT-snPCR protocol. Amplicons were sequenced and sequences subjected to phylogenetic analysis. Fifteen (25% of the 60 samples yielded the expected band size. Of the 15 amplicons sequenced, 12 were exploitable. The remaining 3 had electropherograms with multiple peaks and were unexploitable. Eleven of the 12 exploitable sequences were identified as Coxsackievirus A1 (CVA1, CVA3, CVA4, CVA8, CVA20, echovirus 32 (E32, enterovirus 71 (EV71, EVB80, and EVC99. Subsequently, the last exploitable sequence was identified as enterobacteriophage baseplate gene by nucleotide BLAST. The results of this study document the first description of molecular sequence data on CVA1, CVA8, and E32 strains present in Nigeria. The result further showed that species A enteroviruses were more commonly detected in the region when cell-culture bias is bypassed.

  1. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    International Nuclear Information System (INIS)

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-01-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression

  2. Relationship between methylation status of vitamin D-related genes, vitamin D levels, and methyl-donor biochemistry

    Directory of Open Access Journals (Sweden)

    Emma Louise Beckett

    2016-12-01

    Full Text Available Vitamin D is known for its role in the regulation of gene expression via the vitamin D receptor, a nuclear transcription factor. More recently, a role for vitamin D in regulating DNA methylation has been identified as an additional mechanism of modulation of gene expression. How methylation status influences vitamin D metabolism and response pathways is not yet clear. Therefore, we aimed to assess the relationship between plasma 25-hydroxycholecalciferol (25(OHD and the methylation status of vitamin D metabolism enzyme genes (CYP2R1, CYP27B1 and CYP24A1 and the vitamin D receptor gene (VDR. This analysis was conducted in the context of dietary vitamin D, and background methyl donor related biochemistry, with adjustment for several dietary and lifestyle variables. Percentage methylation at CpG sites was assessed in peripheral blood cells using methylation sensitive and dependent enzymes and qPCR. Standard analytical techniques were used to determine plasma 25(OHD and homocysteine, and serum folate and B12, with the relationship to methylation status assessed using multi-variable regression analysis. CYP2R1 and VDR methylation were found to be independent predictors of plasma 25(OHD, when adjusted for vitamin D intake and other lifestyle variables. CYP24A1 was related to plasma 25(OHD directly, but not in the context of vitamin D intake. Methyl-group donor biochemistry was associated with the methylation status of some genes, but did not alter the relationship between methylation and plasma 25(OHD. Modulation of methylation status of CYP2R1, CYP24A1 and VDR in response to plasma 25(OHD may be part of feedback loops involved in maintaining vitamin D homeostasis, and may explain a portion of the variance in plasma 25(OHD levels in response to intake and sun exposure. Methyl-group donor biochemistry, while a potential independent modulator, did not alter this effect.

  3. JC Virus T-Antigen in Colorectal Cancer Is Associated with p53 Expression and Chromosomal Instability, Independent of CpG Island Methylator Phenotype

    Directory of Open Access Journals (Sweden)

    Katsuhiko Nosho

    2009-01-01

    Full Text Available JC virus has a transforming gene encoding JC virus T-antigen (JCVT. JCVT may inactivate wild-type p53, cause chromosomal instability (CIN, and stabilize β-catenin. A link between JCVT and CpG island methylator phenotype (CIMP has been suggested. However, no large-scale study has examined the relations of JCVT with molecular alterations, clinical outcome, or prognosis in colon cancer. We detected JCVT expression (by immunohistochemistry in 271 (35% of 766 colorectal cancers. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1 and eight other loci (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, WRN by MethyLight. We examined loss of heterozygosity in 2p, 5q, 17q, and 18q. JCVT was significantly associated with p53 expression (P < .0001, p21 loss (P < .0001, CIN (≥2 chromosomal segments with LOH; P < .0001, nuclear β-catenin (P = .006, LINE-1 hypomethylation (P = .002, and inversely with CIMP-high (P = .0005 and microsatellite instability (MSI (P < .0001, but not with PIK3CA mutation. In multivariate logistic regression analysis, the associations of JCVT with p53 [adjusted odds ratio (OR, 8.45; P < .0001], CIN (adjusted OR, 2.53; P = .003, cyclin D1 (adjusted OR, 1.57; P = .02, LINE-1 hypomethylation (adjusted OR, 1.97 for a 30% decline as a unit; P = .03, BRAF mutation (adjusted OR, 2.20; P = .04, and family history of colorectal cancer (adjusted OR, 0.64; P = .04 remained statistically significant. However, JCVT was no longer significantly associated with CIMP, MSI, β-catenin, or cyclooxygenase-2 expression in multivariate analysis. JCVT was unrelated with patient survival. In conclusion, JCVT expression in colorectal cancer is independently associated with p53 expression and CIN, which may lead to uncontrolled cell proliferation.

  4. Activation of protein kinase A and clustering of cell surface receptors by N-methyl-N'-nitro-N-nitrosoguanidine are independent of genomic DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang Zheng; Wang Guliang; Yang Jun; Guo Lei; Yu Yingnian

    2003-07-25

    Alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces cellular stress leading to chromosomal aberrations, mutations and cell death. Previous reports from our laboratory have shown that low concentration of MNNG induces untargeted mutation (UTM), which occurs on intact DNA in mammalian cells through changes in gene expression profile. It also causes the activation of cAMP-protein kinase A (PKA) and up-regulation of POL-{beta}, which is demonstrated to play a role in DNA repair system. In order to find out the possible initial signal involved in UTM, we try to investigate whether the activation of PKA-CREB signal pathway is closely related to DNA damage. Our data shows that the treatment of low concentration MNNG (0.2 {mu}M) activates PKA-CREB pathway in a comparable level both in a nuclear and enucleated cell system. And similar to the cell response caused by UV, the clustering of cell surface receptors of epidermal growth factor (EGF) and tumor necrosis factor {alpha} (TNF{alpha}) was also observed in cells exposed to MNNG. It was further demonstrated that the clustering of the surface receptors is independent of the genomic DNA damage, as this phenomenon was also observed in enucleated cells. These observations indicate that the initiation of signal cascades induced by low concentration of MNNG might be associated with its interaction with cell surface receptors and/or direct activation of related signal proteins but not its DNA damaging property.

  5. DNA methylation affects the lifespan of honey bee (Apis mellifera L.) workers - Evidence for a regulatory module that involves vitellogenin expression but is independent of juvenile hormone function.

    Science.gov (United States)

    Cardoso-Júnior, Carlos A M; Guidugli-Lazzarini, Karina R; Hartfelder, Klaus

    2018-01-01

    The canonic regulatory module for lifespan of honey bee (Apis mellifera) workers involves a mutual repressor relationship between juvenile hormone (JH) and vitellogenin (Vg). Compared to vertebrates, however, little is known about a possible role of epigenetic factors. The full genomic repertoire of DNA methyltransferases (DNMTs) makes the honey bee an attractive emergent model for studying the role of epigenetics in the aging process of invertebrates, and especially so in social insects. We first quantified the transcript levels of the four DNMTs encoding genes in the head thorax and abdomens of workers of different age, showing that dnmt1a and dnmt3 expression is up-regulated in abdomens of old workers, whereas dnmt1b and dnmt2 are down-regulated in heads of old workers. Pharmacological genome demethylation by RG108 treatment caused an increase in worker lifespan. Next, we showed that the genomic DNA methylation status indirectly affects vitellogenin gene expression both in vitro and in vivo in young workers, and that this occurs independent of caloric restriction or JH levels, suggesting that a non-canonical circuitry may be acting in parallel with the JH/Vg module to regulate the adult life cycle of honey bee workers. Our data provide evidence that epigenetic factors play a role in regulatory networks associated with complex life history traits of a social insect. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics.

    Science.gov (United States)

    Panopoulos, Athanasia D; Smith, Erin N; Arias, Angelo D; Shepard, Peter J; Hishida, Yuriko; Modesto, Veronica; Diffenderfer, Kenneth E; Conner, Clay; Biggs, William; Sandoval, Efren; D'Antonio-Chronowska, Agnieszka; Berggren, W Travis; Izpisua Belmonte, Juan Carlos; Frazer, Kelly A

    2017-04-06

    Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming. Published by Elsevier Inc.

  7. Application of real-time PCR of sex-independent insertion-deletion polymorphisms to determine fetal sex using cell-free fetal DNA from maternal plasma.

    Science.gov (United States)

    Ho, Sherry Sze Yee; Barrett, Angela; Thadani, Henna; Asibal, Cecille Laureano; Koay, Evelyn Siew-Chuan; Choolani, Mahesh

    2015-07-01

    Prenatal diagnosis of sex-linked disorders requires invasive procedures, carrying a risk of miscarriage of up to 1%. Cell-free fetal DNA (cffDNA) present in cell-free DNA (cfDNA) from maternal plasma offers a non-invasive source of fetal genetic material for analysis. Detection of Y-chromosome sequences in cfDNA indicates presence of a male fetus; in the absence of a Y-chromosome signal a female fetus is inferred. We aimed to validate the clinical utility of insertion-deletion polymorphisms (INDELs) to confirm presence of a female fetus using cffDNA. Quantitative real-time PCR (qPCR) for the Y-chromosome-specific sequence, SRY, was performed on cfDNA from 82 samples at 6-39 gestational weeks. In samples without detectable SRY, qPCRs for eight INDELs were performed on maternal genomic DNA and cfDNA. Detection of paternally inherited fetal alleles in cfDNA negative for SRY confirmed a female fetus. Fetal sex was correctly determined in 77/82 (93.9%) cfDNA samples. SRY was detected in all 39 samples from male-bearing pregnancies, and none of the 43 female-bearing pregnancies (sensitivity and specificity of SRY qPCR is therefore 100%; 95% CI 91%-100%). Paternally inherited fetal alleles were detected in 38/43 samples with no SRY signal, confirming the presence of a female fetus (INDEL assay sensitivity is therefore 88.4%; 95% CI 74.1%-95.6%). Since paternally inherited fetal INDELs were not used in women bearing male fetuses, the specificity of INDELs cannot be calculated. Five cfDNA samples were negative for both SRY and INDELS. We have validated a non-invasive prenatal test to confirm fetal sex as early as 6 gestational weeks using cffDNA from maternal plasma.

  8. The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline.

    Science.gov (United States)

    Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J; Nee, Kevin; Clark, Amander T

    2015-07-01

    Removal of cytosine methylation from the genome is critical for reprogramming and transdifferentiation and plays a central role in our understanding of the fundamental principles of embryo lineage development. One of the major models for studying cytosine demethylation is the mammalian germ line during the primordial germ cell (PGC) stage of embryo development. It is now understood that oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is required to remove cytosine methylation in a locus-specific manner in PGCs; however, the mechanisms downstream of 5hmC are controversial and hypothesized to involve either active demethylation or replication-coupled loss. In the current study, we used the aorta-gonad-mesonephros (AGM) organ culture model to show that this model recapitulates germ line reprogramming, including 5hmC reorganization and loss of cytosine methylation from Snrpn and H19 imprinting control centers (ICCs). To directly address the hypothesis that cell proliferation is required for cytosine demethylation, we blocked PI3-kinase-dependent PGC proliferation and show that this leads to a G1 and G2/M cell cycle arrest in PGCs, together with retained levels of cytosine methylation at the Snrpn ICC, but not at the H19 ICC. Taken together, the AGM organ culture model is an important tool to evaluate mechanisms of locus-specific demethylation and the role of PI3-kinase-dependent PGC proliferation in the locus-specific removal of cytosine methylation from the genome.

  9. PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor

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    Leyla Esfandiari

    2016-07-01

    Full Text Available A PCR-free, optics-free device is used for the detection of Escherichia coli (E. coli 16S rRNA at 10 fM, which corresponds to ~100–1000 colony forming units/mL (CFU/mL depending on cellular rRNA levels. The development of a rapid, sensitive, and cost-effective nucleic acid detection platform is sought for the detection of pathogenic microbes in food, water and body fluids. Since 16S rRNA sequences are species specific and are present at high copy number in viable cells, these nucleic acids offer an attractive target for microbial pathogen detection schemes. Here, target 16S rRNA of E. coli at 10 fM concentration was detected against a total RNA background using a conceptually simple approach based on electromechanical signal transduction, whereby a step change reduction in ionic current through a pore indicates blockage by an electrophoretically mobilized bead-peptide nucleic acid probe conjugate hybridized to target nucleic acid. We investigated the concentration detection limit for bacterial species-specific 16S rRNA at 1 pM to 1 fM and found a limit of detection of 10 fM for our device, which is consistent with our previous finding with single-stranded DNA of similar length. In addition, no false positive responses were obtained with control RNA and no false negatives with target 16S rRNA present down to the limit of detection (LOD of 10 fM. Thus, this detection scheme shows promise for integration into portable, low-cost systems for rapid detection of pathogenic microbes in food, water and body fluids.

  10. Q-PCR Based Culture-Independent Enumeration and Detection of Enterobacter: An Emerging Environmental Human Pathogen in Riverine Systems and Potable Water

    Science.gov (United States)

    Patel, Chandra B.; Shanker, Rishi; Gupta, Vijai K.; Upadhyay, Ram S.

    2016-01-01

    The availability of safe and pristine water is a global challenge when large numbers of natural and anthropogenic water resources are being depleted with faster rate. The remaining water resources are severely contaminated with various kinds of contaminants including microorganisms. Enterobacter is one of the fecal coliform bacteria of family Enterobacteriaceae. Enterobacter was earlier used as an indicator bacterium along with other fecal Coliforms namely Escherichia coli, Citrobacter, and Klebsiella, but it is now known to cause various diseases in human beings. In this study, we have collected 55 samples from potable water and riverine system and proved their presence using their conserved sequences of 16S rRNA and 23S rRNA genes with the help of SYBR green real-time PCR, which showed very high specificity for the detection of Enterobacter. The Enterobacter counts in potable water were found to 1290 ± 32.89 to 1460 ± 39.42 cfu/100 ml. The Enterobacter levels in surface water were 1.76 × 104 ± 492, 1.33 × 104 ± 334, 1.15 × 104 ± 308, 2.56 × 104 ± 802, 2.89 × 104 ± 962, 8.16 × 104 ± 3443 cfu/100 ml; the levels of Enterobacter contamination associated with hydrophytes were 4.80 × 104 ± 1804, 3.48 × 104 ± 856, 8.50 × 104 ± 2074, 8.09 × 104 ± 1724, 6.30 × 104 ± 1738, 3.68 × 104 ± 949 cfu/10 g and the Enterobacter counts in sediments of the river, were 2.36 × 104 ± 703, 1.98 × 104 ± 530, 9.92 × 104 ± 3839, 6.80 × 104 ± 2230, 8.76 × 104 ± 3066 and 2.34 × 104 ± 732 cfu/10 g at the sampling Site #1, Site #2, Site #3, Site #4, Site #5, and Site #6, respectively. The assay could be used for the regular monitoring of potable water and other water reservoirs to check waterborne outbreaks. PMID:26925044

  11. Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

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    Řičicová Markéta

    2010-04-01

    Full Text Available Abstract Background Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein and of genes belonging to the ALS (agglutinin-like sequence, SAP (secreted aspartyl protease, PLB (phospholipase B and LIP (lipase gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP or in the Centres for Disease Control (CDC reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR model, and on mucosal surfaces in the reconstituted human epithelium (RHE model. Results HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model. Conclusions Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression

  12. Prenatal Exposure to Maternal Cigarette Smoking and DNA Methylation: Epigenome-Wide Association in a Discovery Sample of Adolescents and Replication in an Independent Cohort at Birth through 17 Years of Age

    Science.gov (United States)

    Lee, Ken W.K.; Richmond, Rebecca; Hu, Pingzhao; French, Leon; Shin, Jean; Bourdon, Celine; Reischl, Eva; Waldenberger, Melanie; Zeilinger, Sonja; Gaunt, Tom; McArdle, Wendy; Ring, Susan; Woodward, Geoff; Bouchard, Luigi; Gaudet, Daniel; Smith, George Davey; Relton, Caroline; Paus, Tomas

    2014-01-01

    , Pausova Z. 2015. Prenatal exposure to maternal cigarette smoking and DNA methylation: epigenome-wide association in a discovery sample of adolescents and replication in an independent cohort at birth through 17 years of age. Environ Health Perspect 123:193–199; http://dx.doi.org/10.1289/ehp.1408614 PMID:25325234

  13. Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide).

    Science.gov (United States)

    Pandith, Arshad A; Qasim, Iqbal; Zahoor, Wani; Shah, Parveen; Bhat, Abdul R; Sanadhya, Dheera; Shah, Zafar A; Naikoo, Niyaz A

    2018-04-30

    O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients with respect to lower Hazard Ratio (HR) for better OS and PFS) [p < 0.05]. Interestingly concordant MGMT methylation and lack of protein showed better response in TMZ therapy treated patient subgroups with HR of 2.02 and 0.76 (p < 0.05). We found the merits of prognostication of MGMT parameters, methylation as well as loss of its protein as predictive factors for favorable outcome in terms of better survival for TMZ therapy.

  14. MGMT, GATA6, CD81, DR4, and CASP8 gene promoter methylation in glioblastoma

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    Skiriute Daina

    2012-06-01

    Full Text Available Abstract Background Methylation of promoter region is the major mechanism affecting gene expression in tumors. Recent methylome studies of brain tumors revealed a list of new epigenetically modified genes. Our aim was to study promoter methylation of newly identified epigenetically silenced genes together with already known epigenetic markers and evaluate its separate and concomitant role in glioblastoma genesis and patient outcome. Methods The methylation status of MGMT, CD81, GATA6, DR4, and CASP8 in 76 patients with primary glioblastomas was investigated. Methylation-specific PCR reaction was performed using bisulfite treated DNA. Evaluating glioblastoma patient survival time after operation, patient data and gene methylation effect on survival was estimated using survival analysis. Results The overwhelming majority (97.3% of tumors were methylated in at least one of five genes tested. In glioblastoma specimens gene methylation was observed as follows: MGMT in 51.3%, GATA6 in 68.4%, CD81 in 46.1%, DR4 in 41.3% and CASP8 in 56.8% of tumors. Methylation of MGMT was associated with younger patient age (p CASP8 with older (p MGMT methylation was significantly more frequent event in patient group who survived longer than 36 months after operation (p CASP8 was more frequent in patients who survived shorter than 36 months (p MGMT, GATA6 and CASP8 as independent predictors for glioblastoma patient outcome (p MGMT and GATA6 were independent predictors for patient survival in younger patients’ group, while there were no significant associations observed in older patients’ group when adjusted for therapy. Conclusions High methylation frequency of tested genes shows heterogeneity of glioblastoma epigenome and the importance of MGMT, GATA6 and CASP8 genes methylation in glioblastoma patient outcome.

  15. Glucose-independent persistence of PAI-1 gene expression and H3K4 tri-methylation in type 1 diabetic mouse endothelium: implication in metabolic memory.

    Science.gov (United States)

    Takizawa, Fumihiko; Mizutani, Shuki; Ogawa, Yoshihiro; Sawada, Naoki

    2013-03-29

    Clinical trials with type 1 and type 2 diabetes have identified a phenomenon known as "metabolic memory" in which previous periods of hyperglycemia result in the long-lasting deleterious impact on cardiovascular events. Emerging evidence shows that transient hyperglycemic exposure of human endothelial cells induces histone 3 lysine 4 mono-methylation (H3K4me1) on the promoter and persistent mRNA expression of RelA and IL-8 genes, suggesting that epigenetic histone modification and chromatin structure remodeling is a key event underlying metabolic memory. This burgeoning hypothesis, however, critically remains to be tested for relevance in the disease process of diabetes in vivo, and for broader applicability to an array of genes involved in endothelial dysfunction. To address this, we used type 1 diabetes mouse model induced by streptozocin to be hyperglycemic for 8 weeks, and isolated endothelial cells that were used either freshly after isolation or after 2 to 3-week cell culture in normoglycemic conditions. mRNA expression profiling in diabetic mouse endothelial cells revealed significant and persistent up-regulation of Serpine1 encoding PAI-1, the hypo-fibrinolytic mediator leading to thrombotic diseases in diabetes, along with Rock2, Fn1 and Ccl2, whereas only Serpine 1 was persistently elevated in high glucose-treated mouse endothelial cells. Chromosome immunoprecipitation assay in type 1 diabetic mouse endothelial cells showed predominant enrichment of H3K4 tri-methylation on Serpine1 promoter, suggesting a unique epigenetic regulation in diabetic mice as opposed to high glucose-treated human ECs. Our study demonstrates the importance of combining in vivo models of diabetes with high glucose-treated cell culture to better assess the epigenetic mechanisms relevant to disease. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. CpG island methylator phenotype is an independent predictor of survival after curative resection for colorectal cancer: A prospective cohort study.

    Science.gov (United States)

    Kim, Chang Hyun; Huh, Jung Wook; Kim, Hyeong Rok; Kim, Young Jin

    2017-08-01

    The CpG island methylator phenotype (CIMP) is found in approximately 30% of colorectal cancer (CRC) cases. However, the role of CIMP status in predicting oncologic outcomes in curatively resected CRC is still unclear. Between January 2006 and December 2006, we retrospectively reviewed 157 consecutive patients who underwent curative surgery for CRC. Prognostic significance of CIMP status was evaluated using reverse transcriptase-polymerase chain reaction. CIMP-high (H) and CIMP-none/low (N/L) tumors were found in 50 cases (31.8%) and 107 cases (68.2%), respectively. CIMP-H tumors were significantly associated with female sex, colonic location, poorly/mucinous histologic type, higher T category, perineural invasion, and MSI-high status (P = 0.001). During a median of 64.5 months, tumor recurrence developed in 47 (29.9%) patients. The 5-year disease-free survival for CIMP-H and CIMP-N/L was 61.4% and 76.3% (P = 0.018). In addition, multivariate analysis showed that CIMP-H was also a significant prognostic factor (P = 0.042). When analysis was performed according to anatomical location, more marked survival differences were observed in patients with colon cancer (P = 0.026) than in patients with rectal cancer (P = 0.210). Similarly, the role of CIMP status as a prognostic indicator was more prominent in patients with stage I/II (P = 0.006) than in patients with stage III/IV CRC (P = 0.65). DNA methylation status can be considered as a useful predictor of survival after CRC surgery, particularly for patients with stage I/II disease or colon cancer. © 2017 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

  17. Digital PCR: A brief history

    OpenAIRE

    Morley, Alexander A.

    2014-01-01

    Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term “limiting dilution PCR” and in 1999 using the term “digital PCR”. It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and...

  18. Real-time colorimetric detection of DNA methylation of the PAX1 gene in cervical scrapings for cervical cancer screening with thiol-labeled PCR primers and gold nanoparticles

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    Huang J

    2016-10-01

    Full Text Available Jin Huang,1,2 Yu-Ligh Liou,1,2 Ya-Nan Kang,3 Zhi-Rong Tan,1,2 Ming-Jing Peng,1,2 Hong-Hao Zhou1,2 1Department of Clinical Pharmacology, Xiangya Hospital, Central South University, 2Institute of Clinical Pharmacology, Hunan Key Laboratory of Pharmacogenetics, Central South University, 3Department of Obstetrics and Gynecology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China Background: DNA methylation can induce carcinogenesis by silencing key tumor suppressor genes. Analysis of aberrant methylation of tumor suppressor genes can be used as a prognostic and predictive biomarker for cancer. In this study, we propose a colorimetric method for the detection of DNA methylation of the paired box gene 1 (PAX1 gene in cervical scrapings obtained from 42 patients who underwent cervical colposcopic biopsy. Methods: A thiolated methylation-specific polymerase chain reaction (MSP primer was used to generate MSP products labeled with the thiol group at one end. After bisulfite conversion and MSP amplification, the unmodified gold nanoparticles (AuNPs were placed in a reaction tube and NaCl was added to induce aggregation of bare AuNPs without generating polymerase chain reaction products. After salt addition, the color of AuNPs remained red in the methylated PAX1 gene samples because of binding to the MSP-amplified products. By contrast, the color of the AuNP colloid solution changed from red to blue in the non-methylated PAX1 gene samples because of aggregation of AuNPs in the absence of the MSP-amplified products. Furthermore, PAX1 methylation was quantitatively detected in cervical scrapings of patients with varied pathological degrees of cervical cancer. Conventional quantitative MSP (qMSP was also performed for comparison. Results: The two methods showed a significant correlation of the methylation frequency of the PAX1 gene in cervical scrapings with severity of cervical cancer (n=42, P<0.05. The results of the

  19. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    Science.gov (United States)

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. Human T-Cell Leukemia Virus Type I-Mediated Repression of PDZ-LIM Domain-Containing Protein 2 Involves DNA Methylation But Independent of the Viral Oncoprotein Tax

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    Pengrong Yan

    2009-10-01

    Full Text Available Human T-cell leukemia virus type I (HTLV-I is the etiological agent of adult T-cell leukemia (ATL. Our recent studies have shown that one important mechanism of HTLV-I-Mediated tumorigenesis is through PDZ-LIM domain-containing protein 2 (PDLIM2 repression, although the involved mechanism remains unknown. Here, we further report that HTLV-I-Mediated PDLIM2 repression was a pathophysiological event and the PDLIM2 repression involved DNA methylation. Whereas DNA methyltransferases 1 and 3b but not 3a were upregulated in HTLV-I-transformed T cells, the hypomethylating agent 5-aza-2′-deoxycytidine (5-aza-dC restored PDLIM2 expression and induced death of these malignant cells. Notably, the PDLIM2 repression was independent of the viral regulatory protein Tax because neither short-term induction nor long-term stable expression of Tax could downregulate PDLIM2 expression. These studies provide important insights into PDLIM2 regulation, HTLV-I leukemogenicity, long latency, and cancer health disparities. Given the efficient antitumor activity with no obvious toxicity of 5-aza-dC, these studies also suggest potential therapeutic strategies for ATL.

  1. Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

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    Victoria Gonzalo

    Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

  2. Promoter methylation of APC and RAR-β genes as prognostic markers in non-small cell lung cancer (NSCLC).

    Science.gov (United States)

    Feng, Hongxiang; Zhang, Zhenrong; Qing, Xin; Wang, Xiaowei; Liang, Chaoyang; Liu, Deruo

    2016-02-01

    Aberrant promoter hypermethylations of tumor suppressor genes are promising markers for lung cancer diagnosis and prognosis. The purpose of this study was to determine methylation status at APC and RAR-β promoters in primary NSCLC, and whether they have any relationship with survival. APC and RAR-β promoter methylation status were determined in 41 NSCLC patients using methylation specific PCR. APC promoter methylation was detectable in 9 (22.0%) tumor samples and 6 (14.6%) corresponding non-tumor samples (P=0.391). RAR-β promoter methylation was detectable in 13 (31.7%) tumor samples and 4 (9.8%) corresponding non-tumor samples (P=0.049) in the NSCLC patients. APC promoter methylation was found to be associated with T stage (P=0.046) and nodal status (P=0.019) in non-tumor samples, and with smoking (P=0.004) in tumor samples. RAR-β promoter methylation was found associated with age (P=0.031) in non-tumor samples and with primary tumor site in tumor samples. Patients with APC promoter methylation in tumor samples showed significantly longer survival than patients without it (Log-rank P=0.014). In a multivariate analysis of prognostic factors, APC methylation in tumor samples was an independent prognostic factor (P=0.012), as were N1 positive lymph node number (P=0.025) and N2 positive lymph node number (P=0.06). Our study shows that RAR-β methylation detected in lung tissue may be used as a predictive marker for NSCLC diagnosis and that APC methylation in tumor sample may be a useful marker for superior survival in NSCLC patients. Copyright © 2015. Published by Elsevier Inc.

  3. Allele-Specific DNA Methylation Detection by Pyrosequencing®

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    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide......-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon....

  4. Reference Materials for Calibration of Analytical Biases in Quantification of DNA Methylation.

    Science.gov (United States)

    Yu, Hannah; Hahn, Yoonsoo; Yang, Inchul

    2015-01-01

    Most contemporary methods for the quantification of DNA methylation employ bisulfite conversion and PCR amplification. However, many reports have indicated that bisulfite-mediated PCR methodologies can result in inaccurate measurements of DNA methylation owing to amplification biases. To calibrate analytical biases in quantification of gene methylation, especially those that arise during PCR, we utilized reference materials that represent exact bisulfite-converted sequences with 0% and 100% methylation status of specific genes. After determining relative quantities using qPCR, pairs of plasmids were gravimetrically mixed to generate working standards with predefined DNA methylation levels at 10% intervals in terms of mole fractions. The working standards were used as controls to optimize the experimental conditions and also as calibration standards in melting-based and sequencing-based analyses of DNA methylation. Use of the reference materials enabled precise characterization and proper calibration of various biases during PCR and subsequent methylation measurement processes, resulting in accurate measurements.

  5. Methylation diet and methyl group genetics in risk for adenomatous polyp occurrence

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    Mark Lucock

    2015-06-01

    Conclusion: A methylation diet influences methyl group synthesis in the regulation of blood homocysteine level, and is modulated by genetic interactions. Methylation-related nutrients also interact with key genes to modify risk of AP, a precursor of colorectal cancer. Independent of diet, two methylation-related genes (A2756G-MS and A66G-MSR were directly associated with AP occurrence.

  6. CDO1 promoter methylation is associated with gene silencing and is a prognostic biomarker for biochemical recurrence-free survival in prostate cancer patients.

    Science.gov (United States)

    Meller, Sebastian; Zipfel, Lisa; Gevensleben, Heidrun; Dietrich, Jörn; Ellinger, Jörg; Majores, Michael; Stein, Johannes; Sailer, Verena; Jung, Maria; Kristiansen, Glen; Dietrich, Dimo

    2016-12-01

    Molecular biomarkers may facilitate the distinction between aggressive and clinically insignificant prostate cancer (PCa), thereby potentially aiding individualized treatment. We analyzed cysteine dioxygenase 1 (CDO1) promoter methylation and mRNA expression in order to evaluate its potential as prognostic biomarker. CDO1 methylation and mRNA expression were determined in cell lines and formalin-fixed paraffin-embedded prostatectomy specimens from a first cohort of 300 PCa patients using methylation-specific qPCR and qRT-PCR. Univariate and multivariate Cox proportional hazards and Kaplan-Meier analyses were performed to evaluate biochemical recurrence (BCR)-free survival. Results were confirmed in an independent second cohort comprising 498 PCa cases. Methylation and mRNA expression data from the second cohort were generated by The Cancer Genome Atlas (TCGA) Research Network by means of Infinium HumanMethylation450 BeadChip and RNASeq. CDO1 was hypermethylated in PCa compared to normal adjacent tissues and benign prostatic hyperplasia (P < 0.001) and was associated with reduced gene expression (ρ = -0.91, P = 0.005). Using two different methodologies for methylation quantification, high CDO1 methylation as continuous variable was associated with BCR in univariate analysis (first cohort: HR = 1.02, P = 0.002, 95% CI [1.01-1.03]; second cohort: HR = 1.02, P = 0.032, 95% CI [1.00-1.03]) but failed to reach statistical significance in multivariate analysis. CDO1 promoter methylation is involved in gene regulation and is a potential prognostic biomarker for BCR-free survival in PCa patients following radical prostatectomy. Further studies are needed to validate CDO1 methylation assays and to evaluate the clinical utility of CDO1 methylation for the management of PCa.

  7. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian

    2012-01-01

    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  8. DNA Methylation and Gene Expression Profiling of Ewing Sarcoma Primary Tumors Reveal Genes That Are Potential Targets of Epigenetic Inactivation

    Directory of Open Access Journals (Sweden)

    Nikul Patel

    2012-01-01

    Full Text Available The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1, and VCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

  9. Evaluating genome-wide DNA methylation changes in mice by Methylation Specific Digital Karyotyping

    Directory of Open Access Journals (Sweden)

    Maruoka Shuichiro

    2008-12-01

    Full Text Available Abstract Background The study of genome-wide DNA methylation changes has become more accessible with the development of various array-based technologies though when studying species other than human the choice of applications are limited and not always within reach. In this study, we adapted and tested the applicability of Methylation Specific Digital Karyotyping (MSDK, a non-array based method, for the prospective analysis of epigenetic changes after perinatal nutritional modifications in a mouse model of allergic airway disease. MSDK is a sequenced based method that allows a comprehensive and unbiased methylation profiling. The method generates 21 base pairs long sequence tags derived from specific locations in the genome. The resulting tag frequencies determine in a quantitative manner the methylation level of the corresponding loci. Results Genomic DNA from whole lung was isolated and subjected to MSDK analysis using the methylation-sensitive enzyme Not I as the mapping enzyme and Nla III as the fragmenting enzyme. In a pair wise comparison of the generated mouse MSDK libraries we identified 158 loci that are significantly differentially methylated (P-value = 0.05 after perinatal dietary changes in our mouse model. Quantitative methylation specific PCR and sequence analysis of bisulfate modified genomic DNA confirmed changes in methylation at specific loci. Differences in genomic MSDK tag counts for a selected set of genes, correlated well with changes in transcription levels as measured by real-time PCR. Furthermore serial analysis of gene expression profiling demonstrated a dramatic difference in expressed transcripts in mice exposed to perinatal nutritional changes. Conclusion The genome-wide methylation survey applied in this study allowed for an unbiased methylation profiling revealing subtle changes in DNA methylation in mice maternally exposed to dietary changes in methyl-donor content. The MSDK method is applicable for mouse models

  10. Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Wolff, Dennis W; Xie, Yan; Deng, Caishu; Gatalica, Zoran; Yang, Mingjie; Wang, Bo; Wang, Jincheng; Lin, Ming-Fong; Abel, Peter W; Tu, Yaping

    2012-04-01

    G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity. Copyright © 2011 UICC.

  11. Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

    Science.gov (United States)

    Rebollo, Rita; Mager, Dixie L

    2016-01-01

    Endogenous retroviruses are repetitive sequences found abundantly in mammalian genomes which are capable of modulating host gene expression. Nevertheless, most endogenous retrovirus copies are under tight epigenetic control via histone-repressive modifications and DNA methylation. Here we describe a common method used in our laboratory to detect, quantify, and compare mammalian endogenous retrovirus DNA methylation. More specifically we describe methylated DNA immunoprecipitation (MeDIP) followed by quantitative PCR.

  12. Eliminating PCR contamination

    International Nuclear Information System (INIS)

    Fox, J.C.; Ait-Khaled, Mounir; Webster, Alison; Emery, V.C.

    1991-01-01

    The sensitivity of polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for detection of human cytomegalovirus CMV and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. Sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, ultimate sensitivity of a PCR system for the amplification of an early gene pro-motor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32 P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worth-wile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system. (author). 11 refs.; 3 figs

  13. MicroRNA-137 promoter methylation in oral lichen planus and oral squamous cell carcinoma

    DEFF Research Database (Denmark)

    Dang, Jun; Bian, Yong-qian; Sun, Jian-yong

    2013-01-01

    and patients with oral squamous cell carcinoma (OSCC). A total of 20 OLP and 12 patients with OSCC as well as 10 healthy subjects were subjected to miR-137 promoter methylation analysis using methylation-specific PCR (MSP). To address the malignancy prediction potential from miR-137 promoter methylation status...

  14. Global DNA methylation analysis using methyl-sensitive amplification polymorphism (MSAP).

    Science.gov (United States)

    Yaish, Mahmoud W; Peng, Mingsheng; Rothstein, Steven J

    2014-01-01

    DNA methylation is a crucial epigenetic process which helps control gene transcription activity in eukaryotes. Information regarding the methylation status of a regulatory sequence of a particular gene provides important knowledge of this transcriptional control. DNA methylation can be detected using several methods, including sodium bisulfite sequencing and restriction digestion using methylation-sensitive endonucleases. Methyl-Sensitive Amplification Polymorphism (MSAP) is a technique used to study the global DNA methylation status of an organism and hence to distinguish between two individuals based on the DNA methylation status determined by the differential digestion pattern. Therefore, this technique is a useful method for DNA methylation mapping and positional cloning of differentially methylated genes. In this technique, genomic DNA is first digested with a methylation-sensitive restriction enzyme such as HpaII, and then the DNA fragments are ligated to adaptors in order to facilitate their amplification. Digestion using a methylation-insensitive isoschizomer of HpaII, MspI is used in a parallel digestion reaction as a loading control in the experiment. Subsequently, these fragments are selectively amplified by fluorescently labeled primers. PCR products from different individuals are compared, and once an interesting polymorphic locus is recognized, the desired DNA fragment can be isolated from a denaturing polyacrylamide gel, sequenced and identified based on DNA sequence similarity to other sequences available in the database. We will use analysis of met1, ddm1, and atmbd9 mutants and wild-type plants treated with a cytidine analogue, 5-azaC, or zebularine to demonstrate how to assess the genetic modulation of DNA methylation in Arabidopsis. It should be noted that despite the fact that MSAP is a reliable technique used to fish for polymorphic methylated loci, its power is limited to the restriction recognition sites of the enzymes used in the genomic

  15. MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

    Science.gov (United States)

    Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

    2016-01-01

    The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

  16. Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo

    International Nuclear Information System (INIS)

    Du, Yantao; Liu, Zhaojun; Gu, Liankun; Zhou, Jing; Zhu, Bu-dong; Ji, Jiafu; Deng, Dajun

    2012-01-01

    Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may

  17. External PCR, ASN's decision

    International Nuclear Information System (INIS)

    Anon.

    2012-01-01

    The French law imposes in some situations the presence of a person skilled in radiation protection (PCR). This article describes the cases when this person must belong to the staff of the enterprise or when this person may be sub-contracted. For instance in most nuclear facilities the PCR must be on the payroll, for enterprises dedicated to nuclear transport the PCR's job can be sub-contracted. A decision given by the ASN (French Nuclear Safety Authority) sets the minimal requests (in terms of training, job contract, activities) of the sub-contracted PCR. (A.C.)

  18. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    Science.gov (United States)

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  19. Colorectal Cancer "Methylator Phenotype": Fact or Artifact?

    Directory of Open Access Journals (Sweden)

    Charles Anacleto

    2005-04-01

    Full Text Available It has been proposed that human colorectal tumors can be classified into two groups: one in which methylation is rare, and another with methylation of several loci associated with a "CpG island methylated phenotype (CIMP," characterized by preferential proximal location in the colon, but otherwise poorly defined. There is considerable overlap between this putative methylator phenotype and the well-known mutator phenotype associated with microsatellite instability (MSI. We have examined hypermethylation of the promoter region of five genes (DAPK, MGMT, hMLH1, p16INK4a, and p14ARF in 106 primary colorectal cancers. A graph depicting the frequency of methylated loci in the series of tumors showed a continuous, monotonically decreasing distribution quite different from the previously claimed discontinuity. We observed a significant association between the presence of three or more methylated loci and the proximal location of the tumors. However, if we remove from analysis the tumors with hMLH1 methylation or those with MSI, the significance vanishes, suggesting that the association between multiple methylations and proximal location was indirect due to the correlation with MSI. Thus, our data do not support the independent existence of the so-called methylator phenotype and suggest that it rather may represent a statistical artifact caused by confounding of associations.

  20. MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

    Science.gov (United States)

    McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

    2016-06-01

    Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

  1. Inverse fusion PCR cloning.

    Directory of Open Access Journals (Sweden)

    Markus Spiliotis

    Full Text Available Inverse fusion PCR cloning (IFPC is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

  2. Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

    OpenAIRE

    Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schr?der, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinh?usel, Andreas; Egger, Gerda

    2015-01-01

    Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-...

  3. Are Independent Probes Truly Independent?

    Science.gov (United States)

    Camp, Gino; Pecher, Diane; Schmidt, Henk G.; Zeelenberg, Rene

    2009-01-01

    The independent cue technique has been developed to test traditional interference theories against inhibition theories of forgetting. In the present study, the authors tested the critical criterion for the independence of independent cues: Studied cues not presented during test (and unrelated to test cues) should not contribute to the retrieval…

  4. Recurrence in oral and pharyngeal cancer is associated with quantitative MGMT promoter methylation

    International Nuclear Information System (INIS)

    Taioli, Emanuela; Ragin, Camille; Wang, Xiao-hong; Chen, Jiangying; Langevin, Scott M; Brown, Ashley R; Gollin, Susanne M; Garte, Seymour; Sobol, Robert W

    2009-01-01

    Biomarkers that predict clinical response, tumor recurrence or patient survival are severely lacking for most cancers, particularly for oral and pharyngeal cancer. This study examines whether gene-promoter methylation of tumor DNA correlates with survival and recurrence rates in a population of patients with oral or pharyngeal cancer. The promoter methylation status of the DNA repair gene MGMT and the tumor suppressor genes CDKN2A and RASSF1 were evaluated by methylation-specific PCR in 88 primary oral and pharyngeal tumors and correlated with survival and tumor recurrence. Quantitative MGMT methylation was also assessed. 29.6% of the tumors presented with MGMT methylation, 11.5% with CDKN2A methylation and 12.1% with RASSF1 methylation. MGMT promoter methylation was significantly associated with poorer overall and disease-free survival. No differences in methylation status of MGMT and RASSF1 with HPV infection, smoking or drinking habits were observed. A significant inverse trend with the amount of MGMT methylation and overall and disease-free survival was observed (p trend = 0.002 and 0.001 respectively). These results implicate MGMT promoter methylation as a possible biomarker for oral and pharyngeal cancer prognosis. The critical role of MGMT in DNA repair suggests that defective DNA repair may be correlative in the observed association between MGMT promoter methylation and tumor recurrence. Follow-up studies should include further quantitative MSP-PCR measurement, global methylation profiling and detailed analysis of downstream DNA repair genes regulated by promoter methylation

  5. Infiltrating leukocytes confound the detection of E-cadherin promoter methylation in tumors

    International Nuclear Information System (INIS)

    Lombaerts, Marcel; Middeldorp, Janneke W.; Weide, Esther van der; Philippo, Katja; Wezel, Tom van; Smit, Vincent T.H.B.M.; Cornelisse, Cees J.; Cleton-Jansen, Anne-Marie

    2004-01-01

    Promoter hypermethylation is known to result in transcriptional downregulation of many genes including the CDH1 gene. In this study we set out to determine CDH1 promoter methylation in breast tumors with decreased or absent E-cadherin protein expression and without CDH1 gene mutations by methylation-specific PCR (MSP). Interestingly, some tumor samples with normal E-cadherin expression yielded a methylation-specific PCR product. We hypothesized that other cells than tumor cells contribute to these products. Since in normal breast tissue no CDH1 promoter methylation is detected, infiltrating leukocytes, often present in tumors, might account for these methylation-specific fragments. Indeed, a methylation-specific fragment is found in all twelve leukocyte samples tested. Furthermore, activated T-cells also yielded a methylation-specific fragment. Sequencing of these fragments reveals two distinct methylation profiles. Leukocytes have only partial methylation of some CpGs, while the tumor-associated methylation profile shows complete methylation of most CpGs. Therefore, to assess whether CDH1 methylation is tumor associated, sequencing of MSP products is a prerequisite. Here we show that out of six lobular tumors lacking E-cadherin protein expression, three have tumor-associated CDH1 promoter methylation while in three other tumors no methylation is detected

  6. A tissue biopsy-based epigenetic multiplex PCR assay for prostate cancer detection

    Directory of Open Access Journals (Sweden)

    Van Neste Leander

    2012-06-01

    Full Text Available Abstract Background PSA-directed prostate cancer screening leads to a high rate of false positive identifications and an unnecessary biopsy burden. Epigenetic biomarkers have proven useful, exhibiting frequent and abundant inactivation of tumor suppressor genes through such mechanisms. An epigenetic, multiplex PCR test for prostate cancer diagnosis could provide physicians with better tools to help their patients. Biomarkers like GSTP1, APC and RASSF1 have demonstrated involvement with prostate cancer, with the latter two genes playing prominent roles in the field effect. The epigenetic states of these genes can be used to assess the likelihood of cancer presence or absence. Results An initial test cohort of 30 prostate cancer-positive samples and 12 cancer-negative samples was used as basis for the development and optimization of an epigenetic multiplex assay based on the GSTP1, APC and RASSF1 genes, using methylation specific PCR (MSP. The effect of prostate needle core biopsy sample volume and age of formalin-fixed paraffin-embedded (FFPE samples was evaluated on an independent follow-up cohort of 51 cancer-positive patients. Multiplexing affects copy number calculations in a consistent way per assay. Methylation ratios are therefore altered compared to the respective singleplex assays, but the correlation with patient outcome remains equivalent. In addition, tissue-biopsy samples as small as 20 μm can be used to detect methylation in a reliable manner. The age of FFPE-samples does have a negative impact on DNA quality and quantity. Conclusions The developed multiplex assay appears functionally similar to individual singleplex assays, with the benefit of lower tissue requirements, lower cost and decreased signal variation. This assay can be applied to small biopsy specimens, down to 20 microns, widening clinical applicability. Increasing the sample volume can compensate the loss of DNA quality and quantity in older samples.

  7. Expression profiling of O6 methylguanine-DNA-methyl transferase in prolactinomas: a correlative study of promoter methylation and pathological features in 136 cases

    International Nuclear Information System (INIS)

    Jiang, Xiao-Bing; Hu, Bin; He, Dong-Sheng; Mao, Zhi-Gang; Wang, Xin; Song, Bing-Bing; Zhu, Yong-Hong; Wang, Hai-Jun

    2015-01-01

    Low-level expression of O 6 methylguanine-DNA-methyl transferase (MGMT) prolactinomas has been noted previously in case reports, although what modulates MGMT expression remains unclear. This study therefore aimed to delineate the factors regulating MGMT expression in prolactinomas. We retrospectively reviewed 136 prolactinoma patients who were treated in our center between January 2000 and September 2013. Expression of MGMT, Ki-67, and p53 protein were examined by immunohistochemical staining, and MGMT promoter methylation evaluated with methylation-specific PCR. MGMT immunopositivity was <25 % in 106/136 tumor specimens (77.94 %). MGMT immunoexpression was positively correlated with age (r = 0.251, p = 0.003), but inversely correlated with p53 staining (r = −0.153, p = 0.021). Moreover, reduced MGMT expression was more frequent in atypical prolactinomas (p = 0.044). Methylated MGMT promoter was confirmed in 10/46 specimens (21.7 %), all of which had low level or absent MGMT staining. Both p53 protein (r = −0.33, p = 0.025) and promoter methylation (r = −0.331, p = 0.025) were negatively associated with MGMT expression. Multivariate logistic analysis indicated that age (odds ratio [OR] = 1.127. 95 % confidence interval [CI] 1.027–1.236, p = 0.012) and p53 (OR = 0.116. 95 % CI 0.018–0.761, p = 0.025) staining were independent determents of MGMT expression. The majority of prolactinomas, especially atypical prolactinomas, showed low-level or no MGMT immunoexpression, providing a rationale for the utility of temozolomide as an alternative to managing prolactinomas. In summary, epigenetic and transcriptional regulation are involved in silencing MGMT expression

  8. Molecular diagnostic PCR handbook

    International Nuclear Information System (INIS)

    Viljoen, G.J.; Crowther, J.R.; Nel, L.H.

    2005-01-01

    The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised PCR protocols to detect animal disease pathogens. Examples of Standard Operating Procedures as used in individual specialist laboratories and an outline of training materials necessary for PCR technology transfer are presented. The difficulties, advantages and disadvantages in PCR applications are explained and placed in context with other test systems. Emphasis is placed on the use of PCR for detection of pathogens, with a particular focus on diagnosticians and scientists from the developing world. It is hoped that this book will enable readers from various disciplines and levels of expertise to better judge the merits of PCR and to increase their skills and knowledge in order to assist in a more logical, efficient and assured use of this technology

  9. Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures.

    Science.gov (United States)

    Schwalbe, E C; Hicks, D; Rafiee, G; Bashton, M; Gohlke, H; Enshaei, A; Potluri, S; Matthiesen, J; Mather, M; Taleongpong, P; Chaston, R; Silmon, A; Curtis, A; Lindsey, J C; Crosier, S; Smith, A J; Goschzik, T; Doz, F; Rutkowski, S; Lannering, B; Pietsch, T; Bailey, S; Williamson, D; Clifford, S C

    2017-10-18

    Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation.

  10. Dissociation dynamics of methylal

    Energy Technology Data Exchange (ETDEWEB)

    Beaud, P; Frey, H -M; Gerber, T; Mischler, B; Radi, P P; Tzannis, A -P [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1999-08-01

    The dissociation of methylal is investigated using mass spectrometry, combined with a pyrolytic radical source and femtosecond pump probe experiments. Based on preliminary results two reaction paths of methylal dissociation are proposed and discussed. (author) 4 fig., 3 refs.

  11. Experimental vapor pressures (from 1 Pa to 100 kPa) of six saturated Fatty Acid Methyl Esters (FAMEs): Methyl hexanoate, methyl octanoate, methyl decanoate, methyl dodecanoate, methyl tetradecanoate and methyl hexadecanoate

    International Nuclear Information System (INIS)

    Sahraoui, Lakhdar; Khimeche, Kamel; Dahmani, Abdallah; Mokbel, Ilham; Jose, Jacques

    2016-01-01

    Highlight: • Vapor-liquid equilibria, Enthalpy of Vaporization, saturated Fatty Acid Methyl Ester. - Abstract: Vapor pressures of six saturated Fatty Acid Methyl Esters (FAMEs), methyl hexanoate (or methyl caproate), methyl octanoate (or methyl caprylate), Methyl decanoate (or methyl caprate), methyl dodecanoate (or methyl laurate), methyl tetradecanoate (or methyl myristate), and methyl hexadecanoate (or methyl palmitate) were measured from 1 Pa to 100 kPa and at temperature range between 262 and 453 K using a static apparatus. The experimental data (P-T) were compared with the available literature data.

  12. DNA methylation in states of cell physiology and pathology.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2007-10-01

    Full Text Available DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation. The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences five new genes which are potential biomarkers of lung cancer have been presented.

  13. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

    2015-11-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

  14. Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

    International Nuclear Information System (INIS)

    Koczor, Christopher A.; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

    2015-01-01

    This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

  15. MethPrimer: designing primers for methylation PCRs.

    Science.gov (United States)

    Li, Long-Cheng; Dahiya, Rajvir

    2002-11-01

    DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.

  16. DNA methylation patterns provide insight into epigenetic regulation in the Pacific oyster (Crassostrea gigas

    Directory of Open Access Journals (Sweden)

    Gavery Mackenzie R

    2010-08-01

    Full Text Available Abstract Background DNA methylation is an epigenetic mechanism with important regulatory functions in animals. While the mechanism itself is evolutionarily ancient, the distribution and function of DNA methylation is diverse both within and among phylogenetic groups. Although DNA methylation has been well studied in mammals, there are limited data on invertebrates, particularly molluscs. Here we characterize the distribution and investigate potential functions of DNA methylation in the Pacific oyster (Crassostrea gigas. Results Methylation sensitive PCR and bisulfite sequencing PCR approaches were used to identify CpG methylation in C. gigas genes and demonstrated that this species possesses intragenic methylation. In silico analysis of CpGo/e ratios in publicly available sequence data suggests that DNA methylation is a common feature of the C. gigas genome, and that specific functional categories of genes have significantly different levels of methylation. Conclusions The Pacific oyster genome displays intragenic DNA methylation and contains genes necessary for DNA methylation in animals. Results of this investigation suggest that DNA methylation has regulatory functions in Crassostrea gigas, particularly in gene families that have inducible expression, including those involved in stress and environmental responses.

  17. Predictive value of CpG island methylator phenotype for tumor recurrence in hepatitis B virus-associated hepatocellular carcinoma following liver transplantation

    Directory of Open Access Journals (Sweden)

    Zheng Shu-Sen

    2010-08-01

    Full Text Available Abstract Background CpG island methylator phenotype (CIMP, in which multiple genes concordantly methylated, has been demonstrated to be associated with progression, recurrence, as well as overall survival in some types of cancer. Methods We examined the promoter methylation status of seven genes including P16, CDH1, GSTP1, DAPK, XAF1, SOCS1 and SYK in 65 cases of HCC treated with LT by methylation-specific PCR. CIMP+ was defined as having three or more genes that are concordantly methylated. The relationship between CIMP status and clinicopathological parameters, as well as tumor recurrence was further analyzed. Results CIMP+ was more frequent in HCC with AFP > 400 ng/ml than those with AFP ≤ 400 ng/ml (P = 0.017. In addition, patients with CIMP+ were prone to have multiple tumor numbers than those with CIMP- (P = 0.007. Patients with CIMP+ tumors had significantly worse recurrence-free survival (RFS than patients with CIMP-tumors by Kaplan-Meier estimates (P = 0.004. Multivariate analysis also revealed that CIMP status might be a novel independent prognostic factor of RFS for HCC patients treated with LT (HR: 3.581; 95% CI: 1.473-8.710, P = 0.005. Conclusion Our results suggested that CIMP could serve as a new prognostic biomarker to predict the risk of tumor recurrence in HCC after transplantation.

  18. Predictive value of CpG island methylator phenotype for tumor recurrence in hepatitis B virus-associated hepatocellular carcinoma following liver transplantation

    International Nuclear Information System (INIS)

    Wu, Li-Ming; Zhang, Feng; Zhou, Lin; Yang, Zhe; Xie, Hai-Yang; Zheng, Shu-Sen

    2010-01-01

    CpG island methylator phenotype (CIMP), in which multiple genes concordantly methylated, has been demonstrated to be associated with progression, recurrence, as well as overall survival in some types of cancer. We examined the promoter methylation status of seven genes including P16, CDH1, GSTP1, DAPK, XAF1, SOCS1 and SYK in 65 cases of HCC treated with LT by methylation-specific PCR. CIMP+ was defined as having three or more genes that are concordantly methylated. The relationship between CIMP status and clinicopathological parameters, as well as tumor recurrence was further analyzed. CIMP+ was more frequent in HCC with AFP > 400 ng/ml than those with AFP ≤ 400 ng/ml (P = 0.017). In addition, patients with CIMP+ were prone to have multiple tumor numbers than those with CIMP- (P = 0.007). Patients with CIMP+ tumors had significantly worse recurrence-free survival (RFS) than patients with CIMP-tumors by Kaplan-Meier estimates (P = 0.004). Multivariate analysis also revealed that CIMP status might be a novel independent prognostic factor of RFS for HCC patients treated with LT (HR: 3.581; 95% CI: 1.473-8.710, P = 0.005). Our results suggested that CIMP could serve as a new prognostic biomarker to predict the risk of tumor recurrence in HCC after transplantation

  19. Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

    Science.gov (United States)

    Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

    2017-03-09

    It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

  20. Impact of IGF-1, IGF-1R, and IGFBP-3 promoter methylation on the risk and prognosis of esophageal carcinoma.

    Science.gov (United States)

    Ye, Peng; Qu, Chang-Fa; Hu, Xue-Lin

    2016-05-01

    The aim of this study is to investigate IGF-1, IGF-1R, and IGFBP-3 methylations in esophageal carcinoma (EC) patients and their relationship with the development and prognosis of EC. This study population consisted of 264 patients (case group) whom EC radical resection was performed and 283 healthy individuals (control group). Methylation-specific PCR (MSP) detected the methylation status of IGF-1, IGF-1R, and IGFBP-3 in the peripheral blood in both groups. The expressions of IGF-1, IGF-1R, and IGFBP-3 in EC and adjacent normal tissues were detected by immunohistochemistry (IHC). The methylation rates of IGF-1, IGF-1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 in the case group were higher than those in the control group (all P IGF-1, IGF-1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 IGF-1 among patients of different clinicopathological features (all P IGF-1 and IGF-1R in EC were significantly higher than those in adjacent normal tissues (both P IGF-1 and IGF1R gene promoter methylation was positively correlated with the positive expressions of IGF-1 (r = 0.139, P = 0.024) and IGF-1R (r = 0.135, P = 0.028), while the IGFBP3 methylation was negatively correlated with the positive expression of IGFBP3 (r = -0.133, P = 0.031). The positive expressions of IGF-1, IGF-1R, and IGFBP-3 were related to different clinicopathological features (all P IGF-1, IGF-1R, and IGF-1 + IGF1R + IGFBP3 ; expressions of IGF-1 and IGF-1R protein; infiltration depth; and lymph node metastasis (LNM) were independent factors of EC prognosis. Our study demonstrated that methylation of IGF-1, IGF1R, IGFBP3, and IGF-1 + IGF1R + IGFBP3 was closely linked with the occurrence of EC and patients' clinicopathological features. Besides, the methylation status of the target genes and the expressions of IGF-1 and IGF-1R protein were independent factors of EC prognosis, which could provide a direction for the prognosis and treatment of EC.

  1. Independent preferences

    DEFF Research Database (Denmark)

    Vind, Karl

    1991-01-01

    A simple mathematical result characterizing a subset of a product set is proved and used to obtain additive representations of preferences. The additivity consequences of independence assumptions are obtained for preferences which are not total or transitive. This means that most of the economic ...... theory based on additive preferences - expected utility, discounted utility - has been generalized to preferences which are not total or transitive. Other economic applications of the theorem are given...

  2. PCR in forensic genetics

    DEFF Research Database (Denmark)

    Morling, Niels

    2009-01-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...... and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.......Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...

  3. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

    Directory of Open Access Journals (Sweden)

    Danielle C. Leal

    2011-07-01

    Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

  4. High CpG island methylation of p16 gene and loss of p16 protein

    Indian Academy of Sciences (India)

    Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region andWestern blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression.

  5. Independent Directors

    DEFF Research Database (Denmark)

    Ringe, Wolf-Georg

    2013-01-01

    This paper re-evaluates the corporate governance concept of ‘board independence’ against the disappointing experiences during the 2007-08 financial crisis. Independent or outside directors had long been seen as an essential tool to improve the monitoring role of the board. Yet the crisis revealed...... that they did not prevent firms' excessive risk taking; further, these directors sometimes showed serious deficits in understanding the business they were supposed to control, and remained passive in addressing structural problems. A closer look reveals that under the surface of seemingly unanimous consensus...

  6. Disclosing bias in bisulfite assay: MethPrimers underestimate high DNA methylation.

    Directory of Open Access Journals (Sweden)

    Andrea Fuso

    Full Text Available Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs, having degenerated bases (G/A to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

  7. Whole-genome methylation caller designed for methyl- DNA ...

    African Journals Online (AJOL)

    etchie

    2013-02-20

    Feb 20, 2013 ... Our method uses a single-CpG-resolution, whole-genome methylation ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, ...... methylation is prevalent in embryonic stem cells andmaybe mediated.

  8. Global DNA methylation of ischemic stroke subtypes.

    Directory of Open Access Journals (Sweden)

    Carolina Soriano-Tárraga

    Full Text Available Ischemic stroke (IS, a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA, small-artery disease (SAD, and cardio-aortic embolism (CE. A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204 were included, distributed across 3 IS subtypes: LAA (78/281, 59/204, SAD (97/281, 53/204, and CE (106/281, 89/204. In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.

  9. Correlation between the methylation of APC gene promoter and colon cancer.

    Science.gov (United States)

    Li, Bing-Qiang; Liu, Peng-Peng; Zhang, Cai-Hua

    2017-08-01

    The present study was planned to explore the correlation between the methylation of APC (adenomatous polyposis coli) and colon carcinogenesis. Colon cancer tissues and tumor-adjacent normal tissues of 60 colon cancer patients (who received surgical operation in our hospital from January 2012 to December 2014) were collected. SW1116 cells in human colon cancer tissues were selected for culturing. 5-aza-2c-deoxycytidine (5-aza-dC) was utilized as an inhibitor of the methylation for APC gene. Methylation specific PCR (MSP) was utilized for detection of APC methylation in SW1116 cells. The MTT and Transwell assays were performed to detect the effect of the methylation of APC gene on the proliferation and invasive abilities of SW1116 cells. The correlation between the methylation of APC gene and pathological parameters of colon cancer patients was analyzed. MSP results revealed that 41 cases (68.33%) showed methylation of APC gene in colon cancer tissues. No methylation of APC gene was found in tumor-adjacent normal tissues. 5-aza-dC was able to inhibit the methylation of APC gene in SW1116 cells. APC gene methylation was correlated with tumor size, differentiation degree, lymph node metastasis and Dukes staging. In conclusion, the levels of the methylation of APC in colon cancer tissues and SW1116 cells are relatively high. The methylation of APC promoted the proliferation and invasion abilities of SW1116 cells. Furthermore, methylation is correlated with a variety of clinicopathological features of colon cancer patients.

  10. Methylation pathways in schizophrenia

    International Nuclear Information System (INIS)

    Sargent, T.W. III.

    1982-01-01

    Research on the biochemical causes of human psychosis concentrates on investigating whether schizophremia is linked to abnormalities in the metabolism of methyl carbon groups in the body. The metabolism of C-14 labeled methyl groups in methionine is studied in animals, normal subjects and patient volunteers

  11. Heterogeneity of DNA methylation in multifocal prostate cancer.

    Science.gov (United States)

    Serenaite, Inga; Daniunaite, Kristina; Jankevicius, Feliksas; Laurinavicius, Arvydas; Petroska, Donatas; Lazutka, Juozas R; Jarmalaite, Sonata

    2015-01-01

    Most prostate cancer (PCa) cases are multifocal, and separate foci display histological and molecular heterogeneity. DNA hypermethylation is a frequent alteration in PCa, but interfocal heterogeneity of these changes has not been extensively investigated. Ten pairs of foci from multifocal PCa and 15 benign prostatic hyperplasia (BPH) samples were obtained from prostatectomy specimens, resulting altogether in 35 samples. Methylation-specific PCR (MSP) was used to evaluate methylation status of nine tumor suppressor genes (TSGs), and a set of selected TSGs was quantitatively analyzed for methylation intensity by pyrosequencing. Promoter sequences of the RASSF1 and ESR1 genes were methylated in all paired PCa foci, and frequent (≥75 %) DNA methylation was detected in RARB, GSTP1, and ABCB1 genes. MSP revealed different methylation status of at least one gene in separate foci in 8 out of 10 multifocal tumors. The mean methylation level of ESR1, GSTP1, RASSF1, and RARB differed between the paired foci of all PCa cases. The intensity of DNA methylation in these TSGs was significantly higher in PCa cases than in BPH (p epigenetic profile of recurrent tumors can be inferred from our data.

  12. Female human pluripotent stem cells rapidly lose X chromosome inactivation marks and progress to a skewed methylation pattern during culture.

    Science.gov (United States)

    Geens, M; Seriola, A; Barbé, L; Santalo, J; Veiga, A; Dée, K; Van Haute, L; Sermon, K; Spits, C

    2016-04-01

    Does a preferential X chromosome inactivation (XCI) pattern exist in female human pluripotent stem cells (hPSCs) and does the pattern change during long-term culture or upon differentiation? We identified two independent phenomena that lead to aberrant XCI patterns in female hPSC: a rapid loss of histone H3 lysine 27 trimethylation (H3K27me3) and long non-coding X-inactive specific transcript (XIST) expression during culture, often accompanied by erosion of XCI-specific methylation, and a frequent loss of random XCI in the cultures. Variable XCI patterns have been reported in female hPSC, not only between different hPSC lines, but also between sub-passages of the same cell line, however the reasons for this variability remain unknown. Moreover, while non-random XCI-linked DNA methylation patterns have been previously reported, their origin and extent have not been investigated. We investigated the XCI patterns in 23 human pluripotent stem cell (hPSC) lines, during long-term culture and after differentiation, by gene expression analysis, histone modification assessment and study of DNA methylation. The presence and location of H3K27me3 was studied by immunofluorescence, XIST expression by real-time PCR, and mono- or bi-allelic expression of X-linked genes was studied by sequencing of cDNA. XCI-specific DNA methylation was analysed using methylation-sensitive restriction and PCR, and more in depth by massive parallel bisulphite sequencing. All hPSC lines showed XCI, but we found a rapid loss of XCI marks during the early stages of in vitro culture. While this loss of XCI marks was accompanied in several cases by an extensive erosion of XCI-specific methylation, it did not result in X chromosome reactivation. Moreover, lines without strong erosion of methylation frequently displayed non-random DNA methylation, which occurred independently from the loss of XCI marks. This bias in X chromosome DNA methylation did not appear as a passenger event driven by clonal culture

  13. PCR, exit stage left ...

    CERN Multimedia

    2004-01-01

    The Prevessin Control Room during LEP's start up in 1989. The Prévessin Control Room (PCR) was recently engulfed in a wave of nostalgia. The PCR, scene of some of the greatest moments in CERN's history, is being dismantled to prepare for a complete overhaul. In February 2006, a new combined control centre for all the accelerators will open its doors on the same site, together with a new building currently under construction (see Bulletin issue 27/2004 of 28 June 2004). This marks the end of an important chapter in CERN's history. The Prévessin Control Room saw its first momentous event 28 years ago when the 400 GeV beam for the SPS was commissioned in the presence of Project Leader John Adams. It was also here that the first proton-antiproton collisions were observed, in 1981. Eight years later, in 1989, operators and directors alike jumped for joy at the announcement of the first electron-positron collisions at the start up of LEP, the biggest accelerator in the world. Today the 80 terminals and PCs have b...

  14. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  15. Kismeth: Analyzer of plant methylation states through bisulfite sequencing

    Directory of Open Access Journals (Sweden)

    Martienssen Robert A

    2008-09-01

    Full Text Available Abstract Background There is great interest in probing the temporal and spatial patterns of cytosine methylation states in genomes of a variety of organisms. It is hoped that this will shed light on the biological roles of DNA methylation in the epigenetic control of gene expression. Bisulfite sequencing refers to the treatment of isolated DNA with sodium bisulfite to convert unmethylated cytosine to uracil, with PCR converting the uracil to thymidine followed by sequencing of the resultant DNA to detect DNA methylation. For the study of DNA methylation, plants provide an excellent model system, since they can tolerate major changes in their DNA methylation patterns and have long been studied for the effects of DNA methylation on transposons and epimutations. However, in contrast to the situation in animals, there aren't many tools that analyze bisulfite data in plants, which can exhibit methylation of cytosines in a variety of sequence contexts (CG, CHG, and CHH. Results Kismeth http://katahdin.mssm.edu/kismeth is a web-based tool for bisulfite sequencing analysis. Kismeth was designed to be used with plants, since it considers potential cytosine methylation in any sequence context (CG, CHG, and CHH. It provides a tool for the design of bisulfite primers as well as several tools for the analysis of the bisulfite sequencing results. Kismeth is not limited to data from plants, as it can be used with data from any species. Conclusion Kismeth simplifies bisulfite sequencing analysis. It is the only publicly available tool for the design of bisulfite primers for plants, and one of the few tools for the analysis of methylation patterns in plants. It facilitates analysis at both global and local scales, demonstrated in the examples cited in the text, allowing dissection of the genetic pathways involved in DNA methylation. Kismeth can also be used to study methylation states in different tissues and disease cells compared to a reference sequence.

  16. Methyl-Analyzer--whole genome DNA methylation profiling.

    Science.gov (United States)

    Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

    2011-08-15

    Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

  17. Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample.

    Directory of Open Access Journals (Sweden)

    Katsuhiko Nosho

    Full Text Available The CpG island methylator phenotype (CIMP is a distinct phenotype associated with microsatellite instability (MSI and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. METHOLODOLOGY/PRINCIPAL FINDINGS: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16, CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight. In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1, CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers, multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1 activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also

  18. The Clinical Implications of Methylated p15 and p73 Genes in Adult Acute Lymphoblastic Leukemia

    International Nuclear Information System (INIS)

    ABD EL-HAMID, Th.M.; SHERISHER, M.A.; MOSSALLAM, Gh.I.

    2010-01-01

    Aberrant methylation of promoter associated CpG islands is an epigenetic modification of DNA which is associated with gene silencing. It plays an important role in the leukemia pathogenesis. This phenomenon is frequently observed in acute lymphoblastic leukemia (ALL) and results in the functional inactivation of its associated genes. The aim of this study is to investigate the frequency and the prognostic impact of p15 and p73 genes methylation in adult acute lymphoblastic leukemia patients. Patients and Methods: Methylation-specific polymerase chain reaction (PCR) was used to analyze methylation of the p15 and p73 genes in 51 newly diagnosed adult ALL patients. Results: The methylation frequencies of p15 and p73 genes at diagnosis were 41.2% and 27.5% respectively, while concomitant methylation was detected in 14% of the patients. Concomitant methylation of p15 and p73 genes was associated with significant lower rate of CR compared to patients without methylation (57% versus 90%), p=0.008. Overall survival (OS) was not affected by p15 methylation, but was poorer with p73 methylation and the difference was near significant (p=0.059). For patients without meyhylation, the survival benefit was significant when compared to patients with p15, p73 or both genes methylation (p=0.047). The leukemia free survival was not affected by the methylation status of single gene p15 or p73, but tended to be worse in patients with methylated p15, p73 or both genes when compared to patients without methylation (p= 0.08). Conclusion: Aberrant p73 promoter methylation is a potential prognostic factor in adult ALL patients. P15 methylation is frequent in Egyptian adult ALL patients, its concomitant methylation with p73 is of poor prognostic significance. Identification of these molecular targets improve risk assessment and selection of appropriate therapy.

  19. Identification of a Novel Methylated Gene in Nasopharyngeal Carcinoma: TTC40

    Directory of Open Access Journals (Sweden)

    Wajdi Ayadi

    2014-01-01

    Full Text Available To further explore the epigenetic changes in nasopharyngeal carcinoma (NPC, methylation-sensitive arbitrarily primed PCR was performed on NPC biopsies and nontumor nasopharyngeal samples. We have shown mainly two DNA fragments that appeared to be differentially methylated in NPCs versus nontumors. The first, defined as hypermethylated, corresponds to a CpG island at the 5′-end of the tetratricopeptide repeat domain 40 (TTC40 gene, whereas the second, defined as hypo-methylated, is located on repetitive sequences at chromosomes 16p11.1 and 13.1. Thereafter, the epigenetic alteration on the 5′-TTC40 gene was confirmed by methylation-specific PCR, showing a significant aberrant methylation in NPCs, compared to nontumors. In addition, the bisulfite sequencing analysis has shown a very high density of methylated cytosines in C15, C17, and X666 NPC xenografts. To assess whether TTC40 gene is silenced by aberrant methylation, we examined the gene expression by reverse transcription-PCR. Our analysis showed that the mRNA expression was significantly lower in tumors than in nontumors, which is associated with 5′-TTC40 gene hypermethylation. In conclusion, we found that the 5′-TTC40 gene is frequently methylated and is associated with the loss of mRNA expression in NPCs. Hypermethylation of 5′-TTC40 gene might play a role in NPC development; nevertheless, other studies are needed.

  20. Quantitative correlation between promoter methylation and messenger RNA levels of the reduced folate carrier

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    Kheradpour Albert

    2008-05-01

    Full Text Available Abstract Background Methotrexate (MTX uptake is mediated by the reduced folate carrier (RFC. Defective drug uptake in association with decreased RFC expression is a common mechanism of MTX resistance in many tumor types. Heavy promoter methylation was previously identified as a basis for the complete silencing of RFC in MDA-MB-231 breast cancer cells, its role and prevalence in RFC transcription regulation are, however, not widely studied. Methods In the current study, RFC promoter methylation was assessed using methylation specific PCR in a panel of malignant cell lines (n = 8, including MDA-MB-231, and M805, a MTX resistant cell line directly established from the specimen of a patient with malignant fibrohistocytoma, whom received multiple doses of MTX. A quantitative approach of real-time PCR for measuring the extent of RFC promoter methylation was developed, and was validated by direct bisulfite genomic sequencing. RFC mRNA levels were determined by quantitative real-time RT-PCR and were related to the extent of promoter methylation in these cell lines. Results A partial promoter methylation and RFC mRNA down-regulation were observed in M805. Using the quantitative approach, a reverse correlation (correlation coefficient = -0.59, p Conclusion This study further suggests that promoter methylation is a potential basis for MTX resistance. The quantitative correlation identified in this study implies that promoter methylation is possibly a mechanism involved in the fine regulation of RFC transcription.

  1. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  2. Epigenetic regulation during fetal femur development: DNA methylation matters.

    Directory of Open Access Journals (Sweden)

    María C de Andrés

    Full Text Available Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs, adult chondrocytes and STRO-1(+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2 and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5--methyltransferase 1 (DNMT1 in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development

  3. Methylcap-seq reveals novel DNA methylation markers for the diagnosis and recurrence prediction of bladder cancer in a Chinese population.

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    Yangxing Zhao

    Full Text Available PURPOSE: There is a need to supplement or supplant the conventional diagnostic tools, namely, cystoscopy and B-type ultrasound, for bladder cancer (BC. We aimed to identify novel DNA methylation markers for BC through genome-wide profiling of BC cell lines and subsequent methylation-specific PCR (MSP screening of clinical urine samples. EXPERIMENTAL DESIGN: The methyl-DNA binding domain (MBD capture technique, methylCap/seq, was performed to screen for specific hypermethylated CpG islands in two BC cell lines (5637 and T24. The top one hundred hypermethylated targets were sequentially screened by MSP in urine samples to gradually narrow the target number and optimize the composition of the diagnostic panel. The diagnostic performance of the obtained panel was evaluated in different clinical scenarios. RESULTS: A total of 1,627 hypermethylated promoter targets in the BC cell lines was identified by Illumina sequencing. The top 104 hypermethylated targets were reduced to eight genes (VAX1, KCNV1, ECEL1, TMEM26, TAL1, PROX1, SLC6A20, and LMX1A after the urine DNA screening in a small sample size of 8 normal control and 18 BC subjects. Validation in an independent sample of 212 BC patients enabled the optimization of five methylation targets, including VAX1, KCNV1, TAL1, PPOX1, and CFTR, which was obtained in our previous study, for BC diagnosis with a sensitivity and specificity of 88.68% and 87.25%, respectively. In addition, the methylation of VAX1 and LMX1A was found to be associated with BC recurrence. CONCLUSIONS: We identified a promising diagnostic marker panel for early non-invasive detection and subsequent BC surveillance.

  4. Methylation-Sensitive High Resolution Melting (MS-HRM).

    Science.gov (United States)

    Hussmann, Dianna; Hansen, Lise Lotte

    2018-01-01

    Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

  5. DNA damage, homology-directed repair, and DNA methylation.

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    Concetta Cuozzo

    2007-07-01

    Full Text Available To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP genes (DR-GFP. A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

  6. One-stop polymerase chain reaction (PCR): An improved PCR ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... membrane filtration was carried out with a commercial PCR product purification kit (Generay, Shanghai), according to the manufacture's instruction. In brief, 50 µl PCR product was mixed thoroughly with binding buffer, and the resultant mixture was loaded directly onto a silica membrane Gelclean column.

  7. A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

    Science.gov (United States)

    Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

    2011-10-01

    DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

  8. DNA methylation modulates H19 and IGF2 expression in porcine female eye

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    Dongxu Wang

    2017-03-01

    Full Text Available Abstract The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR and bisulfite sequencing PCR (BSP. We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively. We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs. Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye.

  9. DNA methylation modulates H19 and IGF2 expression in porcine female eye

    Science.gov (United States)

    Wang, Dongxu; Wang, Guodong; Yang, Hao; Liu, Haibo; Li, Cuie; Li, Xiaolan; Lin, Chao; Song, Yuning; Li, Zhanjun; Liu, Dianfeng

    2017-01-01

    Abstract The sexually dimorphic expression of H19/IGF2 is evolutionarily conserved. To investigate whether the expression of H19/IGF2 in the female porcine eye is sex-dependent, gene expression and methylation status were evaluated using quantitative real-time PCR (qPCR) and bisulfite sequencing PCR (BSP). We hypothesized that H19/IGF2 might exhibit a different DNA methylation status in the female eye. In order to evaluate our hypothesis, parthenogenetic (PA) cells were used for analysis by qPCR and BSP. Our results showed that H19 and IGF2 were over-expressed in the female eye compared with the male eye (3-fold and 2-fold, respectively). We observed a normal monoallelic methylation pattern for H19 differentially methylated regions (DMRs). Compared with H19 DMRs, IGF2 DMRs showed a different methylation pattern in the eye. Taken together, these results suggest that elevated expression of H19/IGF2 is caused by a specific chromatin structure that is regulated by the DNA methylation status of IGF2 DMRs in the female eye. PMID:28266684

  10. Synthesis of N-methyl and N-11C-methyl spiperone by phase transfer catalysis in anhydrous solvent

    International Nuclear Information System (INIS)

    Omokawa, Hiroyoshi; Tanaka, Akira; Iio, Mayumi; Nishihara, Yoshiaki; Inoue, Osamu; Yamazaki, Toshio.

    1985-01-01

    Spiperone, a butyrophenone neuroleptic drug, has been used in binding studies of dopamine receptors. Langstrom et al. developed N- 11 C-methyl spiperone, and, in cooperate with Wagner et al., made it possible to visualize the distribution of dopamine receptors in the human brain in vivo. In this paper, we independently developed another synthetic method of N- 11 C-methyl spiperone using the phase transfer catalyst in an anhydrous solvent. Separation of the product is feasible only by passing the reactant solution through a Millipore filter and injecting it onto high pressure liquid chromatography (HPLC). The time required for the synthesis and purification of N- 11 C-methyl spiperone from 11 C-methyl iodide and spiperone was 20 min. Radiochemical yield exceeded 35 % against 11 C-methyl iodide without correcting decay of the radioactivity. (author)

  11. Identification of Differentially Methylated Sites with Weak Methylation Effects

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    Hong Tran

    2018-02-01

    Full Text Available Deoxyribonucleic acid (DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same

  12. MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults.

    Science.gov (United States)

    Alvarez, Marisa C; Santos, Juliana C; Maniezzo, Nathália; Ladeira, Marcelo S; da Silva, Artur L C; Scaletsky, Isabel C A; Pedrazzoli, José; Ribeiro, Marcelo L

    2013-05-28

    To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ(2) test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t-test. Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.

  13. DNA methylation in obesity

    Directory of Open Access Journals (Sweden)

    Małgorzata Pokrywka

    2014-11-01

    Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

  14. IGFBP3 Promoter Methylation in Colorectal Cancer: Relationship with Microsatellite Instability, CpG Island Methylator Phenotype, p53

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    Takako Kawasaki

    2007-12-01

    Full Text Available Insulin-like growth factor binding protein 3 (IGFBP3, which is induced by wild-type p53, regulates IGF and interacts with the TGF-β pathway. IGFBP3 promoter methylation may occur in colorectal cancer with or without the CpG island methylator phenotype (CIMP, which is associated with microsatellite instability (MSI and TGFBR2 mutation. We examined the relationship between IGFBP3 methylation, p53 expression, CIMP and MSI in 902 population-based colorectal cancers. Utilizing real-time PCR (MethyLight, we quantified promoter methylation in IGFBP3 and eight other CIMP-high-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1. IGFBP3 methylation was far more frequent in non-MSI-high CIMP-high tumors (85% = 35/41 than in MSI-high CIMPhigh (49% = 44/90, P < .0001, MSI-high non-CIMP-high (17% = 6/36, P < .0001, non-MSI-high non-CIMP-high tumors (22% = 152/680, P < .0001. Among CIMPhigh tumors, the inverse relationship between MSI and IGFBP3 methylation persisted in p53-negative tumors (P < .0001, but not in p53-positive tumors. IGFBP3 methylation was associated inversely with TGFBR2 mutation in MSI-high non-CIMP-high tumors (P = .02. In conclusion, IGFBP3 methylation is inversely associated with MSI in CIMP-high colorectal cancers, this relationship is limited to p53-negative tumors. Our data suggest complex relationship between global genomic/epigenomic phenomena (such as MSI/ CIMP, single molecular events (e.g., IGFBP3 methylation, TP53 mutation, TGFBR2 mutation, the related pathways.

  15. Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

    DEFF Research Database (Denmark)

    Malorny, B.; Hoorfar, Jeffrey; Hugas, M.

    2003-01-01

    A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained...... presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole......-based diagnostic methods and is currently proposed as international standard document....

  16. Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines

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    Yongwen LI

    2015-05-01

    Full Text Available Background and objective It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. Methods Real-time quantitative PCR and methylation-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D. DNA sequencing was used to confirm the methylation results. Results The level of miR-182 expression significantly differs among these lung cancer cell lines. The highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines significantly increases when treated with 10 μM 5’-Aza-dC. Conclusion DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occurrence and development.

  17. Methylated β-Cyclodextrins

    DEFF Research Database (Denmark)

    Schönbeck, Jens Christian Sidney; Westh, Peter; Madsen, Jens Christian

    2011-01-01

    The complexation of 6 bile salts with various methylated β-cyclodextrins was studied to elucidate how the degree and pattern of substitution affects the binding. The structures of the CDs were determined by mass spectrometry and NMR techniques, and the structures of the inclusion complexes were...

  18. A genome-wide methylation study on obesity: differential variability and differential methylation.

    Science.gov (United States)

    Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Hidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

    2013-05-01

    Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common complex disease. We analyzed genome-wide methylation profiles of over 470,000 CpGs in peripheral blood samples from 48 obese and 48 lean African-American youth aged 14-20 y old. A substantial number of differentially variable CpG sites (DVCs), using statistics based on variances, as well as a substantial number of differentially methylated CpG sites (DMCs), using statistics based on means, were identified. Similar to the findings in cancers, DVCs generally exhibited an outlier structure and were more variable in cases than in controls. By randomly splitting the current sample into a discovery and validation set, we observed that both the DVCs and DMCs identified from the first set could independently predict obesity status in the second set. Furthermore, both the genes harboring DMCs and the genes harboring DVCs showed significant enrichment of genes identified by genome-wide association studies on obesity and related diseases, such as hypertension, dyslipidemia, type 2 diabetes and certain types of cancers, supporting their roles in the etiology and pathogenesis of obesity. We generalized the recent finding on methylation variability in cancer research to obesity and demonstrated that differential variability is also an important feature of obesity-related methylation changes. Future studies on the epigenetics of obesity will benefit from both statistics based on means and statistics based on variances.

  19. Methylation profiling identified novel differentially methylated markers including OPCML and FLRT2 in prostate cancer.

    Science.gov (United States)

    Wu, Yu; Davison, Jerry; Qu, Xiaoyu; Morrissey, Colm; Storer, Barry; Brown, Lisha; Vessella, Robert; Nelson, Peter; Fang, Min

    2016-04-02

    To develop new methods to distinguish indolent from aggressive prostate cancers (PCa), we utilized comprehensive high-throughput array-based relative methylation (CHARM) assay to identify differentially methylated regions (DMRs) throughout the genome, including both CpG island (CGI) and non-CGI regions in PCa patients based on Gleason grade. Initially, 26 samples, including 8 each of low [Gleason score (GS) 6] and high (GS ≥7) grade PCa samples and 10 matched normal prostate tissues, were analyzed as a discovery cohort. We identified 3,567 DMRs between normal and cancer tissues, and 913 DMRs distinguishing low from high-grade cancers. Most of these DMRs were located at CGI shores. The top 5 candidate DMRs from the low vs. high Gleason comparison, including OPCML, ELAVL2, EXT1, IRX5, and FLRT2, were validated by pyrosequencing using the discovery cohort. OPCML and FLRT2 were further validated in an independent cohort consisting of 20 low-Gleason and 33 high-Gleason tissues. We then compared patients with biochemical recurrence (n=70) vs. those without (n=86) in a third cohort, and they showed no difference in methylation at these DMR loci. When GS 3+4 cases and GS 4+3 cases were compared, OPCML-DMR methylation showed a trend of lower methylation in the recurrence group (n=30) than in the no-recurrence (n=52) group. We conclude that whole-genome methylation profiling with CHARM revealed distinct patterns of differential DNA methylation between normal prostate and PCa tissues, as well as between different risk groups of PCa as defined by Gleason scores. A panel of selected DMRs may serve as novel surrogate biomarkers for Gleason score in PCa.

  20. Aberrantly methylated genes in human papillary thyroid cancer and their association with BRAF/RAS mutation.

    Directory of Open Access Journals (Sweden)

    Yasuko eKikuchi

    2013-12-01

    Full Text Available Cancer arises through accumulation of epigenetic and genetic alteration. Aberrant promoter methylation is a common epigenetic mechanism of gene silencing in cancer cells. We here performed genome-wide analysis of DNA methylation of promoter regions by Infinium HumanMethylation27 BeadChip, using 14 clinical papillary thyroid cancer samples and 10 normal thyroid samples. Among the 14 papillary cancer cases, 11 showed frequent aberrant methylation, but the other three cases showed no aberrant methylation at all. Distribution of the hypermethylation among cancer samples was non-random, which implied existence of a subset of preferentially methylated papillary thyroid cancer. Among 25 frequently methylated genes, methylation status of six genes (HIST1H3J, POU4F2, SHOX2, PHKG2, TLX3, HOXA7 was validated quantitatively by pyrosequencing. Epigenetic silencing of these genes in methylated papillary thyroid cancer cell lines was confirmed by gene re-expression following treatment with 5-aza-2'-deoxycytidine and trichostatin A, and detected by real-time RT-PCR. Methylation of these six genes was validated by analysis of additional 20 papillary thyroid cancer and 10 normal samples. Among the 34 cancer samples in total, 26 cancer samples with preferential methylation were significantly associated with mutation of BRAF/RAS oncogene (P=0.04, Fisher’s exact test. Thus we identified new genes with frequent epigenetic hypermethylation in papillary thyroid cancer, two subsets of either preferentially methylated or hardly methylated papillary thyroid cancer, with a concomitant occurrence of oncogene mutation and gene methylation. These hypermethylated genes may constitute potential biomarkers for papillary thyroid cancer.

  1. Quantitative Methylation Profiles for Multiple Tumor Suppressor Gene Promoters in Salivary Gland Tumors

    Science.gov (United States)

    Durr, Megan L.; Mydlarz, Wojciech K.; Shao, Chunbo; Zahurak, Marianna L.; Chuang, Alice Y.; Hoque, Mohammad O.; Westra, William H.; Liegeois, Nanette J.; Califano, Joseph A.; Sidransky, David; Ha, Patrick K.

    2010-01-01

    Background Methylation profiling of tumor suppressor gene (TSGs) promoters is quickly becoming a powerful diagnostic tool for the early detection, prognosis, and even prediction of clinical response to treatment. Few studies address this in salivary gland tumors (SGTs); hence the promoter methylation profile of various TSGs was quantitatively assessed in primary SGT tissue to determine if tumor-specific alterations could be detected. Methodology DNA isolated from 78 tumor and 17 normal parotid gland specimens was assayed for promoter methylation status of 19 TSGs by fluorescence-based, quantitative methylation-specific PCR (qMSP). The data were utilized in a binary fashion as well as quantitatively (using a methylation quotient) allowing for better profiling and interpretation of results. Principal Findings The average number of methylation events across the studied genes was highest in salivary duct carcinoma (SDC), with a methylation value of 9.6, compared to the normal 4.5 (ptrend for increasing methylation in APC, Mint 1, PGP9.5, RAR-β, and Timp3. Conclusions/Significance Screening promoter methylation profiles in SGTs showed considerable heterogeneity. The methylation status of certain markers was surprisingly high in even normal salivary tissue, confirming the need for such controls. Several TSGs were found to be associated with malignant SGTs, especially SDC. Further study is needed to evaluate the potential use of these associations in the detection, prognosis, and therapeutic outcome of these rare tumors. PMID:20520817

  2. Protein methylation in pea chloroplasts

    International Nuclear Information System (INIS)

    Niemi, K.J.; Adler, J.; Selman, B.R.

    1990-01-01

    The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with [ 3 H-methyl]-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. One methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile [ 3 H]methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the [ 3 H]methyl group

  3. Methylation of food commodities during fumigation with methyl bromide

    International Nuclear Information System (INIS)

    Starratt, A.N.; Bond, E.J.

    1990-01-01

    Sites of methylation in several commodities (wheat, oatmeal, peanuts, almonds, apples, oranges, maize, alfalfa and potatoes) during fumigation with 14 C-methyl bromide were studied. Differences were observed in levels of the major volatiles: methanol, dimethyl sulphide and methyl mercaptan, products of O- and S-methylation, resulting from treatment of the fumigated materials with 1N sodium hydroxide. In studies of maize and wheat, histidine was the amino acid which underwent the highest level of N-methylation. (author). 24 refs, 3 tabs

  4. DNA methylation analysis from saliva samples for epidemiological studies.

    Science.gov (United States)

    Nishitani, Shota; Parets, Sasha E; Haas, Brian W; Smith, Alicia K

    2018-06-18

    Saliva is a non-invasive, easily accessible tissue, which is regularly collected in large epidemiological studies to examine genetic questions. Recently, it is becoming more common to use saliva to assess DNA methylation. However, DNA extracted from saliva is a mixture of both bacterial and human DNA derived from epithelial and immune cells in the mouth. Thus, there are unique challenges to using salivary DNA in methylation studies that can influence data quality. This study assesses: (1) quantification of human DNA after extraction; (2) delineation of human and bacterial DNA; (3) bisulfite conversion (BSC); (4) quantification of BSC DNA; (5) PCR amplification of BSC DNA from saliva and; (6) quantitation of DNA methylation with a targeted assay. The framework proposed will allow saliva samples to be more widely used in targeted epigenetic studies.

  5. Altered promoter methylation of PDK4, IL1 B, IL6, and TNF after Roux-en Y gastric bypass

    DEFF Research Database (Denmark)

    Kirchner, Henriette; Nylen, Carolina; Laber, Samantha

    2014-01-01

    methylation of selected promoter regions was measured in whole blood before and after VLCD. A subgroup of seven patients was studied 1–2 days and 12± 3 months after RYGB. Promoter methylation was measured using methylated DNA capture and quantitative real-time polymerase chain reaction (PCR). Results VLCD....... The objective of this study was to test whether promoter methylation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1 A), pyruvate dehydrogenase kinase isozyme-4 (PDK4), transcription factor A (TFAM), interleukin-1 beta (IL1 B), interleukin-6 (IL6) and tumor necrosis factor...... decreased promoter methylation of PPARGC1 A. Methylation of PPARGC1 A, TFAM, IL1 B, IL6, and TNF promoters was changed two days after RYGB. Similar changes were also seen on day one after cholecystectomy. Moreover, methylation increased in PDK4, IL1 B, IL6, and TNF promoters 12 months after RYGB. Conclusion...

  6. The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

    International Nuclear Information System (INIS)

    Sernbo, Sandra; Gustavsson, Elin; Brennan, Donal J; Gallagher, William M; Rexhepaj, Elton; Rydnert, Frida; Jirström, Karin; Borrebaeck, Carl AK; Ek, Sara

    2011-01-01

    The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking. SOX11 expression and clinicopathological data was compared using χ 2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation. SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5'-Aza-2'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11. SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours

  7. The tumour suppressor SOX11 is associated with improved survival among high grade epithelial ovarian cancers and is regulated by reversible promoter methylation

    LENUS (Irish Health Repository)

    Sernbo, Sandra

    2011-09-24

    Abstract Background The neural transcription factor SOX11 has been described as a prognostic marker in epithelial ovarian cancers (EOC), however its role in individual histological subtypes and tumour grade requires further clarification. Furthermore, methylation-dependent silencing of SOX11 has been reported for B cell lymphomas and indicates that epigenetic drugs may be used to re-express this tumour suppressor, but information on SOX11 promoter methylation in EOC is still lacking. Methods SOX11 expression and clinicopathological data was compared using χ2 test in a cohort of 154 cases of primary invasive EOC. Kaplan-Meier analysis and the log rank test were applied to evaluate ovarian cancer-specific survival (OCSS) and overall survival (OS) in strata, according to SOX11 expression. Also, the methylation status of the SOX11 promoter was determined by sodium bisulfite sequencing and methylation specific PCR (MSP). Furthermore, the effect of ectopic overexpression of SOX11 on proliferation was studied through [3H]-thymidine incorporation. Results SOX11 expression was associated with an improved survival of patients with high grade EOC, although not independent of stage. Further analyses of EOC cell lines showed that SOX11 mRNA and protein were expressed in two of five cell lines, correlating with promoter methylation status. Demethylation was successfully performed using 5\\'-Aza-2\\'deoxycytidine (5-Aza-dC) resulting in SOX11 mRNA and protein expression in a previously negative EOC cell line. Furthermore, overexpression of SOX11 in EOC cell lines confirmed the growth regulatory role of SOX11. Conclusions SOX11 is a functionally associated protein in EOC with prognostic value for high-grade tumours. Re-expression of SOX11 in EOC indicates a potential use of epigenetic drugs to affect cellular growth in SOX11-negative tumours.

  8. Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers

    International Nuclear Information System (INIS)

    Liu, Yang; Gao, Weimin; Siegfried, Jill M; Weissfeld, Joel L; Luketich, James D; Keohavong, Phouthone

    2007-01-01

    Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors. Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies. The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene. Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of

  9. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    International Nuclear Information System (INIS)

    Nasab, E. E.; Nasab, E. E.; Hashemi, M.; Rafighdoost, F.

    2016-01-01

    Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent non neoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent non neoplastic tissue (OR=2.30, 95% CI=0.95-5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P=0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients’ clinical characteristics (P>0.05).

  10. Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma.

    Science.gov (United States)

    Ogino, Shuji; Odze, Robert D; Kawasaki, Takako; Brahmandam, Mohan; Kirkner, Gregory J; Laird, Peter W; Loda, Massimo; Fuchs, Charles S

    2006-09-01

    Extensive gene promoter methylation in colorectal carcinoma has been termed the CpG island methylator phenotype (CIMP). Previous studies on CIMP used primarily methylation-specific polymerase chain reaction (PCR), which, unfortunately, may detect low levels of methylation that has little or no biological significance. Utilizing quantitative real-time PCR (MethyLight), we measured DNA methylation in a panel of 5 CIMP-specific gene promoters (CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1) in 459 colorectal carcinomas obtained from 2 large prospective cohort studies. CIMP was defined as tumors that showed methylation in >or=4/5 promoters. CIMP was significantly associated with the presence of mucinous or signet ring cell morphology, marked Crohn's-like lymphoid reaction, tumor infiltrating lymphocytes, marked peritumoral lymphocytic reaction, tumor necrosis, tumor cell sheeting, and poor differentiation. All these features have previously been associated with microsatellite instability (MSI). Therefore, we divided the 459 colorectal carcinomas into 6 subtypes, namely, MSI-high (MSI-H)/CIMP, MSI-H/non-CIMP, MSI-low (MSI-L)/CIMP, MSI-L/non-CIMP, microsatellite stable/CIMP, and micro satellite sstable/non-CIMP. Compared with MSI-H/non-CIMP, MSI-H/CIMP was associated with marked tumor infiltrating lymphocytes, tumor necrosis, sheeting, and poor differentiation (all PCIMP, MSI-L/CIMP was associated with tumors that had CIMP. Both MSI and CIMP appear to play a role in the pathogenesis of specific morphologic patterns of colorectal carcinoma.

  11. PRMT1-mediated arginine methylation controls ATXN2L localization

    Energy Technology Data Exchange (ETDEWEB)

    Kaehler, Christian; Guenther, Anika; Uhlich, Anja; Krobitsch, Sylvia, E-mail: krobitsc@molgen.mpg.de

    2015-05-15

    Arginine methylation is a posttranslational modification that is of importance in diverse cellular processes. Recent proteomic mass spectrometry studies reported arginine methylation of ataxin-2-like (ATXN2L), the paralog of ataxin-2, a protein that is implicated in the neurodegenerative disorder spinocerebellar ataxia type 2. Here, we investigated the methylation state of ATXN2L and its significance for ATXN2L localization. We first confirmed that ATXN2L is asymmetrically dimethylated in vivo, and observed that the nuclear localization of ATXN2L is altered under methylation inhibition. We further discovered that ATXN2L associates with the protein arginine-N-methyltransferase 1 (PRMT1). Finally, we showed that neither mutation of the arginine–glycine-rich motifs of ATXN2L nor methylation inhibition alters ATXN2L localization to stress granules, suggesting that methylation of ATXN2L is probably not mandatory. - Highlights: • ATXN2L is asymmetrically dimethylated in vivo. • ATXN2L interacts with PRMT1 under normal and stress conditions. • PRMT1-mediated dimethylation of ATXN2L controls its nuclear localization. • ATXN2L localization to stress granules appears independent of its methylation state.

  12. Epigenetics in Alzheimer's Disease: Perspective of DNA Methylation.

    Science.gov (United States)

    Qazi, Talal Jamil; Quan, Zhenzhen; Mir, Asif; Qing, Hong

    2018-02-01

    Research over the years has shown that causes of Alzheimer's disease are not well understood, but over the past years, the involvement of epigenetic mechanisms in the developing memory formation either under pathological or physiological conditions has become clear. The term epigenetics represents the heredity of changes in phenotype that are independent of altered DNA sequences. Different studies validated that cytosine methylation of genomic DNA decreases with age in different tissues of mammals, and therefore, the role of epigenetic factors in developing neurological disorders in aging has been under focus. In this review, we summarized and reviewed the involvement of different epigenetic mechanisms especially the DNA methylation in Alzheimer's disease (AD), late-onset Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and autosomal dominant Alzheimer's disease (ADAD). Down to the minutest of details, we tried to discuss the methylation patterns like mitochondrial DNA methylation and ribosomal DNA (rDNA) methylation. Additionally, we mentioned some therapeutic approaches related to epigenetics, which could provide a potential cure for AD. Moreover, we reviewed some recent studies that validate DNA methylation as a potential biomarker and its role in AD. We hope that this review will provide new insights into the understanding of AD pathogenesis from the epigenetic perspective especially from the perspective of DNA methylation.

  13. DNA Methylation as a Biomarker for Body Fluid Identification

    Directory of Open Access Journals (Sweden)

    Rania Gomaa

    2017-12-01

    Full Text Available Currently, available identification techniques for forensic samples are either enzyme or protein based, which can be subjected to degradation, thus limiting its storage potentials. Epigenetic changes arising due to DNA methylation and histone acetylation can be used for body fluid identification. Markers DACT1, USP49, ZC3H12D, FGF7, cg23521140, cg17610929, chromosome 4 (25287119–25287254, chromosome 11 (72085678–72085798, 57171095–57171236, 1493401–1493538, and chromosome 19 (47395505–47395651 are currently being used for semen identification. Markers cg26107890, cg20691722, cg01774894 and cg14991487 are used to differentiate saliva and vaginal secretions from other body fluids. However, such markers show overlapping methylation pattern. This review article aimed to highlight the feasibility of using DNA methylation of certain genetic markers in body fluid identification and its implications for forensic investigations. The reviewed articles have employed molecular genetics techniques such as Bisulfite sequencing PCR (BSP, methylation specific PCR (MSP, Pyrosequencing, Combined Bisulfite Restriction Analysis (COBRA, Methylation-sensitive Single Nucleotide Primer Extension (SNuPE, and Multiplex SNaPshot Microarray. Bioinformatics software such as MATLAB and BiQ Analyzer has been used. Biological fluids have different methylation patterns and thus, this difference can be used to identify the nature of the biological fluid found at the crime scene. Using DNA methylation to identify the body fluids gives accurate results without consumption of the trace evidence and requires a minute amount of DNA for analysis. Recent studies have incorporated next-generation sequencing aiming to find out more reliable markers that can differentiate between different body fluids. Nonetheless, new DNA methylation markers are yet to be discovered to accurately differentiate between saliva and vaginal secretions with high confidence. Epigenetic changes are

  14. A panel of genes methylated with high frequency in colorectal cancer

    International Nuclear Information System (INIS)

    Mitchell, Susan M; Beetson, Iain; Rand, Keith N; McEvoy, Aidan; Thomas, Melissa L; Baker, Rohan T; Wattchow, David A; Young, Graeme P; Lockett, Trevor J; Pedersen, Susanne K; LaPointe, Lawrence C; Ross, Jason P; Molloy, Peter L; Drew, Horace R; Ho, Thu; Brown, Glenn S; Saunders, Neil FW; Duesing, Konsta R; Buckley, Michael J; Dunne, Rob

    2014-01-01

    The development of colorectal cancer (CRC) is accompanied by extensive epigenetic changes, including frequent regional hypermethylation particularly of gene promoter regions. Specific genes, including SEPT9, VIM1 and TMEFF2 become methylated in a high fraction of cancers and diagnostic assays for detection of cancer-derived methylated DNA sequences in blood and/or fecal samples are being developed. There is considerable potential for the development of new DNA methylation biomarkers or panels to improve the sensitivity and specificity of current cancer detection tests. Combined epigenomic methods – activation of gene expression in CRC cell lines following DNA demethylating treatment, and two novel methods of genome-wide methylation assessment – were used to identify candidate genes methylated in a high fraction of CRCs. Multiplexed amplicon sequencing of PCR products from bisulfite-treated DNA of matched CRC and non-neoplastic tissue as well as healthy donor peripheral blood was performed using Roche 454 sequencing. Levels of DNA methylation in colorectal tissues and blood were determined by quantitative methylation specific PCR (qMSP). Combined analyses identified 42 candidate genes for evaluation as DNA methylation biomarkers. DNA methylation profiles of 24 of these genes were characterised by multiplexed bisulfite-sequencing in ten matched tumor/normal tissue samples; differential methylation in CRC was confirmed for 23 of these genes. qMSP assays were developed for 32 genes, including 15 of the sequenced genes, and used to quantify methylation in tumor, adenoma and non-neoplastic colorectal tissue and from healthy donor peripheral blood. 24 of the 32 genes were methylated in >50% of neoplastic samples, including 11 genes that were methylated in 80% or more CRCs and a similar fraction of adenomas. This study has characterised a panel of 23 genes that show elevated DNA methylation in >50% of CRC tissue relative to non-neoplastic tissue. Six of these genes

  15. Patterns of DNMT1 Promoter Methylation in Patients with Acute Lymphoblastic Leukemia.

    Science.gov (United States)

    Rahmani, Tirdad; Azad, Mehdi; Chahardouli, Bahram; Nasiri, Hajar; Vatanmakanian, Mousa; Kaviani, Saeid

    2017-07-01

    Background: Acute lymphoblastic leukemia (ALL) is a clonal malignant disorder characterized by an uncontrolled proliferation of immature T or B lymphocytes. Extensive studies have shown that the epigenetic changes, especially modified DNA methylation patterns in the regulatory regions through the DNA methyltransferase (DNMTs), play an important role in the development of genetic disorders and abnormal growth and maturation capacity of leukemic stem cells (LSCs).The aim of this study was to evaluate the changes in DNMT1 promoter methylation and its expression pattern in patients with ALL. Materials and Methods: In this experimental study, methylation specific PCR (MSP) was used to assess the methylation status of DNMT1 promoter regions in samples collected from ALL patients (n=45) and healthy control subjects. According to this method, un-methylated cytosine nucleotides are converted to uracil by sodium bisulfite and the proliferation of methylated and un-methylated regions are performed using specific primers for target sequences. Results: None of the patients with B and T-ALL showed methylated promoter regions of the DNMT1 gene, while the methylation pattern of both pre-B ALL patients and the control group showed a relative promoter methylation. Conclusion: Analysis of promoter methylation patterns in various subgroups of ALL has revealed the importance of DNMT1 in the regulation of gene expression. Likewise, extensive data have also highlighted the methylation-based mechanisms exerted by DNAM1 as one of the main participants regulating gene expression in B-ALL and T-ALL patients. Investigation of the overall DNA methylation pattern offers significant improvements in the prediction of disease prognosis and treatment response.

  16. A mutant screening method by critical annealing temperature-PCR for site-directed mutagenesis.

    Science.gov (United States)

    Liu, Ying; Wu, Ting; Song, Jian; Chen, Xuelian; Zhang, Yu; Wan, Yu

    2013-03-11

    Distinguishing desired mutants from parental templates and undesired mutants is a problem not well solved in Quikchange™ mutagenesis. Although Dpn I digestion can eliminate methylated parental (WT) DNA, the efficiency is not satisfying due to the existence of hemi-methylated DNA in the PCR products, which is resistant to Dpn I. The present study designed a novel critical annealing temperature (T(c))-PCR to replace Dpn I digestion for more perfect mutant distinguishing, in which part-overlapping primers containing mutation(s) were used to reduce initial concentration of template DNA in mutagenic PCR. A T(c)-PCR with the same mutagenic primers was performed without Dpn I digestion. The T(c) for each pair of the primers was identified by gradient PCR. The relationship between PCR-identified T(c) and T(m) of the primers was analyzed and modeled with correlation and regression. Gradient PCR identified a T(c) for each of 14 tested mutagenic primers, which could discriminate mismatched parental molecules and undesired mutants from desired mutants. The PCR-identified T(c) was correlated to the primer's T(m) (r = 0.804, P<0.0001). Thus, in practical applications, the T(c) can be easily calculated with a regression equation, T(c)= 48.81 + 0.253*T(m). The new protocol introduced a novel T(c)-PCR method for mutant screening which can more efficiently and accurately select against parental molecules and undesired mutations in mutagenic sequence segments.

  17. Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

    OpenAIRE

    MORRILL, Benson H.; COX, Lindsay; WARD, Anika; HEYWOOD, Sierra; PRATHER, Randall S.; ISOM, S. Clay

    2013-01-01

    Abstract The purpose of this experiment was to implement and evaluate the effectiveness of a next-generation sequencing-based method for DNA methylation analysis in porcine embryonic samples. Fourteen discrete genomic regions were amplified by PCR using bisulfite-converted genomic DNA derived from day 14 in vivo-derived (IVV) and parthenogenetic (PA) porcine embryos as template DNA. Resulting PCR products were subjected to high-throughput sequencing using the Illumina Genome Analyzer IIx plat...

  18. Diagnostic markers of urothelial cancer based on DNA methylation analysis

    International Nuclear Information System (INIS)

    Chihara, Yoshitomo; Hirao, Yoshihiko; Kanai, Yae; Fujimoto, Hiroyuki; Sugano, Kokichi; Kawashima, Kiyotaka; Liang, Gangning; Jones, Peter A; Fujimoto, Kiyohide; Kuniyasu, Hiroki

    2013-01-01

    Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation. In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers. Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity. Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect

  19. The application of methylation specific electrophoresis (MSE to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    Directory of Open Access Journals (Sweden)

    Yokoyama Seiya

    2012-02-01

    Full Text Available Abstract Background Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Methods Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE. The MSE method is comprised of the following steps: (a bisulfite treatment of genomic DNA, (b amplification of the target DNA by a nested PCR approach and (c applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. Result The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is Conclusions The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical samples, and this may facilitate identification of new risk markers.

  20. Methylation in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Regina M. Santella

    2007-02-01

    Full Text Available

    The development of HCC is a complex, multistep, multistage process. The molecular pathogenesis of HCC appears to involve multiple genetic aberrations in the molecular control of hepatocyte proliferation, differentiation and death and the maintenance of genomic integrity. This process is influenced by the cumulative activation and inactivation of oncogenes, tumor suppressor genes and other genes. p53, a tumor suppressor gene, is the most frequently mutated gene in human cancers. There is also a striking sequence specific binding and induction of mutations by AFB1 at codon 249 of p53 in HCC.

    Epigenetic alterations are also involved in cancer development and progression. Methylation of promoter CpG islands is associated with inhibition of transcriptional initiation and permanent silencing of downstream genes.

    It is now known that most important tumor suppressor genes are inactivated, not only by mutations and deletions but also by promoter methylation. Several studies indicated that p16, p15, RASSF1A, MGMT, and GSTP1 promoter hypermethylation are prevalent in HCC. In addition, geographic variation in the methylation status of tumor DNA indicates that environmental factors may influence the frequent and concordant degree of hypermethylation in multiple genes in HCC and that epigeneticenvironmental interactions may be involved in hepatocarcinogenesis. We have found significant relationships between promoter methylation and AFB1-DNA adducts confirming the impact of environmental exposures on gene methylation.

    DNA isolated from serum or plasma of cancer patients frequently contains the same genetic and

  1. Lysine methyltransferase G9a is not required for DNMT3A/3B anchoring to methylated nucleosomes and maintenance of DNA methylation in somatic cells

    Directory of Open Access Journals (Sweden)

    Sharma Shikhar

    2012-01-01

    Full Text Available Abstract Background DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear. Results Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation. Conclusions Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.

  2. Methylation of Promoter Regions of Genes of the Human Intrauterine Renin Angiotensin System and Their Expression

    Directory of Open Access Journals (Sweden)

    Shane D. Sykes

    2015-01-01

    Full Text Available The intrauterine renin angiotensin system (RAS is implicated in placentation and labour onset. Here we investigate whether promoter methylation of RAS genes changes with gestation or labour and if it affects gene expression. Early gestation amnion and placenta were studied, as were term amnion, decidua, and placenta collected before labour (at elective caesarean section or after spontaneous labour and delivery. The expression and degree of methylation of the prorenin receptor (ATP6AP2, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AGTR1, and two proteases that can activate prorenin (kallikrein, KLK1, and cathepsin D, CTSD were measured by qPCR and a DNA methylation array. There was no effect of gestation or labour on the methylation of RAS genes and CTSD. Amnion and decidua displayed strong correlations between the percent hypermethylation of RAS genes and CTSD, suggestive of global methylation. There were no correlations between the degree of methylation and mRNA abundance of any genes studied. KLK1 was the most methylated gene and the proportion of hypermethylated KLK1 alleles was lower in placenta than decidua. The presence of intermediate methylated alleles of KLK1 in early gestation placenta and in amnion after labour suggests that KLK1 methylation is uniquely dynamic in these tissues.

  3. Promoter methylation of Wnt-antagonists in polypoid and nonpolypoid colorectal adenomas

    International Nuclear Information System (INIS)

    Voorham, Quirinus JM; Mulder, Chris JJ; Engeland, Manon van; Meijer, Gerrit A; Steenbergen, Renske DM; Carvalho, Beatriz; Janssen, Jerry; Tijssen, Marianne; Snellenberg, Suzanne; Mongera, Sandra; Grieken, Nicole CT van; Grabsch, Heike; Kliment, Martin; Rembacken, Bjorn J

    2013-01-01

    Nonpolypoid adenomas are a subgroup of colorectal adenomas that have been associated with a more aggressive clinical behaviour compared to their polypoid counterparts. A substantial proportion of nonpolypoid and polypoid adenomas lack APC mutations, APC methylation or chromosomal loss of the APC locus on chromosome 5q, suggesting the involvement of other Wnt-pathway genes. The present study investigated promoter methylation of several Wnt-pathway antagonists in both nonpolypoid and polypoid adenomas. Quantitative methylation-specific PCR (qMSP) was used to evaluate methylation of four Wnt-antagonists, SFRP2, WIF-1, DKK3 and SOX17 in 18 normal colorectal mucosa samples, 9 colorectal cancer cell lines, 18 carcinomas, 44 nonpolypoid and 44 polypoid adenomas. Results were integrated with previously obtained data on APC mutation, methylation and chromosome 5q status from the same samples. Increased methylation of all genes was found in the majority of cell lines, adenomas and carcinomas compared to normal controls. WIF-1 and DKK3 showed a significantly lower level of methylation in nonpolypoid compared to polypoid adenomas (p < 0.01). Combining both adenoma types, a positive trend between APC mutation and both WIF-1 and DKK3 methylation was observed (p < 0.05). Methylation of Wnt-pathway antagonists represents an additional mechanism of constitutive Wnt-pathway activation in colorectal adenomas. Current results further substantiate the existence of partially alternative Wnt-pathway disruption mechanisms in nonpolypoid compared to polypoid adenomas, in line with previous observations

  4. Position-specific prediction of methylation sites from sequence conservation based on information theory.

    Science.gov (United States)

    Shi, Yinan; Guo, Yanzhi; Hu, Yayun; Li, Menglong

    2015-07-23

    Protein methylation plays vital roles in many biological processes and has been implicated in various human diseases. To fully understand the mechanisms underlying methylation for use in drug design and work in methylation-related diseases, an initial but crucial step is to identify methylation sites. The use of high-throughput bioinformatics methods has become imperative to predict methylation sites. In this study, we developed a novel method that is based only on sequence conservation to predict protein methylation sites. Conservation difference profiles between methylated and non-methylated peptides were constructed by the information entropy (IE) in a wider neighbor interval around the methylation sites that fully incorporated all of the environmental information. Then, the distinctive neighbor residues were identified by the importance scores of information gain (IG). The most representative model was constructed by support vector machine (SVM) for Arginine and Lysine methylation, respectively. This model yielded a promising result on both the benchmark dataset and independent test set. The model was used to screen the entire human proteome, and many unknown substrates were identified. These results indicate that our method can serve as a useful supplement to elucidate the mechanism of protein methylation and facilitate hypothesis-driven experimental design and validation.

  5. Comparison of quantification algorithms for circulating cell-free DNA methylation biomarkers in blood plasma from cancer patients.

    Science.gov (United States)

    de Vos, Luka; Gevensleben, Heidrun; Schröck, Andreas; Franzen, Alina; Kristiansen, Glen; Bootz, Friedrich; Dietrich, Dimo

    2017-01-01

    SHOX2 and SEPT9 methylation in circulating cell-free DNA (ccfDNA) in blood are established powerful and clinically valuable biomarkers for diagnosis, staging, prognosis, and monitoring of cancer patients. The aim of the present study was to evaluate different quantification algorithms (relative quantification, absolute quantification, quasi-digital PCR) with regard to their clinical performance. Methylation analyses were performed in a training cohort (141 patients with head and neck squamous cell carcinoma [HNSCC], 170 control cases) and a testing cohort (137 HNSCC cases, 102 controls). DNA was extracted from plasma samples, bisulfite-converted, and analyzed via quantitative real-time PCR. SHOX2 and SEPT9 methylations were assessed separately and as panel [mean SEPT9 / SHOX2 ] using the ΔCT method for absolute quantification and the ΔΔCT-method for relative quantification. Quasi-digital PCR was defined as the number of amplification-positive PCR replicates. The diagnostic (sensitivity, specificity, area under the curve (AUC) of the receiver operating characteristic (ROC)) and prognostic accuracy (hazard ratio (HR) from Cox regression) were evaluated. Sporadic methylation in control samples necessitated the introduction of cutoffs resulting in 61-63% sensitivity/90-92% specificity ( SEPT9 /training), 53-57% sensitivity/87-90% specificity ( SHOX2 /training), and 64-65% sensitivity/90-91% specificity (mean SEPT9 / SHOX2 /training). Results were confirmed in a testing cohort with 54-56% sensitivity/88-90% specificity ( SEPT9 /testing), 43-48% sensitivity/93-95% specificity ( SHOX2 /testing), and 49-58% sensitivity/88-94% specificity (mean SEPT9 / SHOX2 /testing). All algorithms showed comparable cutoff-independent diagnostic accuracy with largely overlapping 95% confidence intervals ( SEPT9 : AUC training  = 0.79-0.80; AUC testing  = 0.74-0.75; SHOX2 : AUC training  = 0.78-0.81, AUC testing  = 0.77-0.79; mean SEPT9 / SHOX2 : AUC training  = 0

  6. pcr

    African Journals Online (AJOL)

    DR. AMINU

    Keywords: Polymerase chain reaction, Diagnosis, Bacteria, Infections. INTRODUCTION ... used to amplify a piece of DNA by in-vitro enzymatic replication (David and .... receiving antimicrobial treatment, or when the causative agents are small ...

  7. DNA methylation and memory formation.

    Science.gov (United States)

    Day, Jeremy J; Sweatt, J David

    2010-11-01

    Memory formation and storage require long-lasting changes in memory-related neuronal circuits. Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for an epigenetic mechanism in learning and long-term memory maintenance and raise apparent conundrums and questions. For example, it is unclear how DNA methylation might be reversed during the formation of a memory, how changes in DNA methylation alter neuronal function to promote memory formation, and how DNA methylation patterns differ between neuronal structures to enable both consolidation and storage of memories. Here we evaluate the existing evidence supporting a role for DNA methylation in memory, discuss how DNA methylation may affect genetic and neuronal function to contribute to behavior, propose several future directions for the emerging subfield of neuroepigenetics, and begin to address some of the broader implications of this work.

  8. Absolute photoionization cross-section of the methyl radical.

    Science.gov (United States)

    Taatjes, Craig A; Osborn, David L; Selby, Talitha M; Meloni, Giovanni; Fan, Haiyan; Pratt, Stephen T

    2008-10-02

    The absolute photoionization cross-section of the methyl radical has been measured using two completely independent methods. The CH3 photoionization cross-section was determined relative to that of acetone and methyl vinyl ketone at photon energies of 10.2 and 11.0 eV by using a pulsed laser-photolysis/time-resolved synchrotron photoionization mass spectrometry method. The time-resolved depletion of the acetone or methyl vinyl ketone precursor and the production of methyl radicals following 193 nm photolysis are monitored simultaneously by using time-resolved synchrotron photoionization mass spectrometry. Comparison of the initial methyl signal with the decrease in precursor signal, in combination with previously measured absolute photoionization cross-sections of the precursors, yields the absolute photoionization cross-section of the methyl radical; sigma(CH3)(10.2 eV) = (5.7 +/- 0.9) x 10(-18) cm(2) and sigma(CH3)(11.0 eV) = (6.0 +/- 2.0) x 10(-18) cm(2). The photoionization cross-section for vinyl radical determined by photolysis of methyl vinyl ketone is in good agreement with previous measurements. The methyl radical photoionization cross-section was also independently measured relative to that of the iodine atom by comparison of ionization signals from CH3 and I fragments following 266 nm photolysis of methyl iodide in a molecular-beam ion-imaging apparatus. These measurements gave a cross-section of (5.4 +/- 2.0) x 10(-18) cm(2) at 10.460 eV, (5.5 +/- 2.0) x 10(-18) cm(2) at 10.466 eV, and (4.9 +/- 2.0) x 10(-18) cm(2) at 10.471 eV. The measurements allow relative photoionization efficiency spectra of methyl radical to be placed on an absolute scale and will facilitate quantitative measurements of methyl concentrations by photoionization mass spectrometry.

  9. HIGHLY METHYL ESTERIFIED SEEDS is a pectin methyl esterase involved in embryo development.

    Science.gov (United States)

    Levesque-Tremblay, Gabriel; Müller, Kerstin; Mansfield, Shawn D; Haughn, George W

    2015-03-01

    Homogalacturonan pectin domains are synthesized in a highly methyl-esterified form that later can be differentially demethyl esterified by pectin methyl esterase (PME) to strengthen or loosen plant cell walls that contain pectin, including seed coat mucilage, a specialized secondary cell wall of seed coat epidermal cells. As a means to identify the active PMEs in seed coat mucilage, we identified seven PMEs expressed during seed coat development. One of these, HIGHLY METHYL ESTERIFIED SEEDS (HMS), is abundant during mucilage secretion, peaking at 7 d postanthesis in both the seed coat and the embryo. We have determined that this gene is required for normal levels of PME activity and homogalacturonan methyl esterification in the seed. The hms-1 mutant displays altered embryo morphology and mucilage extrusion, both of which are a consequence of defects in embryo development. A significant decrease in the size of cells in the embryo suggests that the changes in embryo morphology are a consequence of lack of cell expansion. Progeny from a cross between hms-1 and the previously characterized PME inhibitor5 overexpression line suggest that HMS acts independently from other cell wall-modifying enzymes in the embryo. We propose that HMS is required for cell wall loosening in the embryo to facilitate cell expansion during the accumulation of storage reserves and that its role in the seed coat is masked by redundancy. © 2015 American Society of Plant Biologists. All Rights Reserved.

  10. MIRA: An R package for DNA methylation-based inference of regulatory activity.

    Science.gov (United States)

    Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

    2018-03-01

    DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

  11. Independence and Product Systems

    OpenAIRE

    Skeide, Michael

    2003-01-01

    Starting from elementary considerations about independence and Markov processes in classical probability we arrive at the new concept of conditional monotone independence (or operator-valued monotone independence). With the help of product systems of Hilbert modules we show that monotone conditional independence arises naturally in dilation theory.

  12. Whole-genome methylation caller designed for methyl- DNA ...

    African Journals Online (AJOL)

    etchie

    2013-02-20

    Feb 20, 2013 ... Key words: Methyl-DNA immunoprecipitation, next-generation sequencing, Hidden ... its response to environmental cues. .... have a great potential to become the most cost-effective ... hg18 reference genome (set to 0 if not present in retrieved reads). ..... DNA methylation patterns and epigenetic memory.

  13. The altered promoter methylation of oxytocin receptor gene in autism.

    Science.gov (United States)

    Elagoz Yuksel, Mine; Yuceturk, Betul; Karatas, Omer Faruk; Ozen, Mustafa; Dogangun, Burak

    Autism spectrum disorder (ASD) is one of the lifelong existing disorders. Abnormal methylation status of gene promoters of oxytonergic system has been implicated as among the etiologic factors of ASDs. We, therefore, investigated the methylation frequency of oxytocin receptor gene (OXTR) promoter from peripheral blood samples of children with autistic features. Our sample includes 66 children in total (22-94 months); 27 children with ASDs according to the DSM-IV-TR and the Childhood Autism Rating Scale (CARS) and 39 children who do not have any autistic like symptoms as the healthy control group. We investigated the DNA methylation status of OXTR promoter by methylation specific enzymatic digestion of genomic DNA and polymerase chain reaction. A significant relationship has been found between ASDs and healthy controls for the reduction of methylation frequency of the regions MT1 and MT3 of OXTR. We could not find any association in the methylation frequency of MT2 and MT4 regions of OXTR. Although our findings indicate high frequency of OXTR promoter hypomethylation in ASDs, there is need for independent replication of the results for a bigger sample set. We expect that future studies with the inclusion of larger, more homogeneous samples will attempt to disentangle the causes of ASDs.

  14. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

    Science.gov (United States)

    Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

  15. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    Directory of Open Access Journals (Sweden)

    Karolina Chwialkowska

    2017-11-01

    Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

  16. Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    Science.gov (United States)

    Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

    2017-01-01

    Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

  17. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    Science.gov (United States)

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  18. Hepatocellular carcinoma displays distinct DNA methylation signatures with potential as clinical predictors.

    Directory of Open Access Journals (Sweden)

    Hector Hernandez-Vargas

    Full Text Available BACKGROUND: Hepatocellular carcinoma (HCC is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be the cause of its molecular and clinicopathological heterogeneity. A better understanding of the global deregulation of methylation states and how they correlate with disease progression will aid in the design of strategies for earlier detection and better therapeutic decisions. METHODS AND FINDINGS: We characterized the changes in promoter methylation in a series of 30 HCC tumors and their respective surrounding tissue and identified methylation signatures associated with major risk factors and clinical correlates. A wide panel of cancer-related gene promoters was analyzed using Illumina bead array technology, and CpG sites were then selected according to their ability to classify clinicopathological parameters. An independent series of HCC tumors and matched surrounding tissue was used for validation of the signatures. We were able to develop and validate a signature of methylation in HCC. This signature distinguished HCC from surrounding tissue and from other tumor types, and was independent of risk factors. However, aberrant methylation of an independent subset of promoters was associated with tumor progression and etiological risk factors (HBV or HCV infection and alcohol consumption. Interestingly, distinct methylation of an independent panel of gene promoters was strongly correlated with survival after cancer therapy. CONCLUSION: Our study shows that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression stage, with potential clinical applications in diagnosis and prognosis.

  19. RT-PCR Detection of HIV in Republic of Macedonia

    Directory of Open Access Journals (Sweden)

    Golubinka Bosevska

    2008-11-01

    Full Text Available The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works.The total of 33 examined persons were divided in two groups: 1 13 persons seropositive for HIV; and 2 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5.ELFA test for combined detection of HIV p24 antigen and anti HIV-1 + 2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly.In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70°C, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20°C for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  20. Exploring the Link between Nucleosome Occupancy and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Cecilia Lövkvist

    2018-01-01

    Full Text Available Near promoters, both nucleosomes and CpG sites form characteristic spatial patterns. Previously, nucleosome depleted regions were observed upstream of transcription start sites and nucleosome occupancy was reported to correlate both with CpG density and the level of CpG methylation. Several studies imply a causal link where CpG methylation might induce nucleosome formation, whereas others argue the opposite, i.e., that nucleosome occupancy might influence CpG methylation. Correlations are indeed evident between nucleosomes, CpG density and CpG methylation—at least near promoter sites. It is however less established whether there is an immediate causal relation between nucleosome occupancy and the presence of CpG sites—or if nucleosome occupancy could be influenced by other factors. In this work, we test for such causality in human genomes by analyzing the three quantities both near and away from promoter sites. For data from the human genome we compare promoter regions with given CpG densities with genomic regions without promoters but of similar CpG densities. We find the observed correlation between nucleosome occupancy and CpG density, respectively CpG methylation, to be specific to promoter regions. In other regions along the genome nucleosome occupancy is statistically independent of the positioning of CpGs or their methylation levels. Anti-correlation between CpG density and methylation level is however similarly strong in both regions. On promoters, nucleosome occupancy is more strongly affected by the level of gene expression than CpG density or CpG methylation—calling into question any direct causal relation between nucleosome occupancy and CpG organization. Rather, our results suggest that for organisms with cytosine methylation nucleosome occupancy might be primarily linked to gene expression, with no strong impact on methylation.

  1. DELETION AND 5'CPG ISLAND METHYLATION OF p15 GENE IN BRAIN GLIOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective: To investigate the abnormality of p15 gene in brain glioma and the correlation of it with occurrence or malignant progression of brain glioma. Methods: Deletion and 5'CPG island methylation of p15 gene were detected by the methods of PCR and PCR-based methylation in 56 cases of brain glioma. Results: Out of 43 cases of high grade glioma, 14 cases were found to have homozygous deletion of p15E1, while none of the 13 cases of low grade glioma was found to have deletion of p15E1 (P<0.05). Methylation of 5'CPG Island of p15 gene was found only in four cases of glioma. Conclusion: Abnormality of p15 gene may involved in the occurrence and malignant progression of brain glioma. Homozygous deletion of gene is the major mechanism of inactivation for p15 gene in brain glioma.

  2. Promoter methylation inhibits BRD7 expression in human nasopharyngeal carcinoma cells

    International Nuclear Information System (INIS)

    Liu, Huaying; Li, Guiyuan; Zhang, Liming; Niu, Zhaoxia; Zhou, Ming; Peng, Cong; Li, Xiayu; Deng, Tan; Shi, Lei; Tan, Yixin

    2008-01-01

    Nasopharyngeal carcinoma (NPC) is a head and neck malignancy with high occurrence in South-East Asia and Southern China. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumourigenesis of NPC. BRD7 is a NPC-associated bromodomain gene that exhibits a much higher-level of mRNA expression in normal than in NPC biopsies and cell lines. In this study, we explored the role of DNA methylation in regulation of BRD7 transcription. The presence of CpG islands within BRD7 promoter was predicted by EMBOSS CpGplot and Softberry CpGFinder, respectively. Nested methylation-specific PCR and RT-PCR were employed to detect the methylation status of BRD7 promoter and the mRNA expression of BRD7 gene in tumor cell lines as well as clinical samples. Electrophoretic mobility shift assays (EMSA) and luciferase assay were used to detect the effects of cytosine methylation on the nuclear protein binding to BRD7 promoter. We found that DNA methylation suppresses BRD7 expression in NPC cells. In vitro DNA methylation in NPC cells silenced BRD7 promoter activity and inhibited the binding of the nuclear protein (possibly Sp1) to Sp1 binding sites in the BRD7 promoter. In contrast, inhibition of DNA methylation augments induction of endogenous BRD7 mRNA in NPC cells. We also found that methylation frequency of BRD7 promoter is much higher in the tumor and matched blood samples from NPC patients than in the blood samples from normal individuals. BRD7 promoter demethylation is a prerequisite for high level induction of BRD7 gene expression. DNA methylation of BRD7 promoter might serve as a diagnostic marker in NPC

  3. DNA methylation regulates expression of VEGF-C, and S-adenosylmethionine is effective for VEGF-C methylation and for inhibiting cancer growth

    Energy Technology Data Exchange (ETDEWEB)

    Da, M.X. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Zhang, Y.B. [Department of Surgery, Ningxia Medical University, Yinchuan (China); Yao, J.B. [Department of Surgical Oncology, Gansu Provincial Hospital, Lanzhou (China); Duan, Y.X. [Department of Surgery, Ningxia Medical University, Yinchuan (China)

    2014-09-30

    DNA hypomethylation may activate oncogene transcription, thus promoting carcinogenesis and tumor development. S-adenosylmethionine (SAM) is a methyl donor in numerous methylation reactions and acts as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA. The main objectives of this study were to determine whether DNA hypomethylation correlated with vascular endothelial growth factor-C (VEGF-C) expression, and the effect of SAM on VEGF-C methylation and gastric cancer growth inhibition. VEGF-C expression was assayed by Western blotting and RT-qPCR in gastric cancer cells, and by immunohistochemistry in tumor xenografts. VEGF-C methylation was assayed by bisulfite DNA sequencing. The effect of SAM on cell apoptosis was assayed by flow cytometry analyses and its effect on cancer growth was assessed in nude mice. The VEGF-C promoters of MGC-803, BGC-823, and SGC-7901 gastric cancer cells, which normally express VEGF-C, were nearly unmethylated. After SAM treatment, the VEGF-C promoters in these cells were highly methylated and VEGF-C expression was downregulated. SAM also significantly inhibited tumor growth in vitro and in vivo. DNA methylation regulates expression of VEGF-C. SAM can effectively induce VEGF-C methylation, reduce the expression of VEGF-C, and inhibit tumor growth. SAM has potential as a drug therapy to silence oncogenes and block the progression of gastric cancer.

  4. Effect of DNA methylation on identification of aggressive prostate cancer.

    Science.gov (United States)

    Alumkal, Joshi J; Zhang, Zhe; Humphreys, Elizabeth B; Bennett, Christina; Mangold, Leslie A; Carducci, Michael A; Partin, Alan W; Garrett-Mayer, Elizabeth; DeMarzo, Angelo M; Herman, James G

    2008-12-01

    Biochemical (prostate-specific antigen) recurrence of prostate cancer after radical prostatectomy remains a major problem. Better biomarkers are needed to identify high-risk patients. DNA methylation of promoter regions leads to gene silencing in many cancers. In this study, we assessed the effect of DNA methylation on the identification of recurrent prostate cancer. We studied the methylation status of 15 pre-specified genes using methylation-specific polymerase chain reaction on tissue samples from 151 patients with localized prostate cancer and at least 5 years of follow-up after prostatectomy. On multivariate logistic regression analysis, a high Gleason score and involvement of the capsule, lymph nodes, seminal vesicles, or surgical margin were associated with an increased risk of biochemical recurrence. Methylation of CDH13 by itself (odds ratio 5.50, 95% confidence interval [CI] 1.34 to 22.67; P = 0.02) or combined with methylation of ASC (odds ratio 5.64, 95% CI 1.47 to 21.7; P = 0.01) was also associated with an increased risk of biochemical recurrence. The presence of methylation of ASC and/or CDH13 yielded a sensitivity of 72.3% (95% CI 57% to 84.4%) and negative predictive value of 79% (95% CI 66.8% to 88.3%), similar to the weighted risk of recurrence (determined from the lymph node status, seminal vesicle status, surgical margin status, and postoperative Gleason score), a powerful clinicopathologic prognostic score. However, 34% (95% CI 21% to 49%) of the patients with recurrence were identified by the methylation profile of ASC and CDH13 rather than the weighted risk of recurrence. The results of our study have shown that methylation of CDH13 alone or combined with methylation of ASC is independently associated with an increased risk of biochemical recurrence after radical prostatectomy even considering the weighted risk of recurrence score. These findings should be validated in an independent, larger cohort of patients with prostate cancer who have

  5. Potential of DNA methylation in rectal cancer as diagnostic and prognostic biomarkers

    Science.gov (United States)

    Exner, Ruth; Pulverer, Walter; Diem, Martina; Spaller, Lisa; Woltering, Laura; Schreiber, Martin; Wolf, Brigitte; Sonntagbauer, Markus; Schröder, Fabian; Stift, Judith; Wrba, Fritz; Bergmann, Michael; Weinhäusel, Andreas; Egger, Gerda

    2015-01-01

    Background: Aberrant DNA methylation is more prominent in proximal compared with distal colorectal cancers. Although a number of methylation markers were identified for colon cancer, yet few are available for rectal cancer. Methods: DNA methylation differences were assessed by a targeted DNA microarray for 360 marker candidates between 22 fresh frozen rectal tumour samples and 8 controls and validated by microfluidic high-throughput and methylation-sensitive qPCR in fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples, respectively. The CpG island methylator phenotype (CIMP) was assessed by MethyLight in FFPE material from 78 patients with pT2 and pT3 rectal adenocarcinoma. Results: We identified and confirmed two novel three-gene signatures in fresh frozen samples that can distinguish tumours from adjacent tissue as well as from blood with a high sensitivity and specificity of up to 1 and an AUC of 1. In addition, methylation of individual CIMP markers was associated with specific clinical parameters such as tumour stage, therapy or patients' age. Methylation of CDKN2A was a negative prognostic factor for overall survival of patients. Conclusions: The newly defined methylation markers will be suitable for early disease detection and monitoring of rectal cancer. PMID:26335606

  6. Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

    Directory of Open Access Journals (Sweden)

    Robert Grützmann

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9 hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls. 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48% and 7/102 controls (7%. In the test study 73/126 CRCs (58% and 18/183 control samples (10% were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected while maintaining 90% specificity (19/183 for controls. Positive rates for plasmas from the other cancers (11/96 and non-cancerous conditions (41/315 were low. The rate of polyp detection (>1 cm was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

  7. DNA methylation in metabolic disorders

    DEFF Research Database (Denmark)

    Barres, Romain; Zierath, Juleen R

    2011-01-01

    DNA methylation is a major epigenetic modification that controls gene expression in physiologic and pathologic states. Metabolic diseases such as diabetes and obesity are associated with profound alterations in gene expression that are caused by genetic and environmental factors. Recent reports...... have provided evidence that environmental factors at all ages could modify DNA methylation in somatic tissues, which suggests that DNA methylation is a more dynamic process than previously appreciated. Because of the importance of lifestyle factors in metabolic disorders, DNA methylation provides...... a mechanism by which environmental factors, including diet and exercise, can modify genetic predisposition to disease. This article considers the current evidence that defines a role for DNA methylation in metabolic disorders....

  8. Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Shasha Su

    Full Text Available Leucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5 is a candidate marker for colorectal cancer stem cells (CSC. In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.The methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.The lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169 were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05, as well as better prognosis (p = 0.001 in patients with colorectal cancer.Our data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.

  9. Region of interest methylation analysis: a comparison of MSP with MS-HRM and direct BSP.

    Science.gov (United States)

    Akika, Reem; Awada, Zainab; Mogharbil, Nahed; Zgheib, Nathalie K

    2017-07-01

    The aim of this study was to compare and contrast three DNA methylation methods of a specific region of interest (ROI): methylation-specific PCR (MSP), methylation-sensitive high resolution melting (MS-HRM) and direct bisulfite sequencing (BSP). The methylation of a CpG area in the promoter region of Estrogen receptor alpha (ESR1) was evaluated by these three methods with samples and standards of different methylation percentages. MSP data were neither reproducible nor sensitive, and the assay was not specific due to non-specific binding of primers. MS-HRM was highly reproducible and a step forward into categorizing the methylation status of the samples as percent ranges. Direct BSP was the most informative method regarding methylation percentage of each CpG site. Though not perfect, it was reproducible and sensitive. We recommend the use of either method depending on the research question and target amplicon, and provided that the designed primers and expected amplicons are within recommendations. If the research question targets a limited number of CpG sites and simple yes/no results are enough, MSP may be attempted. For short amplicons that are crowded with CpG sites and of single melting domain, MS-HRM may be the method of choice though it only indicates the overall methylation percentage of the entire amplicon. Although the assay is highly reproducible, being semi-quantitative makes it of lesser interest to study ROI methylation of samples with little methylation differences. Direct BSP is a step forward as it gives information about the methylation percentage at each CpG site.

  10. Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes.

    Science.gov (United States)

    Rodríguez-Dorantes, M; Lizano-Soberón, M; Camacho-Arroyo, I; Calzada-León, R; Morimoto, S; Téllez-Ascencio, N; Cerbón, M A

    2002-03-01

    The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.

  11. DNA methylation of angiotensin II receptor gene in nonalcoholic steatohepatitis-related liver fibrosis.

    Science.gov (United States)

    Asada, Kiyoshi; Aihara, Yosuke; Takaya, Hiroaki; Noguchi, Ryuichi; Namisaki, Tadashi; Moriya, Kei; Uejima, Masakazu; Kitade, Mitsuteru; Mashitani, Tsuyoshi; Takeda, Kosuke; Kawaratani, Hideto; Okura, Yasushi; Kaji, Kosuke; Douhara, Akitoshi; Sawada, Yasuhiko; Nishimura, Norihisa; Seki, Kenichiro; Mitoro, Akira; Yamao, Junichi; Yoshiji, Hitoshi

    2016-10-08

    To clarify whether Agtr1a methylation is involved in the development of nonalcoholic steatohepatitis (NASH)-related liver fibrosis in adult rats. A choline-deficient amino acid (CDAA) diet model was employed for methylation analysis of NASH-related liver fibrosis. Agtr1a methylation levels were measured in the livers of CDAA- and control choline-sufficient amino acid (CSAA)-fed rats for 8 and 12 wk using quantitative methylation-specific PCR. Hepatic stellate cells (HSCs) were isolated by collagenase digestion of the liver, followed by centrifugation of the crude cell suspension through a density gradient. Agtr1a methylation and its gene expression were also analyzed during the activation of HSCs. The mean levels of Agtr1a methylation in the livers of CDAA-fed rats (11.5% and 18.6% at 8 and 12 wk, respectively) tended to be higher ( P = 0.06 and 0.09, respectively) than those in the livers of CSAA-fed rats (2.1% and 5.3% at 8 and 12 wk, respectively). Agtr1a was not methylated at all in quiescent HSCs, but was clearly methylated in activated HSCs (13.8%, P < 0.01). Interestingly, although Agtr1a was hypermethylated, the Agtr1a mRNA level increased up to 2.2-fold ( P < 0.05) in activated HSCs compared with that in quiescent HSCs, suggesting that Agtr1a methylation did not silence its expression but instead had the potential to upregulate its expression. These findings indicate that Agtr1a methylation and its upregulation of gene expression are associated with the development of NASH-related liver fibrosis. This is the first study to show that DNA methylation is potentially involved in the regulation of a renin-angiotensin system-related gene expression during liver fibrosis.

  12. Evaluation of folate receptor 1 (FOLR1) mRNA expression, its specific promoter methylation and global DNA hypomethylation in type I and type II ovarian cancers

    International Nuclear Information System (INIS)

    Notaro, Sara; Reimer, Daniel; Fiegl, Heidi; Schmid, Gabriel; Wiedemair, Annamarie; Rössler, Julia; Marth, Christian; Zeimet, Alain Gustave

    2016-01-01

    In this retrospective study we evaluated the respective correlations and clinical relevance of FOLR1 mRNA expression, FOLR1 promoter specific methylation and global DNA hypomethylation in type I and type II ovarian cancer. Two hundred fifty four ovarian cancers, 13 borderline tumours and 60 samples of healthy fallopian epithelium and normal ovarian epithelium were retrospectively analysed for FOLR1 expression with RT-PCR. FOLR1 DNA promoter methylation and global DNA hypomethylation (measured by means of LINE1 DNA hypomethylation) were evaluated with MethyLight technique. No correlation between FOLR1 mRNA expression and its specific promoter DNA methylation was found neither in type I nor in type II cancers, however, high FOLR1 mRNA expression was found to be correlated with global DNA hypomethylation in type II cancers (p = 0.033). Strong FOLR1 mRNA expression was revealed for Grades 2-3, FIGO stages III-IV, residual disease > 0, and serous histotype. High FOLR1 expression was found to predict increased platinum sensitivity in type I cancers (odds ratio = 3.288; 1.256-10.75; p = 0.020). One-year survival analysis showed in type I cancers an independent better outcome for strong expression of FOLR1 in FIGO stage III and IV. For the entire follow up period no significant independent outcome for FOLR1 expression was revealed. In type I cancers LINE 1 DNA hypomethylation was found to exhibit a worse PFS and OS which were confirmed to be independent in multivariate COX regression model for both PFS (p = 0.026) and OS (p = 0.012). No correlations were found between FOLR1 expression and its specific promoter methylation, however, high FOLR1 mRNA expression was associated with DNA hypomethylation in type II cancers. FOLR1 mRNA expression did not prove to predict clinical outcome in type II cancers, although strong FOLR1 expression generally denotes ovarian cancers with highly aggressive phenotype. In type I cancers, however, strong FOLR1 expression has been found to be a

  13. Are Independent Fiscal Institutions Really Independent?

    Directory of Open Access Journals (Sweden)

    Slawomir Franek

    2015-08-01

    Full Text Available In the last decade the number of independent fiscal institutions (known also as fiscal councils has tripled. They play an important oversight role over fiscal policy-making in democratic societies, especially as they seek to restore public finance stability in the wake of the recent financial crisis. Although common functions of such institutions include a role in analysis of fiscal policy, forecasting, monitoring compliance with fiscal rules or costing of spending proposals, their roles, resources and structures vary considerably across countries. The aim of the article is to determine the degree of independence of such institutions based on the analysis of the independence index of independent fiscal institutions. The analysis of this index values may be useful to determine the relations between the degree of independence of fiscal councils and fiscal performance of particular countries. The data used to calculate the index values will be derived from European Commission and IMF, which collect sets of information about characteristics of activity of fiscal councils.

  14. Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

    Science.gov (United States)

    Xiao, Zhujun; Li, Bingsheng; Wang, Guozhen; Zhu, Weisi; Wang, Zhongqiu; Lin, Jinfeng; Xu, Angao; Wang, Xinying

    2014-04-20

    Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (PHRM shows potential for CRC screening. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas

    2010-05-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  16. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas; Chang, Donald Choy; Sheng, Ping; Wen, Weijia; Wu, Jinbo; Xiao, Kang; Yu, Vivian

    2010-01-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  17. DNA methylation and gene expression of HIF3A

    DEFF Research Database (Denmark)

    Main, Ailsa Maria; Gillberg, Linn; Jacobsen, Anna Louisa

    2016-01-01

    from 48 families, from whom we had SAT and muscle biopsies. DNA methylation of four CpG sites in the HIF3A promoter was analyzed in the blood and SAT by pyrosequencing, and HIF3A gene expression was analyzed in SAT and muscle by qPCR. An index of whole-body insulin sensitivity was estimated from oral...... individuals, and whether HIF3A gene expression in SAT and skeletal muscle biopsies showed associations with BMI and insulin resistance. Furthermore, we aimed to investigate gender specificity and heritability of these traits. METHODS: We studied 137 first-degree relatives of type 2 diabetes (T2D) patients...... glucose tolerance tests. RESULTS: BMI was associated with HIF3A methylation at one CpG site in the blood, and there was a positive association between the blood and SAT methylation levels at a different CpG site within the individuals. The SAT methylation level did not correlate with HIF3A gene expression...

  18. Pathogen Causing Disease of Diagnosis PCR Tecnology

    OpenAIRE

    SEVİNDİK, Emre; KIR, A. Çağrı; BAŞKEMER, Kadir; UZUN, Veysel

    2013-01-01

    Polimerase chain reaction (PCR) with which, the development of recombinant DNA tecnology, a technique commonly used in field of moleculer biology and genetic. Duplication of the target DNA is provided with this technique without the need for cloning. Some fungus species, bacteria, viruses constitutent an important group of pathogenicity in human, animals and plants. There are routinely applied types of PCR in the detection of pathogens infections diseases. These Nested- PCR, Real- Time PCR, M...

  19. Transmission of epi-alleles with MET1-dependent dense methylation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Michael Watson

    Full Text Available DNA methylation in plants targets cytosines in three sequence contexts, CG, CHG and CHH (H representing A, C or T. Each of these patterns has traditionally been associated with distinct DNA methylation pathways with CHH methylation being controlled by the RNA dependent DNA methylation (RdDM pathway employing small RNAs as a guide for the de novo DOMAINS REARRANGED METHYLTRANSFERASE (DRM2, and maintenance DNA METHYLTRANSFERASE1 (MET1 being responsible for faithful propagation of CG methylation. Here we report an unusual 'dense methylation' pattern under the control of MET1, with methylation in all three sequence contexts. We identified epi-alleles of dense methylation at a non coding RNA locus (At4g15242 in Arabidopsis ecotypes, with distinct dense methylation and expression characteristics, which are stably maintained and transmitted in genetic crosses and which can be heritably altered by depletion of MET1. This suggests that, in addition to its classical CG maintenance function, at certain loci MET1 plays a role in creating transcriptional diversity based on the generation of independent epi-alleles. Database inspection identified several other loci with MET1-dependent dense methylation patterns. Arabidopsis ecotypes contain distinct epi-alleles of these loci with expression patterns that inversely correlate with methylation density, predominantly within the transcribed region. In Arabidopsis, dense methylation appears to be an exception as it is only found at a small number of loci. Its presence does, however, highlight the potential for MET1 as a contributor to epigenetic diversity, and it will be interesting to investigate the representation of dense methylation in other plant species.

  20. [Methylation of selected tumor-supressor genes in benign and malignant ovarian tumors].

    Science.gov (United States)

    Cul'bová, M; Lasabová, Z; Stanclová, A; Tilandyová, P; Zúbor, P; Fiolka, R; Danko, J; Visnovský, J

    2011-09-01

    To evaluate the usefullness of examination of methylation status of selected tumor-supressor genes in early diagnosis of ovarian cancer. Prospective clinical study. Department of Gynecology and Obstetrics, Department of Molecular Biology, Jessenius Medical Faculty, Commenius University, Martin, Slovak Republic. In this study we analyzed hypermethylation of 5 genes RASSF1A, GSTP, E-cadherin, p16 and APC in ovarian tumor samples from 34 patients - 13 patients with epithelial ovarian cancer, 2 patients with border-line ovarian tumors, 12 patients with benign lesions of ovaries and 7 patients with healthy ovarian tissue. The methylation status of promoter region of tumor-supressor genes was determined by Methylation Specific Polymerase Chain Reaction (MSP) using a nested two-step approach with bisulfite modified DNA template and specific primers. Gene methylation analysis revealed hypermethylation of gene RASSF1A (46%) and GSTP (8%) only in malignant ovarian tissue samples. Ecad, p16 and APC genes were methylated both in maignant and benign tissue samples. Methylation positivity in observed genes was present independently to all clinical stages of ovarian cancer and to tumor grades. However, there was observed a trend of increased number and selective involvement of methylated genes with increasing disease stages. Furthermore, there was no association between positive methylation status and histological subtypes of ovarian carcinomas. RASSF1A and GSTP promoter methylation positivity is associated with ovarian cancer. The revealed gene-selective methylation positivity and the increased number of methylated genes with advancing disease stages could be considered as a useful molecular marker for early detection of ovarian cancer. However, there is need to find diagnostic approach of specifically and frequently methylated genes to determining a methylation phenotype for early detection of ovarian malignancies.

  1. Principles and technical aspects of PCR amplification

    National Research Council Canada - National Science Library

    Pelt-Verkuil, Elizabeth van; Belkum, Alex van; Hays, John P

    2008-01-01

    ... to illustrate any particularly important concepts or comments. Indeed, all commercial PCR biotechnology companies offer information about their products on internet sites and in online technical manuals. These online resources will be invaluable for any readers requiring more detailed PCR protocols. The authors have provided references for many PCR co...

  2. The PCR revolution: basic technologies and applications

    National Research Council Canada - National Science Library

    Bustin, Stephen A

    2010-01-01

    ... by leading authorities on the many applications of PCR and how this technology has revolutionized their respective areas of interest. This book conveys the ways in which PCR has overcome many obstacles in life science and clinical research and also charts the PCR's development from time-consuming, low throughput, nonquantitative proced...

  3. Central Bank independence

    Directory of Open Access Journals (Sweden)

    Vasile DEDU

    2012-08-01

    Full Text Available In this paper we present the key aspects regarding central bank’s independence. Most economists consider that the factor which positively influences the efficiency of monetary policy measures is the high independence of the central bank. We determined that the National Bank of Romania (NBR has a high degree of independence. NBR has both goal and instrument independence. We also consider that the hike of NBR’s independence played an important role in the significant disinflation process, as headline inflation dropped inside the targeted band of 3% ± 1 percentage point recently.

  4. Highly frequent promoter methylation and PIK3CA amplification in non-small cell lung cancer (NSCLC)

    International Nuclear Information System (INIS)

    Ji, Meiju; Guan, Haixia; Gao, Cuixia; Shi, Bingyin; Hou, Peng

    2011-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Genetic and epigenetic alterations have been identified frequently in lung cancer, such as promoter methylation, gene mutations and genomic amplification. However, the interaction between genetic and epigenetic events and their significance in lung tumorigenesis remains poorly understood. We determined the promoter methylation of 6 genes and PIK3CA amplification using quantitative methylation-specific PCR (Q-MSP) and real-time quantitative PCR, respectively, and explore the association of promoter methylation with PIK3CA amplification in a large cohort of clinically well-characterized non-small cell lung cancer (NSCLC). Highly frequent promoter methylation was observed in NSCLC. With 100% diagnostic specificity, excellent sensitivity, ranging from 45.8 to 84.1%, was found for each of the 6 genes. The promoter methylation was associated with histologic type. Methylation of CALCA, CDH1, DAPK1, and EVX2 was more common in squamous cell carcinomas (SCC) compared to adenocarcinomas (ADC). Conversely, there was a trend toward a higher frequency of RASSF1A methylation in ADC than SCC. In addition, PIK3CA amplification was frequently found in NSCLC, and was associated with certain clinicopathologic features, such as smoking history, histologic type and pleural indentation. Importantly, aberrant promoter methylation of certain genes was significantly associated with PIK3CA amplification. Our data showed highly frequent promoter methylation and PIK3CA amplification in Chinese NSCLC population, and first demonstrated the associations of gene methylation with PIK3CA amplification, suggesting that these epigenetic events may be a consequence of overactivation of PI3K/Akt pathway

  5. Organizing Independent Student Work

    Directory of Open Access Journals (Sweden)

    Zhadyra T. Zhumasheva

    2015-03-01

    Full Text Available This article addresses issues in organizing independent student work. The author defines the term “independence”, discusses the concepts of independent learner work and independent learner work under the guidance of an instructor, proposes a classification of assignments to be done independently, and provides methodological recommendations as to the organization of independent student work. The article discusses the need for turning the student from a passive consumer of knowledge into an active creator of it, capable of formulating a problem, analyzing the ways of solving it, coming up with an optimum outcome, and proving its correctness. The preparation of highly qualified human resources is the primary condition for boosting Kazakhstan’s competitiveness. Independent student work is a means of fostering the professional competence of future specialists. The primary form of self-education is independent work.

  6. Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

    Science.gov (United States)

    Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

    2014-08-01

    Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

  7. Quantitative Detection of ID4 Gene Aberrant Methylation in the Differentiation of Myelodysplastic Syndrome from Aplastic Anemia

    Directory of Open Access Journals (Sweden)

    Mian-Yang Li

    2015-01-01

    Full Text Available Background: The diagnosis of myelodysplastic syndrome (MDS, especially hypoplastic MDS, and MDS with low blast counts or normal karyotype may be problematic. This study characterized ID4 gene methylation in patients with MDS and aplastic anemia (AA. Methods: The methylation status of ID4 was analyzed by bisulfite sequencing polymerase chain reaction (PCR and quantitative real-time methylation-specific PCR (MethyLight PCR in 100 patients with MDS and 31 patients with AA. Results: The MDS group had a higher ID4 gene methylation positivity rate (22.22% and higher methylation levels (0.21 [0-3.79] than the AA group (P < 0.05. Furthermore, there were significant differences between the hypoplastic MDS and AA groups, the MDS with low blast count and the AA groups, and the MDS with normal karyotype and the AA groups. The combination of genetic and epigenetic markers was used in much more patients with MDS (62.5% [35/56] than the use of genetic markers only (51.79% [29/56]. Conclusions: These results showed that the detection of ID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

  8. Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    Directory of Open Access Journals (Sweden)

    Schwarzenbach Heidi

    2010-06-01

    Full Text Available Abstract Background The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. Methods In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Results Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3 at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. Conclusions This study is one

  9. Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    International Nuclear Information System (INIS)

    Müller, Imke; Wischnewski, Frank; Pantel, Klaus; Schwarzenbach, Heidi

    2010-01-01

    The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs) and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3) at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. This study is one of the first to reveal the histone code and MBD profile

  10. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

    International Nuclear Information System (INIS)

    Prior, Sara; Miousse, Isabelle R.; Nzabarushimana, Etienne; Pathak, Rupak; Skinner, Charles; Kutanzi, Kristy R.; Allen, Antiño R.; Raber, Jacob; Tackett, Alan J.; Hauer-Jensen, Martin; Nelson, Gregory A.

    2016-01-01

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

  11. Densely ionizing radiation affects DNA methylation of selective LINE-1 elements

    Energy Technology Data Exchange (ETDEWEB)

    Prior, Sara; Miousse, Isabelle R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nzabarushimana, Etienne [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Department of Bioinformatics, School of Informatics and Computing, Indiana University, Bloomington, IN 47405 (United States); Pathak, Rupak [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Skinner, Charles; Kutanzi, Kristy R. [Department of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Allen, Antiño R. [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Raber, Jacob [Departments of Behavioral Neuroscience, Neurology, and Radiation Medicine, Division of Neuroscience, ONPRC, Oregon Health & Science University, Portland, OR 97239 (United States); Tackett, Alan J. [Department of Biochemistry, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Hauer-Jensen, Martin [Division of Radiation Health, Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205 (United States); Nelson, Gregory A. [Department of Basic Sciences, Division of Radiation Research, Loma Linda University, Loma Linda, CA 92350 (United States); and others

    2016-10-15

    Long Interspersed Nucleotide Element 1 (LINE-1) retrotransposons are heavily methylated and are the most abundant transposable elements in mammalian genomes. Here, we investigated the differential DNA methylation within the LINE-1 under normal conditions and in response to environmentally relevant doses of sparsely and densely ionizing radiation. We demonstrate that DNA methylation of LINE-1 elements in the lungs of C57BL6 mice is dependent on their evolutionary age, where the elder age of the element is associated with the lower extent of DNA methylation. Exposure to 5-aza-2′-deoxycytidine and methionine-deficient diet affected DNA methylation of selective LINE-1 elements in an age- and promoter type-dependent manner. Exposure to densely IR, but not sparsely IR, resulted in DNA hypermethylation of older LINE-1 elements, while the DNA methylation of evolutionary younger elements remained mostly unchanged. We also demonstrate that exposure to densely IR increased mRNA and protein levels of LINE-1 via the loss of the histone H3K9 dimethylation and an increase in the H3K4 trimethylation at the LINE-1 5′-untranslated region, independently of DNA methylation. Our findings suggest that DNA methylation is important for regulation of LINE-1 expression under normal conditions, but histone modifications may dictate the transcriptional activity of LINE-1 in response to exposure to densely IR. - Highlights: • DNA methylation of LINE-1 elements is dependent on their evolutionary age. • Densely ionizing radiation affects DNA methylation of selective LINE-1 elements. • Radiation-induced reactivation of LINE-1 is DNA methylation-independent. • Histone modifications dictate the transcriptional activity of LINE-1.

  12. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  13. Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

    Science.gov (United States)

    Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

    2005-01-01

    We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

  14. SLC9B1 methylation predicts fetal intolerance of labor.

    Science.gov (United States)

    Knight, Anna K; Conneely, Karen N; Kilaru, Varun; Cobb, Dawayland; Payne, Jennifer L; Meilman, Samantha; Corwin, Elizabeth J; Kaminsky, Zachary A; Dunlop, Anne L; Smith, Alicia K

    2018-01-01

    Fetal intolerance of labor is a common indication for delivery by Caesarean section. Diagnosis is based on the presence of category III fetal heart rate tracing, which is an abnormal heart tracing associated with increased likelihood of fetal hypoxia and metabolic acidemia. This study analyzed data from 177 unique women who, during their prenatal visits (7-15 weeks and/or 24-32 weeks) to Atlanta area prenatal care clinics, consented to provide blood samples for DNA methylation (HumanMethylation450 BeadChip) and gene expression (Human HT-12 v4 Expression BeadChip) analyses. We focused on 57 women aged 18-36 (mean 25.4), who had DNA methylation data available from their second prenatal visit. DNA methylation patterns at CpG sites across the genome were interrogated for associations with fetal intolerance of labor. Four CpG sites (P value intolerance of labor. DNA methylation and gene expression were negatively associated when examined longitudinally during pregnancy using a linear mixed-effects model. Positive predictive values of methylation of these four sites ranged from 0.80 to 0.89, while negative predictive values ranged from 0.91 to 0.92. The four CpG sites were also associated with fetal intolerance of labor in an independent cohort (the Johns Hopkins Prospective PPD cohort). Therefore, fetal intolerance of labor could be accurately predicted from maternal blood samples obtained between 24-32 weeks gestation. Fetal intolerance of labor may be accurately predicted from maternal blood samples obtained between 24-32 weeks gestation by assessing DNA methylation patterns of SLC9B1. The identification of pregnant women at elevated risk for fetal intolerance of labor may allow for the development of targeted treatments or management plans.

  15. Naturally occurring methyl salicylate glycosides.

    Science.gov (United States)

    Mao, Ping; Liu, Zizhen; Xie, Meng; Jiang, Rui; Liu, Weirui; Wang, Xiaohong; Meng, Shen; She, Gaimei

    2014-01-01

    As an important part of non steroids anti-inflammation drug (NSAIDs), salicylate has developed from natural substance salicylic acid to natrium salicylicum, to aspirin. Now, methyl salicylate glycoside, a new derivative of salicylic acid, is modified with a -COOH group integrated one methyl radical into formic ether, and a -OH linked with a monosaccharide, a disaccharide or a trisaccharide unit by glycosidic linkage. It has the similar pharmacological activities, anti-inflammatory, analgesic, antipyretic and antithrombotic as the previous salicylates' without resulting in serious side effects, particularly the gastrointestinal toxicity. Owing to the superiority of those significant bioactivities, methyl salicylate glycosides have became a hot research area in NSAIDs for several years. This paper compiles all 9 naturally occurring methyl salicylate glycosides, their distribution of the resource and pharmacological mechanism, which could contribute to the new drug discovery.

  16. Prediction of methyl-side Chain Dynamics in Proteins

    International Nuclear Information System (INIS)

    Ming Dengming; Brueschweiler, Rafael

    2004-01-01

    A simple analytical model is presented for the prediction of methyl-side chain dynamics in comparison with S 2 order parameters obtained by NMR relaxation spectroscopy. The model, which is an extension of the local contact model for backbone order parameter prediction, uses a static 3D protein structure as input. It expresses the methyl-group S 2 order parameters as a function of local contacts of the methyl carbon with respect to the neighboring atoms in combination with the number of consecutive mobile dihedral angles between the methyl group and the protein backbone. For six out of seven proteins the prediction results are good when compared with experimentally determined methyl-group S 2 values with an average correlation coefficient r-bar=0.65±0.14. For the unusually rigid cytochrome c 2 no significant correlation between prediction and experiment is found. The presented model provides independent support for the reliability of current side-chain relaxation methods along with their interpretation by the model-free formalism

  17. Differential DNA Methylation Analysis without a Reference Genome

    Directory of Open Access Journals (Sweden)

    Johanna Klughammer

    2015-12-01

    Full Text Available Genome-wide DNA methylation mapping uncovers epigenetic changes associated with animal development, environmental adaptation, and species evolution. To address the lack of high-throughput methods for DNA methylation analysis in non-model organisms, we developed an integrated approach for studying DNA methylation differences independent of a reference genome. Experimentally, our method relies on an optimized 96-well protocol for reduced representation bisulfite sequencing (RRBS, which we have validated in nine species (human, mouse, rat, cow, dog, chicken, carp, sea bass, and zebrafish. Bioinformatically, we developed the RefFreeDMA software to deduce ad hoc genomes directly from RRBS reads and to pinpoint differentially methylated regions between samples or groups of individuals (http://RefFreeDMA.computational-epigenetics.org. The identified regions are interpreted using motif enrichment analysis and/or cross-mapping to annotated genomes. We validated our method by reference-free analysis of cell-type-specific DNA methylation in the blood of human, cow, and carp. In summary, we present a cost-effective method for epigenome analysis in ecology and evolution, which enables epigenome-wide association studies in natural populations and species without a reference genome.

  18. Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

    Science.gov (United States)

    Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

    2018-01-01

    Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

  19. Methylation of MGMT Is Associated with Poor Prognosis in Patients with Stage III Duodenal Adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Tao Fu

    Full Text Available O6-methylguanine-DNA methyltransferase (MGMT methylation status has not been extensively investigated in duodenal adenocarcinoma (DA. The aim of this study was to evaluate the MGMT methylation status and examine its possible prognostic value in patients with stage III DA.Demographics, tumor characteristics and survival were available for 64 patients with stage III DA. MGMT methylation was detected by using MethyLight. A Cox proportional hazard model was built to predict survival, adjusted for clinicopathological characteristics and tumor molecular features, including the CpG island methylator phenotype (CIMP, microsatellite instability (MSI, and KRAS mutations.MGMT methylation was detected in 17 of 64 (26.6% patients, and was not correlated with sex, age, tumor differentiation, CIMP, MSI, or KRAS mutations. MGMT methylation was the only one factor associated with both overall survival (OS and disease-free survival (DFS on both univariate and multivariate analyses. In patients treated with surgery alone, MGMT-methylated group had worse OS and DFS when compared with MGMT-unmethylated group. However, in patients treated with chemotherapy/radiotherapy, outcomes became comparable between the two groups.Our results demonstrate MGMT methylation is a reliable and independent prognostic factor in DAs. Methylation of MGMT is associated with poor prognosis in patients with stage III DAs.

  20. Promoter DNA methylation pattern identifies prognostic subgroups in childhood T-cell acute lymphoblastic leukemia.

    Directory of Open Access Journals (Sweden)

    Magnus Borssén

    Full Text Available BACKGROUND: Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in T-ALL is hitherto undecided. DESIGN AND METHODS: Genome wide promoter DNA methylation analysis was performed in pediatric T-ALL samples (n = 43 using arrays covering >27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary T-ALL group not tested for methylation (n = 32. RESULTS: Based on CpG island methylator phenotype (CIMP, T-ALL samples were subgrouped as CIMP+ (high methylation and CIMP- (low methylation. CIMP- T-ALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. CONCLUSIONS: We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric T-ALL.

  1. Process for the production of methyl methacrylate

    NARCIS (Netherlands)

    Eastham, G.R.; Johnson, D.W.; Straathof, A.J.J.; Fraaije, Marco; Winter, Remko

    2015-01-01

    A process of producing methyl methacrylate or derivatives thereof is described. The process includes the steps of; (i) converting 2-butanone to methyl propionate using a Baeyer-Villiger monooxygenase, and (ii) treating the methyl propionate produced to obtain methyl methacrylate or derivatives

  2. Genome-wide methylation analysis identifies genes silenced in non-seminoma cell lines.

    Science.gov (United States)

    Noor, Dzul Azri Mohamed; Jeyapalan, Jennie N; Alhazmi, Safiah; Carr, Matthew; Squibb, Benjamin; Wallace, Claire; Tan, Christopher; Cusack, Martin; Hughes, Jaime; Reader, Tom; Shipley, Janet; Sheer, Denise; Scotting, Paul J

    2016-01-01

    Silencing of genes by DNA methylation is a common phenomenon in many types of cancer. However, the genome-wide effect of DNA methylation on gene expression has been analysed in relatively few cancers. Germ cell tumours (GCTs) are a complex group of malignancies. They are unique in developing from a pluripotent progenitor cell. Previous analyses have suggested that non-seminomas exhibit much higher levels of DNA methylation than seminomas. The genomic targets that are methylated, the extent to which this results in gene silencing and the identity of the silenced genes most likely to play a role in the tumours' biology have not yet been established. In this study, genome-wide methylation and expression analysis of GCT cell lines was combined with gene expression data from primary tumours to address this question. Genome methylation was analysed using the Illumina infinium HumanMethylome450 bead chip system and gene expression was analysed using Affymetrix GeneChip Human Genome U133 Plus 2.0 arrays. Regulation by methylation was confirmed by demethylation using 5-aza-2-deoxycytidine and reverse transcription-quantitative PCR. Large differences in the level of methylation of the CpG islands of individual genes between tumour cell lines correlated well with differential gene expression. Treatment of non-seminoma cells with 5-aza-2-deoxycytidine verified that methylation of all genes tested played a role in their silencing in yolk sac tumour cells and many of these genes were also differentially expressed in primary tumours. Genes silenced by methylation in the various GCT cell lines were identified. Several pluripotency-associated genes were identified as a major functional group of silenced genes.

  3. Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

    Science.gov (United States)

    Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

    2006-02-09

    Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

  4. [GSTP1, APC and RASSF1 gene methylation in prostate cancer samples: comparative analysis of MS-HRM method and Infinium HumanMethylation450 BeadChip beadchiparray diagnostic value].

    Science.gov (United States)

    Skorodumova, L O; Babalyan, K A; Sultanov, R; Vasiliev, A O; Govorov, A V; Pushkar, D Y; Prilepskaya, E A; Danilenko, S A; Generozov, E V; Larin, A K; Kostryukova, E S; Sharova, E I

    2016-11-01

    There is a clear need in molecular markers for prostate cancer (PC) risk stratification. Alteration of DNA methylation is one of processes that occur during ÐÑ progression. Methylation-sensitive PCR with high resolution melting curve analysis (MS-HRM) can be used for gene methylation analysis in routine laboratory practice. This method requires very small amounts of DNA for analysis. Numerous results have been accumulated on DNA methylation in PC samples analyzed by the Infinium HumanMethylation450 BeadChip (HM450). However, the consistency of MS-HRM results with chip hybridization results has not been examined yet. The aim of this study was to assess the consistency of results of GSTP1, APC and RASSF1 gene methylation analysis in ÐÑ biopsy samples obtained by MS-HRM and chip hybridization. The methylation levels of each gene determined by MS-HRM were statistically different in the group of PC tissue samples and the samples without signs of tumor growth. Chip hybridization data analysis confirmed the results obtained with the MS-HRM. Differences in methylation levels between tumor tissue and histologically intact tissue of each sample determined by MS-HRM and chip hybridization, were consistent with each other. Thus, we showed that the assessment of GSTP1, APC and RASSF1 gene methylation analysis using MS-HRM is suitable for the design of laboratory assays that will differentiate the PC tissue from the tissue without signs of tumor growth.

  5. Protective effects of folic acid on DNA damage and DNA methylation levels induced by N-methyl- N'-nitro- N-nitrosoguanidine in Kazakh esophageal epithelial cells.

    Science.gov (United States)

    Chen, Y; Feng, H; Chen, D; Abuduwaili, K; Li, X; Zhang, H

    2018-01-01

    The protective effects of folic acid on DNA damage and DNA methylation induced by N-methyl- N'-nitro- N-nitrosoguanidine (MNNG) in Kazakh esophageal epithelial cells were investigated using a 3 × 3 factorial design trial. The cells were cultured in vitro and exposed to media containing different concentrations of folic acid and MNNG, after which growth indices were detected. DNA damage levels were measured using comet assays, and genome-wide DNA methylation levels (MLs) were measured using high-performance liquid chromatography. The DNA methylation of methylenetetrahydrofolate reductase (MTHFR) and folate receptor- α (FR α) genes was detected by bisulfite sequencing polymerase chain reaction (PCR). The results showed significant increases in tail DNA concentration, tail length, and Olive tail moment ( p methylation frequencies of MTHFR and FR α genes. In particular, significant differences were observed in the promoter regions of both genes ( p methylation in Kazakh esophageal epithelial cells upon MNNG exposure. Thus, sufficient folic acid levels could play a protective role against the damage induced by this compound.

  6. Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    You Wanhui

    2012-04-01

    Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

  7. Characterization of the Methylation Status of Pax7 and Myogenic Regulator Factors in Cell Myogenic Differentiation.

    Science.gov (United States)

    Chao, Zhe; Zheng, Xin-Li; Sun, Rui-Ping; Liu, Hai-Long; Huang, Li-Li; Cao, Zong-Xi; Deng, Chang-Yan; Wang, Feng

    2016-07-01

    Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

  8. Accounting for Independent Schools.

    Science.gov (United States)

    Sonenstein, Burton

    The diversity of independent schools in size, function, and mode of operation has resulted in a considerable variety of accounting principles and practices. This lack of uniformity has tended to make understanding, evaluation, and comparison of independent schools' financial statements a difficult and sometimes impossible task. This manual has…

  9. Differential SLC1A2 Promoter Methylation in Bipolar Disorder With or Without Addiction

    Directory of Open Access Journals (Sweden)

    Yun-Fang Jia

    2017-07-01

    Full Text Available While downregulation of excitatory amino acid transporter 2 (EAAT2, the main transporter removing glutamate from the synapse, has been recognized in bipolar disorder (BD, the underlying mechanisms of downregulation have not been elucidated. BD is influenced by environmental factors, which may, via epigenetic modulation of gene expression, differentially affect illness presentation. This study thus focused on epigenetic DNA methylation regulation of SLC1A2, encoding for EAAT2, in BD with variable environmental influences of addiction. High resolution melting PCR (HRM-PCR and thymine–adenine (TA cloning with sequence analysis were conducted to examine methylation of the promoter region of the SLC1A2. DNA was isolated from blood samples drawn from BD patients (N = 150 with or without addiction to alcohol, nicotine, or food, defined as binge eating, and matched controls (N = 32. In comparison to controls, the SLC1A2 promoter region was hypermethylated in BD without addiction but was hypomethylated in BD with addiction. After adjusting for age and sex, the association of methylation levels with nicotine addiction (p = 0.0009 and binge eating (p = 0.0002 remained significant. Consistent with HRM-PCR, direct sequencing revealed increased methylation in CpG site 6 in BD, but decreased methylation in three CpG sites (6, 48, 156 in BD with alcohol and nicotine addictions. These results suggest that individual point methylation within the SLC1A2 promoter region may be modified by exogenous addiction and may have a potential for developing clinically valuable epigenetic biomarkers for BD diagnosis and monitoring.

  10. Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

    International Nuclear Information System (INIS)

    Schneider, Katja U; Liebenberg, Volker; Kneip, Christoph; Seegebarth, Anke; Erdogan, Fikret; Rappold, Gudrun; Schmidt, Bernd; Dietrich, Dimo; Fleischhacker, Michael; Leschber, Gunda; Merk, Johannes; Schäper, Frank; Stapert, Henk R; Vossenaar, Erik R; Weickmann, Sabine

    2011-01-01

    DNA methylation in the SHOX2 locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. SHOX2 expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. A hypermethylation of the SHOX2 locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the SHOX2 gene showed no difference. Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples

  11. Methylation-associated down-regulation of RASSF1A and up-regulation of RASSF1C in pancreatic endocrine tumors

    International Nuclear Information System (INIS)

    Malpeli, Giorgio; Amato, Eliana; Dandrea, Mario; Fumagalli, Caterina; Debattisti, Valentina; Boninsegna, Letizia; Pelosi, Giuseppe; Falconi, Massimo; Scarpa, Aldo

    2011-01-01

    RASSF1A gene silencing by DNA methylation has been suggested as a major event in pancreatic endocrine tumor (PET) but RASSF1A expression has never been studied. The RASSF1 locus contains two CpG islands (A and C) and generates seven transcripts (RASSF1A-RASSF1G) by differential promoter usage and alternative splicing. We studied 20 primary PETs, their matched normal pancreas and three PET cell lines for the (i) methylation status of the RASSF1 CpG islands using methylation-specific PCR and pyrosequencing and (ii) expression of RASSF1 isoforms by quantitative RT-PCR in 13 cases. CpG island A methylation was evaluated by methylation-specific PCR (MSP) and by quantitative methylation-specific PCR (qMSP); pyrosequencing was applied to quantify the methylation of 51 CpGs also encompassing those explored by MSP and qMSP approaches. MSP detected methylation in 16/20 (80%) PETs and 13/20 (65%) normal pancreas. At qMSP, 11/20 PETs (55%) and 9/20 (45%) normals were methylated in at least 20% of RASSF1A alleles. Pyrosequencing showed variable distribution and levels of methylation within and among samples, with PETs having average methylation higher than normals in 15/20 (75%) cases (P = 0.01). The evaluation of mRNA expression of RASSF1 variants showed that: i) RASSF1A was always expressed in PET and normal tissues, but it was, on average, expressed 6.8 times less in PET (P = 0.003); ii) RASSF1A methylation inversely correlated with its expression; iii) RASSF1 isoforms were rarely found, except for RASSF1B that was always expressed and RASSF1C whose expression was 11.4 times higher in PET than in normal tissue (P = 0.001). A correlation between RASSF1A expression and gene methylation was found in two of the three PET cell lines, which also showed a significant increase in RASSF1A expression upon demethylating treatment. RASSF1A gene methylation in PET is higher than normal pancreas in no more than 75% of cases and as such it cannot be considered a marker for this neoplasm

  12. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2012-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

  13. DNA Methylation Changes in the IGF1R Gene in Birth Weight Discordant Adult Monozygotic Twins

    DEFF Research Database (Denmark)

    Tsai, Pei-Chien; Van Dongen, Jenny; Tan, Qihua

    2015-01-01

    persists into adulthood. To investigate this further, we performed epigenome-wide association analyses of blood DNA methylation using Infinium HumanMethylation450 BeadChip profiles in 71 adult monozygotic (MZ) twin pairs who were extremely discordant for birth weight. A signal mapping to the IGF1R gene (cg...... were not significant. However, a meta-analysis across the four independent samples, in total 216 birth-weight discordant MZ twin pairs, showed a significant positive association between birth weight and DNA methylation differences at IGF1R (random-effects meta-analysis p = .04), and the effect...... was particularly pronounced in older twins (random-effects meta-analysis p = .008, 98 older birth-weight discordant MZ twin pairs). The results suggest that severe intra-uterine growth differences (birth weight discordance >20%) are associated with methylation changes in the IGF1R gene in adulthood, independent...

  14. Fluorescence-labeled methylation-sensitive amplified fragment length polymorphism (FL-MS-AFLP) analysis for quantitative determination of DNA methylation and demethylation status.

    Science.gov (United States)

    Kageyama, Shinji; Shinmura, Kazuya; Yamamoto, Hiroko; Goto, Masanori; Suzuki, Koichi; Tanioka, Fumihiko; Tsuneyoshi, Toshihiro; Sugimura, Haruhiko

    2008-04-01

    The PCR-based DNA fingerprinting method called the methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis is used for genome-wide scanning of methylation status. In this study, we developed a method of fluorescence-labeled MS-AFLP (FL-MS-AFLP) analysis by applying a fluorescence-labeled primer and fluorescence-detecting electrophoresis apparatus to the existing method of MS-AFLP analysis. The FL-MS-AFLP analysis enables quantitative evaluation of more than 350 random CpG loci per run. It was shown to allow evaluation of the differences in methylation level of blood DNA of gastric cancer patients and evaluation of hypermethylation and hypomethylation in DNA from gastric cancer tissue in comparison with adjacent non-cancerous tissue.

  15. [Inactivation of PMS2 gene by promoter methylation in nasopharyngeal carcinoma].

    Science.gov (United States)

    Ni, H F; Jiang, B; Zhou, Z; Li, Y; Yuan, X Y; Cao, X L; Huang, G W

    2016-11-23

    Objective: To investigate the inactivation of PMS2 gene mediated by promoter methylation and its regulatory mechanism in nasopharyngeal carcinoma (NPC). Methods: Fifty-four NPC tissues, 16 normal nasopharyngeal epithelia (NNE), 5 NPC cell lines (CNE1, CNE2, TWO3, HNE1 and HONE1) and 1 normal nasopharyngeal epithelial cell line (NP69) were collected.Methylation-specific PCR (MSP) was used to detect the PMS2 promoter methylation, semi-quantitative reverse transcription PCR (qRT-PCR) was applied to determine its mRNA expression, and immunohistochemistry (IHC) was used to detect the protein expression of PMS2. The expressions of PMS2 mRNA in CNE1 and CNE2 cells before and after treated with methyltransferase inhibitor 5-aza-2-deoxycytidine were analyzed by qRT-PCR. The impact of methylation and demethylation on the mRNA expression of PMS2, and the association of mRNA and protein expression of PMS2 with clinicopathological features of nasopharyngeal cancer were analyzed. Results: Methylation of PMS2 gene was detected in all of the five NPC cell lines, but not in normal nasopharyngeal epithelial NP69 cells. The methylation rate of PMS2 gene in NPC tissues was 63% (34/54), significantly higher than that of the normal nasopharyngeal epithelia (0/16, P PMS2 mRNA and protein were significantly down-regulated in the 54 NPC tissues when compared with those in the 16 NNE tissues ( P PMS2 mRNA was restored in the CNE1 and CNE2 cells.However, the expressions of PMS2 mRNA and protein were not significantly correlated with patients' age, gender, TNM stage, histopathologic type or lymph node metastasis ( P >0.05 for all). Conclusions: Promoter methylation-mediated inactivation of PMS2 gene participates in carcinogenesis and development of NPC. PMS2 may be a candidate tumor suppressor in the treatment for patients with inactivation of PMS2 promoter methylation.

  16. A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

    Science.gov (United States)

    Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

    2017-10-07

    We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

  17. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Directory of Open Access Journals (Sweden)

    Livia Schrick

    2016-01-01

    Full Text Available PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  18. Absolute quantification by droplet digital PCR versus analog real-time PCR

    Science.gov (United States)

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  19. Expression and promoter DNA methylation of MLH1 in colorectal cancer and lung cancer.

    Science.gov (United States)

    Ma, Yunxia; Chen, Yuan; Petersen, Iver

    2017-04-01

    Aberrant DNA methylation is a common molecular feature in human cancer. The aims of this study were to analyze the methylation status of MLH1, one of the DNA mismatch repair (MMR) genes, in human colorectal and lung cancer and to evaluate its clinical relevance. The expression of MLH1 was analyzed in 8 colorectal cancer (CRC) and 8 lung cancer cell lines by real-time RT-PCR and western blotting. The MLH1 protein expression was evaluated by immunohistochemistry on tissue microarrays including 121 primary CRC and 90 lung cancer patient samples. In cancer cell lines, the methylation status of MLH1 promoter and exon 2 was investigated by bisulfite sequencing (BS). Methylation-specific-PCR (MSP) was used to evaluate methylation status of MLH1. The expression of MLH1 mRNA was detected in 8 CRC cell lines as well as normal colonic fibroblast cells CCD-33Co. At protein levels, MLH1 was lost in one CRC cell line HCT-116 and normal cells CCD-33Co. No methylation was found in the promoter and exon 2 of MLH1 in CRC cell lines. MLH1 was expressed in 8 lung cancer cell lines at both mRNA and protein levels. Compared to cancer cells, normal bronchial epithelial cells (HBEC) had lower expression of MLH1 protein. In primary CRC, 54.5% of cases exhibited positive staining, while 47.8% of lung tumors were positive for MLH1 protein. MSP analysis showed that 58 out of 92 (63.0%) CRC and 41 out of 73 (56.2%) lung cancer exhibited MLH1 methylation. In CRC, the MLH1 methylation was significantly associated with tumor invasion in veins (P=0.012). However, no significant links were found between MLH1 expression and promoter methylation in both tumor entities. MLH1 methylation is a frequent molecular event in CRC and lung cancer patients. In CRC, methylation of MLH1 could be linked to vascular invasiveness. Copyright © 2017 Elsevier GmbH. All rights reserved.

  20. Methylated Glutathione S-transferase 1 (mGSTP1) is a potential plasma free DNA epigenetic marker of prognosis and response to chemotherapy in castrate-resistant prostate cancer.

    Science.gov (United States)

    Mahon, K L; Qu, W; Devaney, J; Paul, C; Castillo, L; Wykes, R J; Chatfield, M D; Boyer, M J; Stockler, M R; Marx, G; Gurney, H; Mallesara, G; Molloy, P L; Horvath, L G; Clark, S J

    2014-10-28

    Glutathione S-transferase 1 (GSTP1) inactivation is associated with CpG island promoter hypermethylation in the majority of prostate cancers (PCs). This study assessed whether the level of circulating methylated GSTP1 (mGSTP1) in plasma DNA is associated with chemotherapy response and overall survival (OS). Plasma samples were collected prospectively from a Phase I exploratory cohort of 75 men with castrate-resistant PC (CRPC) and a Phase II independent validation cohort (n=51). mGSTP1 levels in free DNA were measured using a sensitive methylation-specific PCR assay. The Phase I cohort identified that detectable baseline mGSTP1 DNA was associated with poorer OS (HR, 4.2 95% CI 2.1-8.2; P<0.0001). A decrease in mGSTP1 DNA levels after cycle 1 was associated with a PSA response (P=0.008). In the Phase II cohort, baseline mGSTP1 DNA was a stronger predictor of OS than PSA change after 3 months (P=0.02). Undetectable plasma mGSTP1 after one cycle of chemotherapy was associated with PSA response (P=0.007). We identified plasma mGSTP1 DNA as a potential prognostic marker in men with CRPC as well as a potential surrogate therapeutic efficacy marker for chemotherapy and corroborated these findings in an independent Phase II cohort. Prospective Phase III assessment of mGSTP1 levels in plasma DNA is now warranted.

  1. Reduced DNA methylation of FKBP5 in Cushing's syndrome.

    Science.gov (United States)

    Resmini, Eugenia; Santos, Alicia; Aulinas, Anna; Webb, Susan M; Vives-Gilabert, Yolanda; Cox, Olivia; Wand, Gary; Lee, Richard S

    2016-12-01

    FKBP5 encodes a co-chaperone of HSP90 protein that regulates intracellular glucocorticoid receptor sensitivity. When it is bound to the glucocorticoid receptor complex, cortisol binds with lower affinity to glucocorticoid receptor. Cushing's syndrome is associated with memory deficits, smaller hippocampal volumes, and wide range of cognitive impairments. We aimed at evaluating blood DNA methylation of FKBP5 and its relationship with memory and hippocampal volumes in Cushing's syndrome patients. Polymorphism rs1360780 in FKBP5 has also been assessed to determine whether genetic variations can also govern CpG methylation. Thirty-two Cushing's syndrome patients and 32 matched controls underwent memory tests, 3-Tesla MRI of the brain, and DNA extraction from total leukocytes. DNA samples were bisulfite treated, PCR amplified, and pyrosequenced to assess a total of 41CpG-dinucleotides in the introns 1, 2, 5, and 7 of FKBP5. Significantly lower intronic FKBP5 DNA methylation in CS patients compared to controls was observed in ten CpG-dinucleotides. DNA methylation at these CpGs correlated with left and right HV (Intron-2-Region-2-CpG-3: LHV, r = 0.73, p = 0.02; RHV, r = 0.58, p = 0.03). Cured and active CS patients showed both lower methylation of intron 2 (92.37, 91.8, and 93.34 %, respectively, p = 0.03 for both) and of intron 7 (77.08, 73.74, and 79.71 %, respectively, p = 0.02 and p < 0.01) than controls. Twenty-two subjects had the CC genotype, 34 had the TC genotype, and eight had the TT genotype. Lower average DNA methylation in intron 7 was observed in the TT subjects compared to CC (72.5vs. 79.5 %, p = 0.02) and to TC (72.5 vs. 79.0 %, p = 0.03). Our data demonstrate, for the first time, a reduction of intronic DNA methylation of FKBP5 in CS patients.

  2. Probabilistic conditional independence structures

    CERN Document Server

    Studeny, Milan

    2005-01-01

    Probabilistic Conditional Independence Structures provides the mathematical description of probabilistic conditional independence structures; the author uses non-graphical methods of their description, and takes an algebraic approach.The monograph presents the methods of structural imsets and supermodular functions, and deals with independence implication and equivalence of structural imsets.Motivation, mathematical foundations and areas of application are included, and a rough overview of graphical methods is also given.In particular, the author has been careful to use suitable terminology, and presents the work so that it will be understood by both statisticians, and by researchers in artificial intelligence.The necessary elementary mathematical notions are recalled in an appendix.

  3. Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours.

    Science.gov (United States)

    Kajiura, Koichiro; Takizawa, Hiromitsu; Morimoto, Yuki; Masuda, Kiyoshi; Tsuboi, Mitsuhiro; Kishibuchi, Reina; Wusiman, Nuliamina; Sawada, Toru; Kawakita, Naoya; Toba, Hiroaki; Yoshida, Mitsuteru; Kawakami, Yukikiyo; Naruto, Takuya; Imoto, Issei; Tangoku, Akira; Kondo, Kazuya

    2017-09-01

    Aberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Genome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. We identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. RASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Methylation and silencing of the retinoic acid receptor-β2 gene in cervical cancer

    International Nuclear Information System (INIS)

    Ivanova, Tatyana; Petrenko, Anatolii; Gritsko, Tatyana; Vinokourova, Svetlana; Eshilev, Ernest; Kobzeva, Vera; Kisseljov, Fjodor; Kisseljova, Natalia

    2002-01-01

    Expression of the retinoic acid receptor β2 (RAR-β2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-β2 expression remains obscure. We examined whether methylation of RAR-β2 gene could be responsible for this silencing in cervical SCC. Expression of RAR-β2 mRNA and methylation status of the 5' region of RAR-β2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively. In 8 out 20 cervical SCC (40%) the levels of RAR-β2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-β2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-β2 transcript. The RAR-β2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-β2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-β2 gene. These findings suggest that methylation of the 5' region of RAR-β2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation

  5. Altered mucosal DNA methylation in parallel with highly active Helicobacter pylori-related gastritis.

    Science.gov (United States)

    Yoshida, Takeichi; Kato, Jun; Maekita, Takao; Yamashita, Satoshi; Enomoto, Shotaro; Ando, Takayuki; Niwa, Tohru; Deguchi, Hisanobu; Ueda, Kazuki; Inoue, Izumi; Iguchi, Mikitaka; Tamai, Hideyuki; Ushijima, Toshikazu; Ichinose, Masao

    2013-10-01

    Chronic inflammation triggered by Helicobacter pylori causes altered DNA methylation in stomach mucosae, which is deeply involved in gastric carcinogenesis. This study aimed to elucidate the correlation between altered mucosal DNA methylation levels and activity of H. pylori-related gastritis, because inflammatory activity shows particular correlations with the development of diffuse-type cancer. Methylation levels in stomach mucosae of 78 healthy volunteers were determined by real-time methylation-specific PCR or bisulfite pyrosequencing. Examined loci were the promoter CpG islands of six genes (FLNc, HAND1, THBD, p41ARC, HRASLS, and LOX) and the CpG sites of non-coding repetitive elements (Alu and Satα) that are reportedly altered by H. pylori infection. Activity of H. pylori-related gastritis was evaluated using two serum markers: H. pylori antibody titer and pepsinogen II. Methylation levels of the six CpG islands were consistently increased, and those of the two repetitive elements were consistently decreased in a stepwise manner with the activity of gastric inflammation as represented by serum marker levels. Each serum marker level was well correlated with the overall DNA methylation status of stomach mucosa, and these two serologic markers were additive in the detection of the mucosa with severely altered DNA methylation. Alteration in mucosal DNA methylation level was closely correlated with activity of H. pylori-related gastritis as evaluated by serum markers. The observed correlation between altered DNA methylation levels and activity of H. pylori-related gastritis appears to be one of the relevant molecular mechanisms underlying the development of diffuse-type cancer.

  6. In silico mining identifies IGFBP3 as a novel target of methylation in prostate cancer.

    LENUS (Irish Health Repository)

    Perry, A S

    2007-05-21

    Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5\\' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49\\/79 primary prostate adenocarcinoma and 7\\/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5\\/37 benign prostatic hyperplasia (P < 0.0001) and in 0\\/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.

  7. Parvovirus b19 DNA CpG dinucleotide methylation and epigenetic regulation of viral expression.

    Directory of Open Access Journals (Sweden)

    Francesca Bonvicini

    Full Text Available CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression.The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections.The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19.

  8. Parvovirus B19 DNA CpG Dinucleotide Methylation and Epigenetic Regulation of Viral Expression

    Science.gov (United States)

    Bonvicini, Francesca; Manaresi, Elisabetta; Di Furio, Francesca; De Falco, Luisa; Gallinella, Giorgio

    2012-01-01

    CpG DNA methylation is one of the main epigenetic modifications playing a role in the control of gene expression. For DNA viruses whose genome has the ability to integrate in the host genome or to maintain as a latent episome, a correlation has been found between the extent of DNA methylation and viral quiescence. No information is available for Parvovirus B19, a human pathogenic virus, which is capable of both lytic and persistent infections. Within Parvovirus B19 genome, the inverted terminal regions display all the characteristic signatures of a genomic CpG island; therefore we hypothesised a role of CpG dinucleotide methylation in the regulation of viral genome expression. The analysis of CpG dinucleotide methylation of Parvovirus B19 DNA was carried out by an aptly designed quantitative real-time PCR assay on bisulfite-modified DNA. The effects of CpG methylation on the regulation of viral genome expression were first investigated by transfection of either unmethylated or in vitro methylated viral DNA in a model cell line, showing that methylation of viral DNA was correlated to lower expression levels of the viral genome. Then, in the course of in vitro infections in different cellular environments, it was observed that absence of viral expression and genome replication were both correlated to increasing levels of CpG methylation of viral DNA. Finally, the presence of CpG methylation was documented in viral DNA present in bioptic samples, indicating the occurrence and a possible role of this epigenetic modification in the course of natural infections. The presence of an epigenetic level of regulation of viral genome expression, possibly correlated to the silencing of the viral genome and contributing to the maintenance of the virus in tissues, can be relevant to the balance and outcome of the different types of infection associated to Parvovirus B19. PMID:22413013

  9. Melatonin-Mediated Development of Ovine Cumulus Cells, Perhaps by Regulation of DNA Methylation

    Directory of Open Access Journals (Sweden)

    Yi Fang

    2018-02-01

    Full Text Available Cumulus cells of pre-pubertal domestic animals are dysfunctional, perhaps due to age-specific epigenetic events. This study was designed to determine effects of melatonin treatment of donors on methylation modification of pre-pubertal cumulus cells. Cumulus cells from germinal vesicle stage cumulus oocyte complexes (COCs were collected from eighteen lambs which were randomly divided into control group (C and melatonin group given an 18 mg melatonin implant subcutaneous (M. Compared to the C group, the M group had higher concentrations of melatonin in plasma and follicular fluid (p < 0.05, greater superovulation, a higher proportion of fully expanded COCs, and a lower proportion of apoptotic cumulus cells (p < 0.05. Real-time PCR results showed that melatonin up-regulated expression of genes MT1, Bcl2, DNMT1, DNMT3a and DNMT3b, but down-regulated expression of genes p53, Caspase 3 and Bax (p < 0.05. Furthermore, melatonin increased FI of FITC (global methylation level on cumulus cells (p < 0.05. To understand the regulation mechanism, the DNMTs promoter methylation sequence were analyzed. Compared to the C group, although there was less methylation at two CpG sites of DNMT1 (p < 0.05 and higher methylation at two CpG sites of DNMT3a (p < 0.05, there were no significant differences in methylation of the detected DNMT1 and DNMT3a promoter regions. However, there were lower methylation levels at five CpG sites of DNMT3b, which decreased methylation of detected DNMT3b promoter region on M group (p < 0.05. In conclusion, alterations of methylation regulated by melatonin may mediate development of cumulus cells in lambs.

  10. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

    Science.gov (United States)

    Smith, Cindy J; Osborn, A Mark

    2009-01-01

    Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

  11. Genomic imprinting of IGF2 in marsupials is methylation dependent

    Directory of Open Access Journals (Sweden)

    Imumorin Ikhide

    2008-05-01

    Full Text Available Abstract Background- Parent-specific methylation of specific CpG residues is critical to imprinting in eutherian mammals, but its importance to imprinting in marsupials and, thus, the evolutionary origins of the imprinting mechanism have been the subject of controversy. This has been particularly true for the imprinted Insulin-like Growth Factor II (IGF2, a key regulator of embryonic growth in vertebrates and a focal point of the selective forces leading to genomic imprinting. The presence of the essential imprinting effector, DNMT3L, in marsupial genomes and the demonstration of a differentially methylated region (DMR in the retrotransposon-derived imprinted gene, PEG10, in tammar wallaby argue for a role for methylation in imprinting, but several studies have found no evidence of parent-specific methylation at other imprinted loci in marsupials. Results- We performed the most extensive search to date for allele-specific patterns of CpG methylation within CpG isochores or CpG enriched segments across a 22 kilobase region surrounding the IGF2 gene in the South American opossum Monodelphis domestica. We identified a previously unknown 5'-untranslated exon for opossum IGF2, which is flanked by sequences defining a putative neonatal promoter, a DMR and an active Matrix Attachment Region (MAR. Demethylation of this DMR in opossum neonatal fibroblasts results in abherrant biallelic expression of IGF2. Conclusion- The demonstration of a DMR and an active MAR in the 5' flank of opossum IGF2 mirrors the regulatory features of the 5' flank of Igf2 in mice. However, demethylation induced activation of the maternal allele of IGF2 in opossum differs from the demethylation induced repression of the paternal Igf2 allele in mice. While it can now be concluded that parent-specific DNA methylation is an epigentic mark common to Marsupialia and Eutheria, the molecular mechanisms of transcriptional silencing at imprinted loci have clearly evolved along independent

  12. Two-temperature LATE-PCR endpoint genotyping

    Directory of Open Access Journals (Sweden)

    Reis Arthur H

    2006-12-01

    Full Text Available Abstract Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of

  13. Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

    Directory of Open Access Journals (Sweden)

    Woodfine Kathryn

    2011-01-01

    Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

  14. The application of methylation specific electrophoresis (MSE) to DNA methylation analysis of the 5' CpG island of mucin in cancer cells

    International Nuclear Information System (INIS)

    Yokoyama, Seiya; Yonezawa, Suguru; Kitamoto, Sho; Yamada, Norishige; Houjou, Izumi; Sugai, Tamotsu; Nakamura, Shin-ichi; Arisaka, Yoshifumi; Takaori, Kyoichi; Higashi, Michiyo

    2012-01-01

    Methylation of CpG sites in genomic DNA plays an important role in gene regulation and especially in gene silencing. We have reported mechanisms of epigenetic regulation for expression of mucins, which are markers of malignancy potential and early detection of human neoplasms. Epigenetic changes in promoter regions appear to be the first step in expression of mucins. Thus, detection of promoter methylation status is important for early diagnosis of cancer, monitoring of tumor behavior, and evaluating the response of tumors to targeted therapy. However, conventional analytical methods for DNA methylation require a large amount of DNA and have low sensitivity. Here, we report a modified version of the bisulfite-DGGE (denaturing gradient gel electrophoresis) using a nested PCR approach. We designated this method as methylation specific electrophoresis (MSE). The MSE method is comprised of the following steps: (a) bisulfite treatment of genomic DNA, (b) amplification of the target DNA by a nested PCR approach and (c) applying to DGGE. To examine whether the MSE method is able to analyze DNA methylation of mucin genes in various samples, we apply it to DNA obtained from state cell lines, ethanol-fixed colonic crypts and human pancreatic juices. The MSE method greatly decreases the amount of input DNA. The lower detection limit for distinguishing different methylation status is < 0.1% and the detectable minimum amount of DNA is 20 pg, which can be obtained from only a few cells. We also show that MSE can be used for analysis of challenging samples such as human isolated colonic crypts or human pancreatic juices, from which only a small amount of DNA can be extracted. The MSE method can provide a qualitative information of methylated sequence profile. The MSE method allows sensitive and specific analysis of the DNA methylation pattern of almost any block of multiple CpG sites. The MSE method can be applied to analysis of DNA methylation status in many different clinical

  15. Predictive value of CHFR and MLH1 methylation in human gastric cancer.

    Science.gov (United States)

    Li, Yazhuo; Yang, Yunsheng; Lu, Youyong; Herman, James G; Brock, Malcolm V; Zhao, Po; Guo, Mingzhou

    2015-04-01

    Gastric carcinoma (GC) has one of the highest mortality rates of cancer diseases and has a high incidence rate in China. Palliative chemotherapy is the main treatment for advanced gastric cancer. It is necessary to compare the effectiveness and toxicities of different regimens. This study explores the possibility of methylation of DNA damage repair genes serving as a prognostic and chemo-sensitive marker in human gastric cancer. The methylation status of five DNA damage repair genes (CHFR, FANCF, MGMT, MLH1, and RASSF1A) was detected by nested methylation-specific PCR in 102 paraffin-embedded gastric cancer samples. Chi-square or Fisher's exact tests were used to evaluate the association of methylation status and clinic-pathological factors. The Kaplan-Meier method and Cox proportional hazards models were employed to analyze the association of methylation status and chemo-sensitivity. The results indicate that CHFR, MLH1, RASSF1A, MGMT, and FANCF were methylated in 34.3% (35/102), 21.6% (22/102), 12.7% (13/102), 9.8% (10/102), and 0% (0/102) of samples, respectively. No association was found between methylation of CHFR, MLH1, RASSF1A, MGMT, or FANCF with gender, age, tumor size, tumor differentiation, lymph node metastasis, and TNM stage. In docetaxel-treated gastric cancer patients, resistance to docetaxel was found in CHFR unmethylated patients by Cox proportional hazards model (HR 0.243, 95% CI, 0.069-0.859, p = 0.028), and overall survival is longer in the CHFR methylated group compared with the CHFR unmethylated group (log-rank, p = 0.036). In oxaliplatin-treated gastric cancer patients, resistance to oxaliplatin was found in MLH1 methylated patients (HR 2.988, 95% CI, 1.064-8.394, p = 0.038), and overall survival was longer in the MLH1 unmethylated group compared with the MLH1 methylated group (log-rank, p = 0.046). CHFR is frequently methylated in human gastric cancer, and CHFR methylation may serve as a docetaxel-sensitive marker. MLH1 methylation was

  16. Bioinformatic tools for PCR Primer design

    African Journals Online (AJOL)

    ES

    reaction (PCR), oligo hybridization and DNA sequencing. Proper primer design is actually one of the most important factors/steps in successful DNA sequencing. Various bioinformatics programs are available for selection of primer pairs from a template sequence. The plethora programs for PCR primer design reflects the.

  17. [E-MTAB-587] PCR_artifacts

    NARCIS (Netherlands)

    Muino Acuna, J.M.

    2011-01-01

    WARNING: This library was yield low amount of material and it was over-amplified by PCR. This libraries are used study the robustness of several statitical methods against PCR artifacts. ChIP experiments were performed on Arabidopsis wildtype inflorescences using an antibody raised against a

  18. Digital PCR for detection of citrus pathogens

    Science.gov (United States)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  19. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  20. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...

  1. PCR specific for Actinobacillus pleuropneumoniae serotype 3

    DEFF Research Database (Denmark)

    Zhou, L.; Jones, S.C.P.; Angen, Øystein

    2008-01-01

    , but the method has liminations, for example, cross-reactions between serotypes 3, 6, and 8. This study describes the development of a serotype 3-specific PCR, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The PCR test was evaluated on 266 strains...

  2. Polymerase chain reaction methods (PCR in agrobiotechnology

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija

    2006-01-01

    Full Text Available The agricultural biotechnology applies polymerase chain reaction (PCR technology at numerous steps throughout product development. The major uses of PCR technology during product development include gene discovery and cloning, vector construction, transformant identification, screening and characterization as well as seed quality control. Commodity and food companies as well as testing laboratories rely on PCR technology to verify the presence or absence of genetically modification (GM in a product or to quantify the amount of GM material present in the product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains and highlights some of areas to which attention must be paid in order to produce reliable test results. The article also discuses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

  3. Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes

    International Nuclear Information System (INIS)

    Kaup, Sahana; Grandjean, Valerie; Mukherjee, Rajarshi; Kapoor, Aparna; Keyes, Edward; Seymour, Colin B.; Mothersill, Carmel E.; Schofield, Paul N.

    2006-01-01

    The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the 'bystander effect') rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations

  4. Hepatitis B virus X protein suppresses caveolin-1 expression in hepatocellular carcinoma by regulating DNA methylation

    International Nuclear Information System (INIS)

    Yan, Jun; Lu, Qian; Dong, Jiahong; Li, Xiaowu; Ma, Kuansheng; Cai, Lei

    2012-01-01

    To understand the molecular mechanisms of caveolin-1 downregulation by hepatitis B virus X protein (HBx). The DNA methylation status of the caveolin-1 promoter was examined by nested methylation-specific PCR of 33 hepatitis B virus (HBV)-infected hepatocellular carcinoma (HCC) samples. The SMMC-7721 hepatoma cell line was transfected with a recombinant HBx adenoviral vector, and the effects of HBx protein on caveolin-1 expression and promoter methylation were examined and confirmed by sequencing. A reporter gene containing the caveolin-1 promoter region was constructed, and the effects of HBx on the transcriptional activity of the promoter were also studied. Methylation of the caveolin-1 promoter was detected in 84.8% (28/33) of HBV-infected HCC samples. Expression of caveolin-1 was significantly downregulated (P = 0.022), and multiple CpG sites in the promoter region of caveolin-1 were methylated in SMMC-7721 cells after HBx transfection. Transfected HBx significantly suppressed caveolin-1 promoter activity (P = 0.001). HBx protein induces methylation of the caveolin-1 promoter region and suppresses its expression

  5. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  6. Real-time PCR in virology.

    Science.gov (United States)

    Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

    2002-03-15

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

  7. Study of the Role of siRNA Mediated Promoter Methylation in DNMT3B Knockdown and Alteration of Promoter Methylation of CDH1, GSTP1 Genes in MDA-MB -453 Cell Line.

    Science.gov (United States)

    Naghitorabi, Mojgan; Mir Mohammad Sadeghi, Hamid; Mohammadi Asl, Javad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas

    2017-01-01

    Promoter methylation is one of the main epigenetic mechanisms that leads to the inactivation of tumor suppressor genes during carcinogenesis. Due to the reversible nature of DNA methylation, many studies have been performed to correct theses epigenetic defects by inhibiting DNA methyltransferases (DNMTs). In this case novel therapeutics especially siRNA oligonucleotides have been used to specifically knock down the DNMTs at mRNA level. Also many studies have focused on transcriptional gene silencing in mammalian cells via siRNA mediated promoter methylation. The present study was designed to assess the role of siRNA mediated promoter methylation in DNMT3B knockdown and alteration of promoter methylation of Cadherin-1 (CDH1), Glutathione S-Transferase Pi 1(GSTP1), and DNMT3B genes in MDA-MB-453 cell line. MDA-MB-453 cells were transfected with siDNMT targeting DNMT3B promoter and harvested at 24 and 48 h post transfection to monitor gene silencing and promoter methylation respectively. DNMT3B expression was monitored by quantitative RT-PCR method. Promoter methylation was quantitatively evaluated using differential high resolution melting analysis. A non-significant 20% reduction in DNMT3B mRNA level was shown only after first transfection with siDNMT, which was not reproducible. Promoter methylation levels of DNMT3B, CDH1, and GSTP1 were detected at about 15%, 70% and 10% respectively, in the MDA-MB-453 cell line, with no significant change after transfection. Our results indicated that siDNMT sequence were not able to affect promoter methylation and silencing of DNMT3B in MDA-MB-453 cells. However, quantitation of methylation confirmed a hypermethylated phenotype at CDH1 and GSTP1 promoters as well as a differential methylation pattern at DNMT3B promoter in breast cancer.

  8. Histone Lysine Methylation and Neurodevelopmental Disorders

    Directory of Open Access Journals (Sweden)

    Jeong-Hoon Kim

    2017-06-01

    Full Text Available Methylation of several lysine residues of histones is a crucial mechanism for relatively long-term regulation of genomic activity. Recent molecular biological studies have demonstrated that the function of histone methylation is more diverse and complex than previously thought. Moreover, studies using newly available genomics techniques, such as exome sequencing, have identified an increasing number of histone lysine methylation-related genes as intellectual disability-associated genes, which highlights the importance of accurate control of histone methylation during neurogenesis. However, given the functional diversity and complexity of histone methylation within the cell, the study of the molecular basis of histone methylation-related neurodevelopmental disorders is currently still in its infancy. Here, we review the latest studies that revealed the pathological implications of alterations in histone methylation status in the context of various neurodevelopmental disorders and propose possible therapeutic application of epigenetic compounds regulating histone methylation status for the treatment of these diseases.

  9. miRNAting control of DNA methylation

    Indian Academy of Sciences (India)

    miRNAting control of DNA methylation. ASHWANI ... function and biological process ... Enrichment analysis of the genes methylated by DRM2 for molecular function and biological ... 39(3), June 2014, 365–380, © Indian Academy of Sciences.

  10. Bacterial production of methyl ketones

    Energy Technology Data Exchange (ETDEWEB)

    Beller, Harry R.; Goh, Ee-Been

    2017-01-31

    The present invention relates to methods and compositions for increasing production of methyl ketones in a genetically modified host cell that overproduces .beta.-ketoacyl-CoAs through a re-engineered .beta.-oxidation pathway and overexpresses FadM.

  11. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

  12. Independent technical review, handbook

    International Nuclear Information System (INIS)

    1994-02-01

    Purpose Provide an independent engineering review of the major projects being funded by the Department of Energy, Office of Environmental Restoration and Waste Management. The independent engineering review will address questions of whether the engineering practice is sufficiently developed to a point where a major project can be executed without significant technical problems. The independent review will focus on questions related to: (1) Adequacy of development of the technical base of understanding; (2) Status of development and availability of technology among the various alternatives; (3) Status and availability of the industrial infrastructure to support project design, equipment fabrication, facility construction, and process and program/project operation; (4) Adequacy of the design effort to provide a sound foundation to support execution of project; (5) Ability of the organization to fully integrate the system, and direct, manage, and control the execution of a complex major project

  13. Independent technical review, handbook

    Energy Technology Data Exchange (ETDEWEB)

    1994-02-01

    Purpose Provide an independent engineering review of the major projects being funded by the Department of Energy, Office of Environmental Restoration and Waste Management. The independent engineering review will address questions of whether the engineering practice is sufficiently developed to a point where a major project can be executed without significant technical problems. The independent review will focus on questions related to: (1) Adequacy of development of the technical base of understanding; (2) Status of development and availability of technology among the various alternatives; (3) Status and availability of the industrial infrastructure to support project design, equipment fabrication, facility construction, and process and program/project operation; (4) Adequacy of the design effort to provide a sound foundation to support execution of project; (5) Ability of the organization to fully integrate the system, and direct, manage, and control the execution of a complex major project.

  14. Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera.

    Directory of Open Access Journals (Sweden)

    Gianpiero Marconi

    Full Text Available Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone and salinity-sensitive (Toccata rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4 and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site

  15. Electronic transport in methylated fragments of DNA

    International Nuclear Information System (INIS)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L.; Albuquerque, E. L.; Freire, V. N.; Caetano, E. W. S.; Moura, F. A. B. F. de; Lyra, M. L.

    2015-01-01

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics

  16. Electronic transport in methylated fragments of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, M. L. de; Oliveira, J. I. N.; Lima Neto, J. X.; Gomes, C. E. M.; Fulco, U. L., E-mail: umbertofulco@gmail.com; Albuquerque, E. L. [Departamento de Biofísica e Farmacologia, Universidade Federal do Rio Grande do Norte, 59072-970 Natal-RN (Brazil); Freire, V. N. [Departamento de Física, Universidade Federal do Ceará, 60455-760 Fortaleza, CE (Brazil); Caetano, E. W. S. [Instituto Federal de Educação, Ciência e Tecnologia do Ceará, 60040-531 Fortaleza, CE (Brazil); Moura, F. A. B. F. de; Lyra, M. L. [Instituto de Física, Universidade Federal de Alagoas, 57072-900 Maceió-AL (Brazil)

    2015-11-16

    We investigate the electronic transport properties of methylated deoxyribonucleic-acid (DNA) strands, a biological system in which methyl groups are added to DNA (a major epigenetic modification in gene expression), sandwiched between two metallic platinum electrodes. Our theoretical simulations apply an effective Hamiltonian based on a tight-binding model to obtain current-voltage curves related to the non-methylated/methylated DNA strands. The results suggest potential applications in the development of novel biosensors for molecular diagnostics.

  17. The prognostic significance of whole blood global and specific DNA methylation levels in gastric adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Mansour S Al-Moundhri

    Full Text Available BACKGROUND: Epigenetics, particularly DNA methylation, has recently been elucidated as important in gastric cancer (GC initiation and progression. We investigated the clinical and prognostic importance of whole blood global and site-specific DNA methylation in GC. METHODS: Genomic DNA was extracted from the peripheral blood of 105 Omani GC patients at diagnosis. DNA methylation was quantified by pyrosequencing of global DNA and specific gene promoter regions at 5 CpG sites for CDH1, 7 CpG sites for p16, 4 CpG sites for p53, and 3 CpG sites for RUNX3. DNA methylation levels in patients were categorized into low, medium, and high tertiles. Associations between methylation level category and clinicopathological features were evaluated using χ(2 tests. Survival analyses were carried out using the Kaplan-Meier method and log rank test. A backward conditional Cox proportional hazards regression model was used to identify independent predictors of survival. RESULTS: Older GC patients had increased methylation levels at specific CpG sites within the CDH1, p53, and RUNX-3 promoters. Male gender was significantly associated with reduced global and increased site-specific DNA methylation levels in CDH1, p16, and p53 promoters. Global DNA low methylation level was associated with better survival on univariate analysis. Patients with high and medium methylation vs. low methylation levels across p16 promoter CpG sites, site 2 in particular, had better survival. Multivariate analysis showed that global DNA hypermethylation was a significant independent predictor of worse survival (hazard ratio (HR = 2.0, 95% CI: 1.1-3.8; p = 0.02 and high methylation mean values across p16 promoter sites 1-7 were associated with better survival with HR of 0.3 (95% CI, 0.1-0.8; p = 0.02 respectively. CONCLUSIONS: Analysis of global and site-specific DNA methylation in peripheral blood by pyrosequencing provides quantitative DNA methylation values that may serve as important

  18. Androgenic effect of honeybee drone milk in castrated rats: roles of methyl palmitate and methyl oleate.

    Science.gov (United States)

    Seres, A B; Ducza, E; Báthori, M; Hunyadi, A; Béni, Z; Dékány, M; Hajagos-Tóth, J; Verli, J; Gáspár, Róbert

    2014-04-28

    Numerous honeybee (Apis mellifera) products have been used in traditional medicine to treat infertility and to increase vitality in both men and women. Drone milk (DM) is a relatively little-known honeybee product with a putative sexual hormone effect. The oestrogenic effect of a fraction of DM has recently been reported in rats. However, no information is available on the androgenic effects of DM. The purpose of the present study was to determine the androgen-like effect of DM in male rats and to identify effective compounds. A modified Hershberger assay was used to investigate the androgenic effect of crude DM, and the plasma level of testosterone was measured. The prostatic mRNA and protein expression of Spot14-like androgen-inducible protein (SLAP) were also examined with real-time PCR and Western blot techniques. GC-MS and NMR spectroscopic investigations were performed to identify the active components gained by bioactivity-guided fractionation. The crude DM increased the relative weights of the androgen-dependent organs and the plasma testosterone level in castrated rats and these actions were flutamide-sensitive. DM increased the tissue mRNA and protein level of SLAP, providing further evidence of its androgen-like character. After bioactivity-guided fractionation, two fatty acid esters, methyl palmitate (MP) and methyl oleate (MO), were identified as active compounds. MP alone showed an androgenic effect, whereas MO increased the weight of androgen-sensitive tissues and the plasma testosterone level only in combination. The experimental data of DM and its active compounds (MO and MP) show androgenic activity confirming the traditional usage of DM. DM or MP or/and MO treatments may project a natural mode for the therapy of male infertility. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Methylation sensitive amplified polymorphism (MSAP) reveals that ...

    African Journals Online (AJOL)

    ajl yemi

    2011-12-19

    Dec 19, 2011 ... Key words: Salt stress, alkali stress, Gossypium hirsutum L., DNA methylation, methylation sensitive amplified polymorphism (MSAP). INTRODUCTION. DNA methylation is one of the key epigenetic mecha- nisms among eukaryotes that can modulate gene expression without the changes of DNA sequence.

  20. miRNAting control of DNA methylation

    Indian Academy of Sciences (India)

    DNA methylation is a type of epigenetic modification where a methyl group is added to the cytosine or adenine residue of a given DNA sequence. It has been observed that DNA methylation is achieved by some collaborative agglomeration of certain proteins and non-coding RNAs. The assembly of IDN2 and its ...

  1. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas; Chang, Donald Choy; Gong, Xiuqing; Wen, Weijia; Wu, Jinbo; Xiao, Kang; Yi, Xin

    2010-01-01

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  2. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas

    2010-04-23

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  3. Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.

    Science.gov (United States)

    Wang, Meng; Yang, Junjie; Gai, Zhongtao; Huo, Shengnan; Zhu, Jianhua; Li, Jun; Wang, Ranran; Xing, Sheng; Shi, Guosheng; Shi, Feng; Zhang, Lei

    2018-02-02

    As a kind of zero-tolerance foodborne pathogens, Salmonella typhimurium poses a great threat to quality of food products and public health. Hence, rapid and efficient approaches to identify Salmonella typhimurium are urgently needed. Combined with PCR and fluorescence technique, real-time PCR (qPCR) and digital PCR (ddPCR) are regarded as suitable tools for detecting foodborne pathogens. To compare the effect between qPCR and ddPCR in detecting Salmonella typhimurium, a series of nucleic acid, pure strain culture and spiking milk samples were applied and the resistance to inhibitors referred in this article as well. Compared with qPCR, ddPCR exhibited more sensitive (10 -4 ng/μl or 10 2 cfu/ml) and less pre-culturing time (saving 2h). Moreover, ddPCR had stronger resistance to inhibitors than qPCR, yet absolute quantification hardly performed when target's concentration over 1ng/μl or 10 6 cfu/ml. This study provides an alternative strategy in detecting foodborne Salmonella typhimurium. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

    DEFF Research Database (Denmark)

    Gillberg, Linn; Jacobsen, Stine; Rönn, Tina

    2014-01-01

    OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High...... and insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA...... methylation was assessed with bisulfite sequencing and mRNA expression with quantitative real-time PCR. RESULTS: Following high-fat overfeeding, increased SAT PPARGC1A DNA methylation was observed in LBW subjects but not in NBW controls. Basal SAT PPARGC1A mRNA expression was unaffected by diet and similar...

  5. SHOX2 gene methylation as a diagnostic marker of lung cancer

    International Nuclear Information System (INIS)

    Konecny, M.; Markus, J.; Dolesova, L.; Waczulikova, I.; Majer, I.; Novosadova, H.

    2016-01-01

    Purpose: In the actual study we have detected in the group of patients with mailgnant and also non-malignant disease the presence of DNA methylation of SHOX2 gene. The basic aim was to evaluate the reliability of detection of SHOX2 methylation in blood plasma as a potential alternative to used bronchial lavage. Further, we also determine the correlation of test results with samples of lavage and blood plasma. Materials and methods: The specimens were achieved of each patient from bronchial lavage and also peripheral blood and from january to december 2014 we analysed 69 parallel samples. DNA from bronchial lavage was purified with commercial kit Epi pro Lung BL DNA preparation kit (Epigenomics) and samples from peripheral blood using Epi pro Colon Plasma Quick kit (Epigenomics). Quantitative PCR analysis was accomplished using Epi pro Lung BL real-time PCR kit (Epigenomics) on the ABI 7500 Fast real-time PCR instrument (Life Technologies). Methylation results were performed with ΔΔCT method. Statistical characteristics of patients and other clinical data we analysed by methods of bi variant and descriptive statistics, like hood ratio by Cohen kappa and significance of observed differences by Maxwell and McNemar chi-quadrant test. Results: Altogether we analysed 69 parallel samples and methylation of SHOX2 gene was identified in 63,8% of samples. Overall the methylation was detected in 31 out of 37 lavage and 20 out of 31 blood samples. Diagnostic performance of the model with bio marker of SHOX2 methylation in lavage samples was determined at the level of 89,19% sensitivity and 84,62% specificity, on the other hand model with bio marker of SHOX2 methylation in plasma samples showed 80,65% sensitivity and 78,57% specificity. Conclusion: Generally, we established that methylation analysis of SHOX2 gene in bronchial lavage samples represents a reliable test, which may be used as an additional marker in the lung cancer diagnostics. Test of blood plasma features

  6. Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver

    Science.gov (United States)

    Di Gioia, Sonia; Bianchi, Paolo; Destro, Annarita; Grizzi, Fabio; Malesci, Alberto; Laghi, Luigi; Levrero, Massimo; Morabito, Alberto; Roncalli, Massimo

    2006-01-01

    Background Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver. Methods To address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia), dysplastic (large regenerative, low and high grade dysplastic nodules) and neoplastic (hepatocellular adenoma and carcinoma) growths. Results In the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC) we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis) to hepatocellular nodules (p < 0.01) to HCC (p < 0.001). In the non-hepatitic liver a consistent pattern of gene methylation was also found in both lesional (focal nodular hyperplasia and hepatocellular adenoma) and non-lesional tissue. Specifically, hepatocellular adenomas (HA) showed a methylation index significantly higher than that detected in focal nodular hyperplasia (FNH) (p < 0.01) and in non-lesional tissue (p < 0.001). In non-lesional liver also the methylation index gradually increased by ageing (p = 0.002), suggesting a progressive spreading of methylated cells over time. As opposed to RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver. Conclusion We have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly

  7. Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

    Directory of Open Access Journals (Sweden)

    Tsang Percy CK

    2006-08-01

    Full Text Available Abstract Background Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. Methods For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ2 or Fisher's exact test. Results APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2, endometrial cancer (CASP8, CDH13, hMLH1 and p73, and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1. The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer to the highest 56

  8. Clinical Significance of Retinoic Acid Receptor Beta Promoter Methylation in Prostate Cancer: A Meta-Analysis.

    Science.gov (United States)

    Dou, MengMeng; Zhou, XueLiang; Fan, ZhiRui; Ding, XianFei; Li, LiFeng; Wang, ShuLing; Xue, Wenhua; Wang, Hui; Suo, Zhenhe; Deng, XiaoMing

    2018-01-01

    Retinoic acid receptor beta (RAR beta) is a retinoic acid receptor gene that has been shown to play key roles during multiple cancer processes, including cell proliferation, apoptosis, migration and invasion. Numerous studies have found that methylation of the RAR beta promoter contributed to the occurrence and development of malignant tumors. However, the connection between RAR beta promoter methylation and prostate cancer (PCa) remains unknown. This meta-analysis evaluated the clinical significance of RAR beta promoter methylation in PCa. We searched all published records relevant to RAR beta and PCa in a series of databases, including PubMed, Embase, Cochrane Library, ISI Web of Science and CNKI. The rates of RAR beta promoter methylation in the PCa and control groups (including benign prostatic hyperplasia and normal prostate tissues) were summarized. In addition, we evaluated the source region of available samples and the methods used to detect methylation. To compare the incidence and variation in RAR beta promoter methylation in PCa and non-PCa tissues, the odds ratio (OR) and 95% confidence interval (CI) were calculated accordingly. All the data were analyzed with the statistical software STATA 12.0. Based on the inclusion and exclusion criteria, 15 articles assessing 1,339 samples were further analyzed. These data showed that the RAR beta promoter methylation rates in PCa tissues were significantly higher than the rates in the non-PCa group (OR=21.65, 95% CI: 9.27-50.57). Subgroup analysis according to the source region of samples showed that heterogeneity in Asia was small (I2=0.0%, P=0.430). Additional subgroup analysis based on the method used to detect RAR beta promoter methylation showed that the heterogeneity detected by MSP (methylation-specific PCR) was relatively small (I2=11.3%, P=0.343). Although studies reported different rates for RAR beta promoter methylation in PCa tissues, the total analysis demonstrated that RAR beta promoter methylation

  9. Clinical Significance of Retinoic Acid Receptor Beta Promoter Methylation in Prostate Cancer: A Meta-Analysis

    Directory of Open Access Journals (Sweden)

    MengMeng Dou

    2018-03-01

    Full Text Available Background/Aims: Retinoic acid receptor beta (RAR beta is a retinoic acid receptor gene that has been shown to play key roles during multiple cancer processes, including cell proliferation, apoptosis, migration and invasion. Numerous studies have found that methylation of the RAR beta promoter contributed to the occurrence and development of malignant tumors. However, the connection between RAR beta promoter methylation and prostate cancer (PCa remains unknown. This meta-analysis evaluated the clinical significance of RAR beta promoter methylation in PCa. Materials and Methods: We searched all published records relevant to RAR beta and PCa in a series of databases, including PubMed, Embase, Cochrane Library, ISI Web of Science and CNKI. The rates of RAR beta promoter methylation in the PCa and control groups (including benign prostatic hyperplasia and normal prostate tissues were summarized. In addition, we evaluated the source region of available samples and the methods used to detect methylation. To compare the incidence and variation in RAR beta promoter methylation in PCa and non-PCa tissues, the odds ratio (OR and 95% confidence interval (CI were calculated accordingly. All the data were analyzed with the statistical software STATA 12.0. Results: Based on the inclusion and exclusion criteria, 15 articles assessing 1,339 samples were further analyzed. These data showed that the RAR beta promoter methylation rates in PCa tissues were significantly higher than the rates in the non-PCa group (OR=21.65, 95% CI: 9.27-50.57. Subgroup analysis according to the source region of samples showed that heterogeneity in Asia was small (I2=0.0%, P=0.430. Additional subgroup analysis based on the method used to detect RAR beta promoter methylation showed that the heterogeneity detected by MSP (methylation-specific PCR was relatively small (I2=11.3%, P=0.343. Conclusion: Although studies reported different rates for RAR beta promoter methylation in PCa

  10. Differential DNA methylation profiles in gynecological cancers and correlation with clinico-pathological data

    International Nuclear Information System (INIS)

    Yang, Hui-Juan; Liu, Vincent WS; Wang, Yue; Tsang, Percy CK; Ngan, Hextan YS

    2006-01-01

    Epigenetic gene silencing is one of the major causes of carcinogenesis. Its widespread occurrence in cancer genome could inactivate many cellular pathways including DNA repair, cell cycle control, apoptosis, cell adherence, and detoxification. The abnormal promoter methylation might be a potential molecular marker for cancer management. For rapid identification of potential targets for aberrant methylation in gynecological cancers, methylation status of the CpG islands of 34 genes was determined using pooled DNA approach and methylation-specific PCR. Pooled DNA mixture from each cancer type (50 cervical cancers, 50 endometrial cancers and 50 ovarian cancers) was made to form three test samples. The corresponding normal DNA from the patients of each cancer type was also pooled to form the other three control samples. Methylated alleles detected in tumors, but not in normal controls, were indicative of aberrant methylation in tumors. Having identified potential markers, frequencies of methylation were further analyzed in individual samples. Markers identified are used to correlate with clinico-pathological data of tumors using χ 2 or Fisher's exact test. APC and p16 were hypermethylated across the three cancers. MINT31 and PTEN were hypermethylated in cervical and ovarian cancers. Specific methylation was found in cervical cancer (including CDH1, DAPK, MGMT and MINT2), endometrial cancer (CASP8, CDH13, hMLH1 and p73), and ovarian cancer (BRCA1, p14, p15, RIZ1 and TMS1). The frequencies of occurrence of hypermethylation in 4 candidate genes in individual samples of each cancer type (DAPK, MGMT, p16 and PTEN in 127 cervical cancers; APC, CDH13, hMLH1 and p16 in 60 endometrial cancers; and BRCA1, p14, p16 and PTEN in 49 ovarian cancers) were examined for further confirmation. Incidence varied among different genes and in different cancer types ranging from the lowest 8.2% (PTEN in ovarian cancer) to the highest 56.7% (DAPK in cervical cancer). Aberrant methylation

  11. Model-Independent Diffs

    DEFF Research Database (Denmark)

    Könemann, Patrick

    just contain a list of strings, one for each line, whereas the structure of models is defined by their meta models. There are tools available which are able to compute the diff between two models, e.g. RSA or EMF Compare. However, their diff is not model-independent, i.e. it refers to the models...

  12. All Those Independent Variables.

    Science.gov (United States)

    Meacham, Merle L.

    This paper presents a case study of a sixth grade remedial math class which illustrates the thesis that only the "experimental attitude," not the "experimental method," is appropriate in the classroom. The thesis is based on the fact that too many independent variables exist in a classroom situation to allow precise measurement. The case study…

  13. Bayesian Independent Component Analysis

    DEFF Research Database (Denmark)

    Winther, Ole; Petersen, Kaare Brandt

    2007-01-01

    In this paper we present an empirical Bayesian framework for independent component analysis. The framework provides estimates of the sources, the mixing matrix and the noise parameters, and is flexible with respect to choice of source prior and the number of sources and sensors. Inside the engine...

  14. Independent safety organization

    International Nuclear Information System (INIS)

    Kato, W.Y.; Weinstock, E.V.; Carew, J.F.; Cerbone, R.J.; Guppy, J.G.; Hall, R.E.; Taylor, J.H.

    1985-01-01

    Brookhaven National Laboratory has conducted a study on the need and feasibility of an independent organization to investigate significant safety events for the Office for Analysis and Evaluation of Operational Data, USNRC. The study consists of three parts: the need for an independent organization to investigate significant safety events, alternative organizations to conduct investigations, and legislative requirements. The determination of need was investigated by reviewing current NRC investigation practices, comparing aviation and nuclear industry practices, and interviewing a spectrum of representatives from the nuclear industry, the regulatory agency, and the public sector. The advantages and disadvantages of alternative independent organizations were studied, namely, an Office of Nuclear Safety headed by a director reporting to the Executive Director for Operations (EDO) of NRC; an Office of Nuclear Safety headed by a director reporting to the NRC Commissioners; a multi-member NTSB-type Nuclear Safety Board independent of the NRC. The costs associated with operating a Nuclear Safety Board were also included in the study. The legislative requirements, both new authority and changes to the existing NRC legislative authority, were studied. 134 references

  15. Aberrant methylation of Polo-like kinase CpG islands in Plk4 heterozygous mice

    International Nuclear Information System (INIS)

    Ward, Alejandra; Morettin, Alan; Shum, David; Hudson, John W

    2011-01-01

    Hepatocellular carcinoma (HCC), one of the most common cancers world-wide occurs twice as often in men compared to women. Predisposing conditions such as alcoholism, chronic viral hepatitis, aflatoxin B1 ingestion, and cirrhosis all contribute to the development of HCC. We used a combination of methylation specific PCR and bisulfite sequencing, qReal-Time PCR (qPCR), and Western blot analysis to examine epigenetic changes for the Polo-like kinases (Plks) during the development of hepatocellular carcinoma (HCC) in Plk4 heterozygous mice and murine embryonic fibroblasts (MEFs). Here we report that the promoter methylation of Plk4 CpG islands increases with age, was more prevalent in males and that Plk4 epigenetic modification and subsequent downregulation of expression was associated with the development of HCC in Plk4 mutant mice. Interestingly, the opposite occurs with another Plk family member, Plk1 which was typically hypermethylated in normal liver tissue but became hypomethylated and upregulated in liver tumours. Furthermore, upon alcohol exposure murine embryonic fibroblasts exhibited increased Plk4 hypermethylation and downregulation along with increased centrosome numbers and multinucleation. These results suggest that aberrant Plk methylation is correlated with the development of HCC in mice

  16. Real-time PCR in virology

    OpenAIRE

    Mackay, Ian M.; Arden, Katherine E.; Nitsche, Andreas

    2002-01-01

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of P...

  17. Quantitative evaluation of RASSF1A methylation in the non-lesional, regenerative and neoplastic liver

    Directory of Open Access Journals (Sweden)

    Laghi Luigi

    2006-04-01

    Full Text Available Abstract Background Epigenetic changes during ageing and their relationship with cancer are under the focus of intense research. RASSF1A and NORE1A are novel genes acting in concert in the proapoptotic pathway of the RAS signalling. While NORE1A has not been previously investigated in the human liver, recent reports have suggested that RASSF1A is frequently epigenetically methylated not only in HCC but also in the cirrhotic liver. Methods To address whether epigenetic changes take place in connection to age and/or to the underlying disease, we investigated RASSF1A and NORE1A gene promoter methylation by conventional methylation specific PCR and Real-Time MSP in a series of hepatitic and non-hepatitic livers harboring regenerative/hyperplastic (cirrhosis/focal nodular hyperplasia, dysplastic (large regenerative, low and high grade dysplastic nodules and neoplastic (hepatocellular adenoma and carcinoma growths. Results In the hepatitic liver (chronic hepatitic/cirrhosis, hepatocellular nodules and HCC we found widespread RASSF1A gene promoter methylation with a methylation index that increased from regenerative conditions (cirrhosis to hepatocellular nodules (p RASSF1A gene promoter methylation, NORE1A gene was never found epigenetically alterated in both hepatitic and non-hepatitic liver. Conclusion We have shown that in non-lesional, regenerative and neoplastic liver the RASSF1A gene is increasingly methylated, that this condition takes place as an age-related phenomenon and that the early setting and spreading over time of an epigenetically methylated hepatocyte subpopulation, might be related to liver tumorigenesis.

  18. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    Directory of Open Access Journals (Sweden)

    Oliveira Jorge

    2007-07-01

    Full Text Available Abstract Background Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. Methods A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC, 13 papillary (pRCC, 10 chromophobe (chRCC, and 10 oncocytomas and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Results Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007, PTGS2 (p = 0.002, and RASSF1A (p = 0.0001. CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively, whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004. RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035. In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031 and nuclear grade (p = 0.022, respectively. Conclusion The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

  19. Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

    International Nuclear Information System (INIS)

    Costa, Vera L; Henrique, Rui; Ribeiro, Franclim R; Pinto, Mafalda; Oliveira, Jorge; Lobo, Francisco; Teixeira, Manuel R; Jerónimo, Carmen

    2007-01-01

    Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses

  20. [Novel Approaches in DNA Methylation Studies - MS-HRM Analysis and Electrochemistry].

    Science.gov (United States)

    Bartošík, M; Ondroušková, E

    Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

  1. IFI44L promoter methylation as a blood biomarker for systemic lupus erythematosus

    Science.gov (United States)

    Zhao, Ming; Zhou, Yin; Zhu, Bochen; Wan, Mengjie; Jiang, Tingting; Tan, Qiqun; Liu, Yan; Jiang, Juqing; Luo, Shuaihantian; Tan, Yixin; Wu, Haijing; Renauer, Paul; Gutiérrez, Maria del Mar Ayala; Palma, Maria Jesús Castillo; Castro, Rafaela Ortega; Fernández-Roldán, Concepción; Raya, Enrique; Faria, Raquel; Carvalho, Claudia; Alarcón-Riquelme, Marta E; Xiang, Zhongyuan; Chen, Jinwei; Li, Fen; Ling, Guanghui; Zhao, Hongjun; Liao, Xiangping; Lin, Youkun; Sawalha, Amr H; Lu, Qianjin

    2016-01-01

    Objective Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease with limited reliable diagnostic biomarkers. We investigated whether gene methylation could meet sensitivity and specificity criteria for a robust biomarker. Methods IFI44L promoter methylation was examined using DNA samples from a discovery set including 377 patients with SLE, 358 healthy controls (HCs) and 353 patients with rheumatoid arthritis (RA). Two independent sets including 1144 patients with SLE, 1350 HCs, 429 patients with RA and 199 patients with primary Sjögren’s syndrome (pSS) were used for validation. Results Significant hypomethylation of two CpG sites within IFI44L promoter, Site1 (Chr1: 79 085 222) and Site2 (Chr1: 79 085 250; cg06872964), was identified in patients with SLE compared with HCs, patients with RA and patients with pSS. In a comparison between patients with SLE and HCs included in the first validation cohort, Site1 methylation had a sensitivity of 93.6% and a specificity of 96.8% at a cut-off methylation level of 75.5% and Site2 methylation had a sensitivity of 94.1% and a specificity of 98.2% at a cut-off methylation level of 25.5%. The IFI44L promoter methylation marker was also validated in an European-derived cohort. In addition, the methylation levels of Site1 and Site2 within IFI44L promoter were significantly lower in patients with SLE with renal damage than those without renal damage. Patients with SLE showed significantly increased methylation levels of Site1 and Site2 during remission compared with active stage. Conclusions The methylation level of IFI44L promoter can distinguish patients with SLE from healthy persons and other autoimmune diseases, and is a highly sensitive and specific diagnostic marker for SLE. PMID:26787370

  2. Promoter Methylation and BDNF and DAT1 Gene Expression Profiles in Patients with Drug Addiction.

    Science.gov (United States)

    Kordi-Tamandani, Dor Mohammad; Tajoddini, Shahrad; Salimi, Farzaneh

    2015-01-01

    Drug addiction is a brain disorder that has negative consequences for individuals and society. Addictions are chronic relapsing diseases of the brain that are caused by direct drug-induced effects and persevering neuroadaptations at the epigenetic, neuropeptide and neurotransmitter levels. Because the dopaminergic system has a significant role in drug abuse, the purpose of this study was to analyze the methylation and expression profile of brain-derived neurotrophic factor (BDNF) and dopamine transporter (DAT1) genes in individuals with drug addiction. BDNF and DAT1 promoter methylation were investigated with a methylation-specific polymerase chain reaction (PCR) technique in blood samples from 75 individuals with drug addiction and 65 healthy controls. The expression levels of BDNF and DAT1 were assessed in 12 mRNA samples from the blood of patients and compared to the samples of healthy controls (n = 12) with real-time quantitative reverse transcription PCR. No significant differences were found in the methylation of BDNF and DAT1 between patients and controls, but the relative levels of expression of BDNF and DAT1 mRNA differed significantly in the patients compared to controls (p drug addiction.

  3. Identification of endometrial cancer methylation features using combined methylation analysis methods.

    Directory of Open Access Journals (Sweden)

    Michael P Trimarchi

    Full Text Available DNA methylation is a stable epigenetic mark that is frequently altered in tumors. DNA methylation features are attractive biomarkers for disease states given the stability of DNA methylation in living cells and in biologic specimens typically available for analysis. Widespread accumulation of methylation in regulatory elements in some cancers (specifically the CpG island methylator phenotype, CIMP can play an important role in tumorigenesis. High resolution assessment of CIMP for the entire genome, however, remains cost prohibitive and requires quantities of DNA not available for many tissue samples of interest. Genome-wide scans of methylation have been undertaken for large numbers of tumors, and higher resolution analyses for a limited number of cancer specimens. Methods for analyzing such large datasets and integrating findings from different studies continue to evolve. An approach for comparison of findings from a genome-wide assessment of the methylated component of tumor DNA and more widely applied methylation scans was developed.Methylomes for 76 primary endometrial cancer and 12 normal endometrial samples were generated using methylated fragment capture and second generation sequencing, MethylCap-seq. Publically available Infinium HumanMethylation 450 data from The Cancer Genome Atlas (TCGA were compared to MethylCap-seq data.Analysis of methylation in promoter CpG islands (CGIs identified a subset of tumors with a methylator phenotype. We used a two-stage approach to develop a 13-region methylation signature associated with a "hypermethylator state." High level methylation for the 13-region methylation signatures was associated with mismatch repair deficiency, high mutation rate, and low somatic copy number alteration in the TCGA test set. In addition, the signature devised showed good agreement with previously described methylation clusters devised by TCGA.We identified a methylation signature for a "hypermethylator phenotype" in

  4. FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

    Science.gov (United States)

    Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

    2014-01-01

    Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

  5. Changes in IL12A methylation pattern in livers from mice fed DDC.

    Science.gov (United States)

    Oliva, J; French, S W

    2012-04-01

    Mallory-Denk body (MDB) formation is a component of alcoholic and non alcoholic hepatitis. Proteins of the TLR pathway were shown to be involved in the formation of MDBs, in mice fed DDC. TLR genes are upregulated and SAMe supplementation prevents this up regulation and prevented the formation of MDBs. DNA of livers from control mice, from mice fed DDC 10weeks, refed 1week with DDC and with DDC+SAMe were extracted and used to study the methylation pattern of genes involves in the TLR pathway. A PCR array was used to analyze it. Using PCR arrays for the mouse TLR pathway,24 genes were found whose expression of IL12A was regulated by the methylation of its gene. DDC fed for 10weeks reduced the methylation of the IL12A gene expression. This expression was also reduced when DDC was refed. However, when SAMe was fed, the intermediate level methylation of IL12A was up regulated to the intermediate level and the methylation of the promoter decreased compared to DDC refeeding or DDC 10weeks. IL12A is known to induce the production of IFNg by NK and L(T). We showed in a previous publication that IFNg is one of the major cytokines involved in the induction of MDB formation. The low expression of IL12A associated with the intermediate methylation of its promoter could explain one step in the mechanism which leads to the formation of MDBs. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Does DNA Methylation of PPARGC1A Influence Insulin Action in First Degree Relatives of Patients with Type 2 Diabetes?

    DEFF Research Database (Denmark)

    Gillberg, Linn; Jacobsen, Stine; Ribel-Madsen, Rasmus

    2013-01-01

    and in muscle from individuals at risk of T2D. This study aimed to investigate DNA promoter methylation and gene expression of PPARGC1A in skeletal muscle from first degree relatives (FDR) of T2D patients, and to determine the association with insulin action as well as the influence of family relation. We...... genetic regulation to play a role. No significant effect of familiality on DNA methylation was found. Taken together, increased DNA methylation of the PPARGC1A promoter is unlikely to play a major causal role for the development of insulin resistance in FDR of patients with T2D....... included 124 Danish FDR of T2D patients from 46 different families. Skeletal muscle biopsies were excised from vastus lateralis and insulin action was assessed by oral glucose tolerance tests. DNA methylation and mRNA expression levels were measured using bisulfite sequencing and quantitative real-time PCR...

  7. MethylMix 2.0: an R package for identifying DNA methylation genes.

    Science.gov (United States)

    Cedoz, Pierre-Louis; Prunello, Marcos; Brennan, Kevin; Gevaert, Olivier

    2018-04-14

    DNA methylation is an important mechanism regulating gene transcription, and its role in carcinogenesis has been extensively studied. Hyper and hypomethylation of genes is a major mechanism of gene expression deregulation in a wide range of diseases. At the same time, high-throughput DNA methylation assays have been developed generating vast amounts of genome wide DNA methylation measurements. We developed MethylMix, an algorithm implemented in R to identify disease specific hyper and hypomethylated genes. Here we present a new version of MethylMix that automates the construction of DNA-methylation and gene expression datasets from The Cancer Genome Atlas (TCGA). More precisely, MethylMix 2.0 incorporates two major updates: the automated downloading of DNA methylation and gene expression datasets from TCGA and the automated preprocessing of such datasets: value imputation, batch correction and CpG sites clustering within each gene. The resulting datasets can subsequently be analyzed with MethylMix to identify transcriptionally predictive methylation states. We show that the Differential Methylation Values created by MethylMix can be used for cancer subtyping. olivier.gevaert@stanford.edu. https://bioconductor.org/packages/release/bioc/manuals/MethylMix/man/MethylMix.pdf. MethylMix 2.0 was implemented as an R package and is available in bioconductor.

  8. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...

  9. Avocado and olive oil methyl esters

    International Nuclear Information System (INIS)

    Knothe, Gerhard

    2013-01-01

    Biodiesel, the mono-alkyl esters of vegetable oils, animal fats or other triacylglycerol-containing materials and an alternative to conventional petroleum-based diesel fuel, has been derived from a variety of feedstocks. Numerous feedstocks have been investigated as potential biodiesel sources, including commodity oils, however, the methyl esters of avocado and olive oil would likely be suitable as biodiesel fuel. In order to expand the database and comprehensive evaluation of the properties of vegetable oil esters, in this work the fuel-related properties of avocado and olive oil methyl esters, which exhibit similar fatty acid profiles including high oleic acid content, are determined. The cetane numbers of avocado oil methyl esters and olive oil methyl esters are relatively high, determined as 59.2 and 62.5, respectively, due to their elevated content of methyl oleate. Other properties are well within the ranges specified in biodiesel standards. The cloud points of both esters are slightly above 0 °C due to their content of saturated esters, especially methyl palmitate. Overall, avocado and olive oil yield methyl esters with fuel properties comparable to methyl esters from other commodity vegetable oils. The 1 H and 13 C NMR spectra of avocado and olive oil methyl esters are reported. -- Highlights: • Methyl esters of avocado and olive oil meet biodiesel fuel standards. • Provides comparison for methyl esters of other vegetable oils with high oleic content. • Discusses and compares present results with prior literature

  10. Transgene detection by digital droplet PCR.

    Directory of Open Access Journals (Sweden)

    Dirk A Moser

    Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  11. Methylated genes as new cancer biomarkers.

    LENUS (Irish Health Repository)

    Duffy, M J

    2012-02-01

    Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

  12. Quantum independent increment processes

    CERN Document Server

    Franz, Uwe

    2005-01-01

    This volume is the first of two volumes containing the revised and completed notes lectures given at the school "Quantum Independent Increment Processes: Structure and Applications to Physics". This school was held at the Alfried-Krupp-Wissenschaftskolleg in Greifswald during the period March 9 – 22, 2003, and supported by the Volkswagen Foundation. The school gave an introduction to current research on quantum independent increment processes aimed at graduate students and non-specialists working in classical and quantum probability, operator algebras, and mathematical physics. The present first volume contains the following lectures: "Lévy Processes in Euclidean Spaces and Groups" by David Applebaum, "Locally Compact Quantum Groups" by Johan Kustermans, "Quantum Stochastic Analysis" by J. Martin Lindsay, and "Dilations, Cocycles and Product Systems" by B.V. Rajarama Bhat.

  13. Quantum independent increment processes

    CERN Document Server

    Franz, Uwe

    2006-01-01

    This is the second of two volumes containing the revised and completed notes of lectures given at the school "Quantum Independent Increment Processes: Structure and Applications to Physics". This school was held at the Alfried-Krupp-Wissenschaftskolleg in Greifswald in March, 2003, and supported by the Volkswagen Foundation. The school gave an introduction to current research on quantum independent increment processes aimed at graduate students and non-specialists working in classical and quantum probability, operator algebras, and mathematical physics. The present second volume contains the following lectures: "Random Walks on Finite Quantum Groups" by Uwe Franz and Rolf Gohm, "Quantum Markov Processes and Applications in Physics" by Burkhard Kümmerer, Classical and Free Infinite Divisibility and Lévy Processes" by Ole E. Barndorff-Nielsen, Steen Thorbjornsen, and "Lévy Processes on Quantum Groups and Dual Groups" by Uwe Franz.

  14. Independent random sampling methods

    CERN Document Server

    Martino, Luca; Míguez, Joaquín

    2018-01-01

    This book systematically addresses the design and analysis of efficient techniques for independent random sampling. Both general-purpose approaches, which can be used to generate samples from arbitrary probability distributions, and tailored techniques, designed to efficiently address common real-world practical problems, are introduced and discussed in detail. In turn, the monograph presents fundamental results and methodologies in the field, elaborating and developing them into the latest techniques. The theory and methods are illustrated with a varied collection of examples, which are discussed in detail in the text and supplemented with ready-to-run computer code. The main problem addressed in the book is how to generate independent random samples from an arbitrary probability distribution with the weakest possible constraints or assumptions in a form suitable for practical implementation. The authors review the fundamental results and methods in the field, address the latest methods, and emphasize the li...

  15. Methyl mercury in terrestrial compartments

    International Nuclear Information System (INIS)

    Stoeppler, M.; Burow, M.; Padberg, S.; May, K.

    1993-09-01

    On the basis of the analytical methodology available at present the state of the art for the determination of total mercury and of various organometallic compounds of mercury in air, precipitation, limnic systems, soils, plants and biota is reviewed. This is followed by the presentation and discussion of examples for the data obtained hitherto for trace and ultratrace levels of total mercury and mainly methyl mercury in terrestrial and limnic environments as well as in biota. The data discussed stem predominantly from the past decade in which, due to significant methodological progress, many new aspects were elucidated. They include the most important results in this area achieved by the Research Centre (KFA) Juelich within the project 'Origin and Fate of Methyl Mercury' (contracts EV4V-0138-D and STEP-CT90-0057) supported by the Commission of the European Communities, Brussels. (orig.) [de

  16. Diagnosis of trichomonas vaginalis infection by PCR

    International Nuclear Information System (INIS)

    Issa, R.M.; Shalaby, M.A.

    2007-01-01

    To compare the sensitivity of PCR, wet preparation and culture in detecting Trichomonas vaginalis in urine and vaginal fluid. A PCR targeting the beta-tubulin genes of T. vaginalis was used for the detection of the organism in both vaginal swab and urine specimens from infected patients. Random urine samples were collected from 30 patients (23 females and 7 males), and tested for T. vaginalis by wet preparation and the Inpouch T. vaginalis culture systeme. Two vaginal swabs were collected by each woman. PCR detection. was carried out on samples negative by first methods. The positive result was found in 28.57% in male urine and 39.13% in female urine samples, 65.21% in 1st swab and 78.26 % in 2nd swab by wet preparation. By culture, the male urine samples showed 42.85% positive, female urine 69.56% while 1st swab showed 86.95% positive and 2nd swab 91.30% positive. All negative cases by culture in urine and vaginal samples were tested by PCR, which showed 2 cases to be positive in male urine samples and 5 cases positive in female urine sample. PCR assay was as good as or more sensitive than wet preparation and culture and resulted in practical advantage of providing results in shorter time. However, PCR test is still very expensive. (author)

  17. International exploration by independents

    International Nuclear Information System (INIS)

    Bertagne, R.G.

    1991-01-01

    Recent industry trends indicate that the smaller US independents are looking at foreign exploration opportunities as one of the alternatives for growth in the new age of exploration. It is usually accepted that foreign finding costs per barrel are substantially lower than domestic because of the large reserve potential of international plays. To get involved overseas requires, however, an adaptation to different cultural, financial, legal, operational, and political conditions. Generally foreign exploration proceeds at a slower pace than domestic because concessions are granted by the government, or are explored in partnership with the national oil company. First, a mid- to long-term strategy, tailored to the goals and the financial capabilities of the company, must be prepared; it must be followed by an ongoing evaluation of quality prospects in various sedimentary basins, and a careful planning and conduct of the operations. To successfully explore overseas also requires the presence on the team of a minimum number of explorationists and engineers thoroughly familiar with the various exploratory and operational aspects of foreign work, having had a considerable amount of onsite experience in various geographical and climatic environments. Independents that are best suited for foreign expansion are those that have been financially successful domestically, and have a good discovery track record. When properly approached foreign exploration is well within the reach of smaller US independents and presents essentially no greater risk than domestic exploration; the reward, however, can be much larger and can catapult the company into the big leagues

  18. International exploration by independent

    International Nuclear Information System (INIS)

    Bertragne, R.G.

    1992-01-01

    Recent industry trends indicate that the smaller U.S. independents are looking at foreign exploration opportunities as one of the alternatives for growth in the new age of exploration. Foreign finding costs per barrel usually are accepted to be substantially lower than domestic costs because of the large reserve potential of international plays. To get involved in overseas exploration, however, requires the explorationist to adapt to different cultural, financial, legal, operational, and political conditions. Generally, foreign exploration proceeds at a slower pace than domestic exploration because concessions are granted by a country's government, or are explored in partnership with a national oil company. First, the explorationist must prepare a mid- to long-term strategy, tailored to the goals and the financial capabilities of the company; next, is an ongoing evaluation of quality prospects in various sedimentary basins, and careful planning and conduct of the operations. To successfully explore overseas also requires the presence of a minimum number of explorationists and engineers thoroughly familiar with the various exploratory and operational aspects of foreign work. Ideally, these team members will have had a considerable amount of on-site experience in various countries and climates. Independents best suited for foreign expansion are those who have been financially successful in domestic exploration. When properly approached, foreign exploration is well within the reach of smaller U.S. independents, and presents essentially no greater risk than domestic exploration; however, the reward can be much larger and can catapult the company into the 'big leagues.'

  19. Agent independent task planning

    Science.gov (United States)

    Davis, William S.

    1990-01-01

    Agent-Independent Planning is a technique that allows the construction of activity plans without regard to the agent that will perform them. Once generated, a plan is then validated and translated into instructions for a particular agent, whether a robot, crewmember, or software-based control system. Because Space Station Freedom (SSF) is planned for orbital operations for approximately thirty years, it will almost certainly experience numerous enhancements and upgrades, including upgrades in robotic manipulators. Agent-Independent Planning provides the capability to construct plans for SSF operations, independent of specific robotic systems, by combining techniques of object oriented modeling, nonlinear planning and temporal logic. Since a plan is validated using the physical and functional models of a particular agent, new robotic systems can be developed and integrated with existing operations in a robust manner. This technique also provides the capability to generate plans for crewmembers with varying skill levels, and later apply these same plans to more sophisticated robotic manipulators made available by evolutions in technology.

  20. International exploration by independents

    International Nuclear Information System (INIS)

    Bertagne, R.G.

    1992-01-01

    Recent industry trends indicate that the smaller U.S. independents are looking at foreign exploration opportunities as one of the alternatives for growth in the new age of exploration. The problems of communications and logistics caused by different cultures and by geographic distances must be carefully evaluated. A mid-term to long-term strategy tailored to the goals and the financial capabilities of the company should be prepared and followed by a careful planning of the operations. This paper addresses some aspects of foreign exploration that should be considered before an independent venture into the foreign field. It also provides some guidelines for conducting successful overseas operations. When properly assessed, foreign exploration is well within the reach of smaller U.S. independents and presents no greater risk than domestic exploration; the rewards, however, can be much larger. Furthermore, the Oil and Gas Journal surveys of the 300 largest U.S. petroleum companies show that companies with a consistent foreign exploration policy have fared better financially during difficult times

  1. Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

    International Nuclear Information System (INIS)

    Dávalos-Salas, Mercedes; Furlan-Magaril, Mayra; González-Buendía, Edgar; Valdes-Quezada, Christian; Ayala-Ortega, Erandi; Recillas-Targa, Félix

    2011-01-01

    Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human retinoblastoma (Rb) gene promoter in different tumoral cell lines. To assess the DNA methylation status of the Rb promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a GFP reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. Rb gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays. We found that the inability of CTCF to bind to the Rb promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation. This study indicates that CTCF plays an important role in maintaining the Rb promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing

  2. Genome-wide DNA methylation profiling identifies ALDH1A3 promoter methylation as a prognostic predictor in G-CIMP- primary glioblastoma.

    Science.gov (United States)

    Zhang, Wei; Yan, Wei; You, Gan; Bao, Zhaoshi; Wang, Yongzhi; Liu, Yanwei; You, Yongping; Jiang, Tao

    2013-01-01

    To date, the aberrations in the DNA methylation patterns that are associated with different prognoses of G-CIMP- primary GBMs remain to be elucidated. Here, DNA methylation profiling of primary GBM tissues from 13 long-term survivors (LTS; overall survival ⩾18months) and 20 short-term survivors (STS; overall survival ⩽9months) was performed. Then G-CIMP+ samples were excluded. The differentially expressed CpG loci were identified between residual 18 STS and 9 LTS G-CIMP- samples. Methylation levels of 11 CpG loci (10genes) were statistically significantly lower, and 43 CpG loci (40genes) were statistically significantly higher in the tumor tissues of LTS than those of STS G-CIMP- samples (PCIMP- samples, 3 CpG loci localized in the promoter of ALDH1A3. Furthermore, using an independent validation cohort containing 37 primary GBM samples without IDH1 mutation and MGMT promoter methylation, the hypermethylation status of ALDH1A3 promoter predicted a better prognosis with an accompanied low expression of ALDH1A3 protein. Taken together, our results defined prognosis-related methylation signatures systematically for the first time in G-CIMP- primary GBMs. ALDH1A3 promoter methylation conferred a favorable prognosis in G-CIMP- primary GBMs. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Thermophysical study of methyl levulinate

    International Nuclear Information System (INIS)

    Lomba, Laura; Lafuente, Carlos; García-Mardones, Mónica; Gascón, Ignacio; Giner, Beatriz

    2013-01-01

    Highlights: • We have carried out a thermophysical characterization of methyl levulinate. • The study has been performed over a temperature range from (278.15 to 328.15) K. • pρT behavior has been studied over a temperature range from (333.15 to 453.15) K. • TRIDEN equation has been used to correlate pρT data. • Results have been compared with of ethyl and butyl levulinate and levulinic acid. -- Abstract: Several thermophysical properties (density, speed of sound, refractive index, surface tension, static permittivity and dynamic viscosity) of methyl levulinate have been measured under atmospheric pressure at temperatures from (278.15 to 338.15) K, while the vapor pressure was determined over a temperature range from (333.15 to 453.15) K. Furthermore, pρT behavior has been also investigated using a high-pressure, high-temperature vibrating tube densimeter over a temperature range from (283.15 to 338.15) K and a pressure range from (0.1 to 60.0) MPa. All these values obtained for methyl levulinate have been compared with other members of the levulinate family and also with levulinic acid

  4. Oxidative stability and ignition quality of algae derived methyl esters containing varying levels of methyl eicosapentaenoate and methyl docosahexaenoate

    Science.gov (United States)

    Bucy, Harrison

    Microalgae is currently receiving strong consideration as a potential biofuel feedstock to help meet the advanced biofuels mandate of the 2007 Energy Independence and Security Act because of its theoretically high yield (gallons/acre/year) in comparison to current terrestrial feedstocks. Additionally, microalgae also do not compete with food and can be cultivated with wastewater on non-arable land. Microalgae lipids can be converted into a variety of biofuels including fatty acid methyl esters (e.g. FAME biodiesel), renewable diesel, renewable gasoline, or jet fuel. For microalgae derived FAME, the fuel properties will be directly related to the fatty acid composition of the lipids produced by the given microalgae strain. Several microalgae species under consideration for wide scale cultivation, such as Nannochloropsis, produce lipids with fatty acid compositions containing substantially higher quantities of long chainpolyunsaturated fatty acids (LC-PUFA) in comparison to terrestrial feedstocks. It is expected that increased levels of LC-PUFA will be problematic in terms of meeting all of the current ASTM specifications for biodiesel. For example, it is known that oxidative stability and cetane number decrease with increasing levels of LC-PUFA. However, these same LC-PUFA fatty acids, such as eicosapentaenoic acid (EPA: C20:5) and docosahexaenoic acid (DHA: C22:6) are known to have high nutritional value thereby making separation of these compounds economically attractive. Given the uncertainty in the future value of these LC-PUFA compounds and the economic viability of the separation process, the goal of this study was to examine the oxidative stability and ignition quality of algae-based FAME with varying levels of EPA and DHA removal. Oxidative stability tests were conducted at a temperature of 110°C and airflow of 10 L/h using a Metrohm 743 Rancimat with automatic induction period determination following the EN 14112 Method from the ASTM D6751 and EN 14214

  5. Epigenomics of Total Acute Sleep Deprivation in Relation to Genome-Wide DNA Methylation Profiles and RNA Expression.

    Science.gov (United States)

    Nilsson, Emil K; Boström, Adrian E; Mwinyi, Jessica; Schiöth, Helgi B

    2016-06-01

    Despite an established link between sleep deprivation and epigenetic processes in humans, it remains unclear to what extent sleep deprivation modulates DNA methylation. We performed a within-subject randomized blinded study with 16 healthy subjects to examine the effect of one night of total sleep deprivation (TSD) on the genome-wide methylation profile in blood compared with that in normal sleep. Genome-wide differences in methylation between both conditions were assessed by applying a paired regression model that corrected for monocyte subpopulations. In addition, the correlations between the methylation of genes detected to be modulated by TSD and gene expression were examined in a separate, publicly available cohort of 10 healthy male donors (E-GEOD-49065). Sleep deprivation significantly affected the DNA methylation profile both independently and in dependency of shifts in monocyte composition. Our study detected differential methylation of 269 probes. Notably, one CpG site was located 69 bp upstream of ING5, which has been shown to be differentially expressed after sleep deprivation. Gene set enrichment analysis detected the Notch and Wnt signaling pathways to be enriched among the differentially methylated genes. These results provide evidence that total acute sleep deprivation alters the methylation profile in healthy human subjects. This is, to our knowledge, the first study that systematically investigated the impact of total acute sleep deprivation on genome-wide DNA methylation profiles in blood and related the epigenomic findings to the expression data.

  6. Integrated analysis of epigenomic and genomic changes by DNA methylation dependent mechanisms provides potential novel biomarkers for prostate cancer.

    Science.gov (United States)

    White-Al Habeeb, Nicole M A; Ho, Linh T; Olkhov-Mitsel, Ekaterina; Kron, Ken; Pethe, Vaijayanti; Lehman, Melanie; Jovanovic, Lidija; Fleshner, Neil; van der Kwast, Theodorus; Nelson, Colleen C; Bapat, Bharati

    2014-09-15

    Epigenetic silencing mediated by CpG methylation is a common feature of many cancers. Characterizing aberrant DNA methylation changes associated with tumor progression may identify potential prognostic markers for prostate cancer (PCa). We treated two PCa cell lines, 22Rv1 and DU-145 with the demethylating agent 5-Aza 2'-deoxycitidine (DAC) and global methylation status was analyzed by performing methylation-sensitive restriction enzyme based differential methylation hybridization strategy followed by genome-wide CpG methylation array profiling. In addition, we examined gene expression changes using a custom microarray. Gene Set Enrichment Analysis (GSEA) identified the most significantly dysregulated pathways. In addition, we assessed methylation status of candidate genes that showed reduced CpG methylation and increased gene expression after DAC treatment, in Gleason score (GS) 8 vs. GS6 patients using three independent cohorts of patients; the publically available The Cancer Genome Atlas (TCGA) dataset, and two separate patient cohorts. Our analysis, by integrating methylation and gene expression in PCa cell lines, combined with patient tumor data, identified novel potential biomarkers for PCa patients. These markers may help elucidate the pathogenesis of PCa and represent potential prognostic markers for PCa patients.

  7. CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer.

    Science.gov (United States)

    Stefansson, Olafur Andri; Hermanowicz, Stefan; van der Horst, Jasper; Hilmarsdottir, Holmfridur; Staszczak, Zuzanna; Jonasson, Jon Gunnlaugur; Tryggvadottir, Laufey; Gudjonsson, Thorkell; Sigurdsson, Stefan

    2017-07-05

    DNA repair of alkylation damage is defective in various cancers. This occurs through somatically acquired inactivation of the MGMT gene in various cancer types, including breast cancers. In addition to MGMT, the two E. coli AlkB homologs ALKBH2 and ALKBH3 have also been linked to direct reversal of alkylation damage. However, it is currently unknown whether ALKBH2 or ALKBH3 are found inactivated in cancer. Methylome datasets (GSE52865, GSE20713, GSE69914), available through Omnibus, were used to determine whether ALKBH2 or ALKBH3 are found inactivated by CpG promoter methylation. TCGA dataset enabled us to then assess the impact of CpG promoter methylation on mRNA expression for both ALKBH2 and ALKBH3. DNA methylation analysis for the ALKBH3 promoter region was carried out by pyrosequencing (PyroMark Q24) in 265 primary breast tumours and 30 proximal normal breast tissue samples along with 8 breast-derived cell lines. ALKBH3 mRNA and protein expression were analysed in cell lines using RT-PCR and Western blotting, respectively. DNA alkylation damage assay was carried out in cell lines based on immunofluorescence and confocal imaging. Data on clinical parameters and survival outcomes in patients were obtained and assessed in relation to ALKBH3 promoter methylation. The ALKBH3 gene, but not ALKBH2, undergoes CpG promoter methylation and transcriptional silencing in breast cancer. We developed a quantitative alkylation DNA damage assay based on immunofluorescence and confocal imaging revealing higher levels of alkylation damage in association with epigenetic inactivation of the ALKBH3 gene (P = 0.029). In our cohort of 265 primary breast cancer, we found 72 cases showing aberrantly high CpG promoter methylation over the ALKBH3 promoter (27%; 72 out of 265). We further show that increasingly higher degree of ALKBH3 promoter methylation is associated with reduced breast-cancer specific survival times in patients. In this analysis, ALKBH3 promoter methylation at >20

  8. Methylation in the promoter regions of WT1, NKX6-1 and DBC1 genes in cervical cancer tissues of Uygur women in Xinjiang

    Directory of Open Access Journals (Sweden)

    Dan Wu

    Full Text Available Abstract This study aimed to explore: 1 DNA methylation in the promoter regions of Wilms tumor gene 1 (WT1, NK6 transcription factor related locus 1 gene (NKX6-1 and Deleted in bladder cancer 1 (DBC1 gene in cervical cancer tissues of Uygur women in Xinjiang, and 2 the correlation of gene methylation with the infection of HPV16/18 viruses. We detected HPV16/18 infection in 43 normal cervical tissues, 30 cervical intraepithelial neoplasia lesions (CIN and 48 cervical cancer tissues with polymerase chain reaction (PCR method. Methylation in the promoter regions of the WT1, NKX6-1 and DBC1 genes in the above-mentioned tissues was measured by methylation-specific PCR (MSP and cloning sequencing. The expression level of these three genes was measured by real-time PCR (qPCR in 10 methylation-positive cervical cancer tissues and 10 methylation-negative normal cervical tissues. We found that the infection of HPV16 in normal cervical tissues, CIN and cervical cancer tissues was 14.0, 36.7 and 66.7%, respectively. The infection of HPV18 was 0, 6.7 and 10.4%, respectively. The methylation rates of WT1, NKX6-1 and DBC1 genes were 7.0, 11.6 and 23.3% in normal cervical tissues, 36.7, 46.7 and 30.0% in CIN tissues, and 89.6, 77.1 and 85.4% in cervical cancer tissues. Furthermore, WT1, NKX6-1 and DBC1 genes were hypermethylated in the high-grade squamous intraepithelial lesion (CIN2, CIN3 and in the cervical cancer tissues with infection of HPV16/18 (both P< 0.05. The expression of WT1, NKX6-1 and DBC1 was significantly lower in the methylation-positive cervical cancer tissues than in methylation-negative normal cervical tissues. Our findings indicated that methylation in the promoter regions of WT1, NKX6-1 and DBC1 is correlated with cervical cancer tumorigenesis in Uygur women. The infection of HPV16/18 might be correlated with methylation in these genes. Gene inactivation caused by methylation might be related to the incidence and development of cervical

  9. Correlation of MLH1 and MGMT expression and promoter methylation with genomic instability in patients with thyroid carcinoma

    International Nuclear Information System (INIS)

    Santos, Juliana Carvalho; Bastos, André Uchimura; Cerutti, Janete Maria; Ribeiro, Marcelo Lima

    2013-01-01

    Gene silencing of the repair genes MLH1 and MGMT was shown to be a mechanism underlying the development of microsatellite instability (MSI), a phenotype frequently associated with various human malignancies. Recently, aberrant methylation of MLH1, MGMT and MSI were shown to be associated with mutations in genes such as BRAF, RAS and IDH1 in colon and brain tumours. Little is known about the methylation status of MLH1 and MGMT in thyroid tumours and its association with MSI and mutational status. In a series of 96 thyroid tumours whose mutational profiles of BRAF, IDH1 and NRAS mutations and RET/PTC were previously determined, we investigated MLH1 and MGMT expression and methylation status by qPCR and methylation-specific PCR after bisulphite treatment, respectively. MSI was determined by PCR using seven standard microsatellite markers. Samples with point mutations (BRAF, IDH1 and NRAS) show a decrease in MLH1 expression when compared to negative samples. Additionally, malignant lesions show a higher MSI pattern than benign lesions. The MSI phenotype was also associated with down-regulation of MLH1. The results of this study allow us to conclude that low expression of MLH1 is associated with BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) in patients with thyroid carcinoma. In addition, a significant relationship between MSI status and histological subtypes was found

  10. Promoter methylation-associated loss of ID4 expression is a marker of tumour recurrence in human breast cancer

    International Nuclear Information System (INIS)

    Noetzel, Erik; Veeck, Jürgen; Niederacher, Dieter; Galm, Oliver; Horn, Felicitas; Hartmann, Arndt; Knüchel, Ruth; Dahl, Edgar

    2008-01-01

    Inhibitor of DNA binding/Inhibitor of differentiation 4 (ID4) is a critical factor for cell proliferation and differentiation in normal vertebrate development. ID4 has regulative functions for differentiation and growth of the developing brain. The role of ID1, ID2 and ID3 are expected to be oncogenic due to their overexpression in pancreatic cancer and colorectal adenocarcinomas, respectively. Aside from these findings, loss of ID3 expression was demonstrated in ovarian cancer. The aim of the present study was to reveal the factual role of ID4 in carcinogenesis in more detail, since its role for the pathogenesis of human breast cancer has been discussed controversially, assigning both oncogenic and tumour suppressive functions. ID4 promoter methylation, ID4 mRNA expression and ID4 protein expression were analysed in primary human breast cancer specimens using methylation-specific PCR (MSP) (n=170), semiquantitative realtime RT-PCR (n=46) and immunhistochemistry (n=3), respectively. In order to demonstrate a functional association of ID4 promoter methylation with its gene silencing, we performed DNA demethylation analysis with four human breast cell lines using MSP and semiquantitative realtime RT-PCR. In addition, we performed correlations of ID4 promoter methylation with ID4 mRNA and ID4 protein expression in matched samples of breast tumour and corresponding normal tissue. We carried out statistical analyses in order to find correlations between ID4 promoter methylation and clinicopathological parameters. Frequent ID4 promoter methylation was observed in primary breast cancer samples (69%, 117/170). We found a tight correlation (P<0.0001) between ID4 promoter methylation and loss of ID4 expression in primary breast cancer 3 specimens. Demethylating treatment with breast cancer cell lines was associated with clear ID4 mRNA re-expression. Tumours with ID4 promoter methylation showed distinct loss of ID4 expression on both transcription and protein level

  11. SASqPCR: robust and rapid analysis of RT-qPCR data in SAS.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available Reverse transcription quantitative real-time PCR (RT-qPCR is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at http://code.google.com/p/sasqpcr/downloads/list.

  12. WetLab-2: Providing Quantitative PCR Capabilities on ISS

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy Kar Chuen; Almeida, Eduardo; Boone, Travis David; Schonfeld, Julie; Tran, Luan Hoang

    2015-01-01

    The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a system capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project has developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage that it uses non-toxic chemicals, alcohols or other organics. The resulting RNA is transferred into a pipette and then dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. These reaction tubes are mounted on rotors to centrifuge the liquid to the reaction window of the tube using a cordless drill. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The resulting process takes less than 30 min to have tubes ready for loading into the qRT-PCR unit.The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, the Cepheid SmartCycler, that will fly in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid thermal ramp times and four-color detection. The ability to detect up to four fluorescent channels will enable multiplex assays that can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system will have the capability to downlink data from the ISS to the ground after a completed run and to uplink new programs. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The

  13. The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

    Science.gov (United States)

    Daugaard, Iben; Kjeldsen, Tina E; Hager, Henrik; Hansen, Lise Lotte; Wojdacz, Tomasz K

    2015-12-01

    Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Independence in appearance

    DEFF Research Database (Denmark)

    Warming-Rasmussen, Bent; Quick, Reiner; Liempd, Dennis van

    2011-01-01

    In the wake of the financial crisis, the EU Commission has published a Green Paper on the future role of the audit function in Europe. The Green Paper lists a number of proposals for tighter rules for audits and auditors in order to contribute to stabilizing the financial system. The present...... article presents research contributions to the question whether the auditor is to continue to provide both audit and non-audit services (NAS) to an audit client. Research results show that this double function for the same audit client is a problem for stakeholders' confidence in auditor independence...

  15. Evolution of DNA Methylation across Insects.

    Science.gov (United States)

    Bewick, Adam J; Vogel, Kevin J; Moore, Allen J; Schmitz, Robert J

    2017-03-01

    DNA methylation contributes to gene and transcriptional regulation in eukaryotes, and therefore has been hypothesized to facilitate the evolution of plastic traits such as sociality in insects. However, DNA methylation is sparsely studied in insects. Therefore, we documented patterns of DNA methylation across a wide diversity of insects. We predicted that underlying enzymatic machinery is concordant with patterns of DNA methylation. Finally, given the suggestion that DNA methylation facilitated social evolution in Hymenoptera, we tested the hypothesis that the DNA methylation system will be associated with presence/absence of sociality among other insect orders. We found DNA methylation to be widespread, detected in all orders examined except Diptera (flies). Whole genome bisulfite sequencing showed that orders differed in levels of DNA methylation. Hymenopteran (ants, bees, wasps and sawflies) had some of the lowest levels, including several potential losses. Blattodea (cockroaches and termites) show all possible patterns, including a potential loss of DNA methylation in a eusocial species whereas solitary species had the highest levels. Species with DNA methylation do not always possess the typical enzymatic machinery. We identified a gene duplication event in the maintenance DNA methyltransferase 1 (DNMT1) that is shared by some Hymenoptera, and paralogs have experienced divergent, nonneutral evolution. This diversity and nonneutral evolution of underlying machinery suggests alternative DNA methylation pathways may exist. Phylogenetically corrected comparisons revealed no evidence that supports evolutionary association between sociality and DNA methylation. Future functional studies will be required to advance our understanding of DNA methylation in insects. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  16. Restoration of CpG Methylation in The Egf Promoter Region during Rat Liver Regeneration

    Science.gov (United States)

    Deming, Li; Ziwei, Li; Xueqiang, Guo; Cunshuan, Xu

    2015-01-01

    Epidermal growth factor (EGF) is an important factor for healing after tissue damage in diverse experimental models. It plays an important role in liver regeneration (LR). The objective of this experiment is to investigate the methylation variation of 10 CpG sites in the Egf promoter region and their relevance to Egf expression during rat liver regenera- tion. As a follow up of our previous study, rat liver tissue was collected after rat 2/3 partial hepatectomy (PH) during the re-organization phase (from days 14 to days 28). Liver DNA was extracted and modified by sodium bisulfate. The methylation status of 10 CpG sites in Egf promoter region was determined using bisulfite sequencing polymerase chain reaction (PCR), as BSP method. The results showed that 3 (sites 3, 4 and 9) out of 10 CpG sites have strikingly methylation changes during the re-organization phase compared to the regeneration phase (from 2 hours to 168 hours, P=0.002, 0.048 and 0.018, respectively). Our results showed that methylation modification of CpGs in the Egf promoter region could be restored to the status before PH operation and changes of methylation didn’t affect Egf mRNA expression during the re-organization phase. PMID:26464832

  17. Methylation of class II transactivator gene promoter IV is not associated with susceptibility to Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Lincoln Matthew R

    2008-07-01

    Full Text Available Abstract Background Multiple sclerosis (MS is a complex trait in which alleles at or near the class II loci HLA-DRB1 and HLA-DQB1 contribute significantly to genetic risk. The MHC class II transactivator (MHC2TA is the master controller of expression of class II genes, and methylation of the promoter of this gene has been previously been shown to alter its function. In this study we sought to assess whether or not methylation of the MHC2TA promoter pIV could contribute to MS disease aetiology. Methods In DNA from peripheral blood mononuclear cells from a sample of 50 monozygotic disease discordant MS twins the MHC2TA promoter IV was sequenced and analysed by methylation specific PCR. Results No methylation or sequence variation of the MHC2TA promoter pIV was found. Conclusion The results of this study cannot support the notion that methylation of the pIV promoter of MHC2TA contributes to MS disease risk, although tissue and timing specific epigenetic modifications cannot be ruled out.

  18. Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss.

    Science.gov (United States)

    Baek, Jong Min; Kim, Ju-Young; Lee, Chang Hoon; Yoon, Kwon-Ha; Lee, Myeung Su

    2017-03-07

    In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP) staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca 2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca 2+ influx intensity (staining with Fluo-3/AM). Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

  19. Methyl Gallate Inhibits Osteoclast Formation and Function by Suppressing Akt and Btk-PLCγ2-Ca2+ Signaling and Prevents Lipopolysaccharide-Induced Bone Loss

    Directory of Open Access Journals (Sweden)

    Jong Min Baek

    2017-03-01

    Full Text Available In the field of bone research, various natural derivatives have emerged as candidates for osteoporosis treatment by targeting abnormally elevated osteoclastic activity. Methyl gallate, a plant-derived phenolic compound, is known to have numerous pharmacological effects against inflammation, oxidation, and cancer. Our purpose was to explore the relation between methyl gallate and bone metabolism. Herein, we performed screening using methyl gallate by tartrate resistant acid phosphatase (TRAP staining and revealed intracellular mechanisms responsible for methyl gallate-mediated regulation of osteoclastogenesis by Western blotting and quantitative reverse transcription polymerase chain reaction (RT-PCR. Furthermore, we assessed the effects of methyl gallate on the characteristics of mature osteoclasts. We found that methyl gallate significantly suppressed osteoclast formation through Akt and Btk-PLCγ2-Ca2+ signaling. The blockade of these pathways was confirmed through transduction of cells with a CA-Akt retrovirus and evaluation of Ca2+ influx intensity (staining with Fluo-3/AM. Indeed, methyl gallate downregulated the formation of actin ring-positive osteoclasts and resorption pit areas. In agreement with in vitro results, we found that administration of methyl gallate restored osteoporotic phenotype stimulated by acute systemic injection of lipopolysaccharide in vivo according to micro-computed tomography and histological analysis. Our data strongly indicate that methyl gallate may be useful for the development of a plant-based antiosteoporotic agent.

  20. Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate.

    Science.gov (United States)

    Olejniczak, E; Lee, H J

    1984-06-01

    The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.

  1. Genome-wide DNA methylation profiling in cultured eutopic and ectopic endometrial stromal cells.

    Directory of Open Access Journals (Sweden)

    Yoshiaki Yamagata

    Full Text Available The objective of this study was to characterize the genome-wide DNA methylation profiles of isolated endometrial stromal cells obtained from eutopic endometria with (euESCa and without endometriosis (euESCb and ovarian endometrial cysts (choESC. Three samples were analyzed in each group. The infinium methylation array identified more hypermethylated and hypomethylated CpGs in choESC than in euESCa, and only a few genes were methylated differently in euESCa and euESCb. A functional analysis revealed that signal transduction, developmental processes, immunity, etc. were different in choESC and euESCa. A clustering analysis and a principal component analysis performed based on the methylation levels segregated choESC from euESC, while euESCa and euESCb were identical. A transcriptome analysis was then conducted and the results were compared with those of the DNA methylation analysis. Interestingly, the hierarchical clustering and principal component analyses showed that choESC were segregated from euESCa and euESCb in the DNA methylation analysis, while no segregation was recognized in the transcriptome analysis. The mRNA expression levels of the epigenetic modification enzymes, including DNA methyltransferases, obtained from the specimens were not significantly different between the groups. Some of the differentially methylated and/or expressed genes (NR5A1, STAR, STRA6 and HSD17B2, which are related with steroidogenesis, were validated by independent methods in a larger number of samples. Our findings indicate that different DNA methylation profiles exist in ectopic ESC, highlighting the benefits of genome wide DNA methylation analyses over transcriptome analyses in clarifying the development and characterization of endometriosis.

  2. SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

    International Nuclear Information System (INIS)

    Schmidt, Bernd; Lewin, Jörn; Tetzner, Reimo; Weickmann, Sabine; Wille, Ulrike; Liloglou, Triantafillos; Raji, Olaide; Walshaw, Martin; Fleischhacker, Michael; Witt, Christian; Field, John K; Liebenberg, Volker; Dietrich, Dimo; Schlegel, Thomas; Kneip, Christoph; Seegebarth, Anke; Flemming, Nadja; Seemann, Stefanie; Distler, Jürgen

    2010-01-01

    This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment. Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance. Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]). Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous

  3. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  4. Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

    Directory of Open Access Journals (Sweden)

    Robinson Claire M

    2012-08-01

    Full Text Available Abstract Background Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu. As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. Methods CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR was used to examine the methylation status of the Thy-1 promoter. Results Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2′-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. Conclusion These data suggest that global and

  5. NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Shen Shourong

    2010-04-01

    Full Text Available Abstract Background Nasopharyngeal carcinoma associated gene 6 (NGX6 is down-regulated in most colon cancer cell lines and tumor tissues when compared with their normal tissue samples. As a novel suppress tumor gene, it could inhibit colon cancer cell growth and cell cycle progression. However, little is known about the transcriptional mechanisms controlling NGX6 gene expression. Recent findings suggest that epigenetic inactivation of multiple tumor suppressor genes plays an important role in the tumorigenesis of colorectal carcinoma (CRC. In this study, we explored the role of DNA methylation in regulation of NGX6 transcription. Methods In the present study, we cloned the NGX6 promoter with characteristics of a CpG island by luciferase reporter assay. Then, the CpG methylation status around the NGX6 promoter region in colon cancer cell lines and colorectal tumor tissues was examined by methylation-specific PCR and bisulfite DNA sequencing. Finally, 5-Aza-2'-deoxycytidine (5-Aza-dC treatment was used to confirm the correlation between NGX6 promoter methylation and its gene inactivation. Results The sequence spanning positions -157 to +276 was identified as the NGX6 promoter, in which no canonical TATA boxes were found, while two CAAT boxes and GC boxes were discovered. Methylation status was observed more frequently in 40 colorectal cancer samples than in 40 adjacent normal mucosa samples (18/40 versus 7/40; P Conclusions Down-regulation of NGX6 gene is related to the promoter methylation. DNA methylation of NGX6 promoter might be a potential molecular marker for diagnosis or prognosis, or serve as a therapeutic target.

  6. Aberrant Methylation-Mediated Suppression of APAF1 in Myelodysplastic Syndrome.

    Science.gov (United States)

    Zaker, Farhad; Nasiri, Nahid; Amirizadeh, Naser; Razavi, Seyed Mohsen; Yaghmaie, Marjan; Teimoori-Toolabi, Ladan; Maleki, Ali; Bakhshayesh, Masoumeh

    2017-04-01

    Background: Myelodysplastic syndromes (MDSs) include a diverse group of clonal bone marrow disorders characterized by ineffective hematopoiesis and pancytopenia. It was found that down regulation of APAF1, a putative tumor suppressor gene (TSG), leads to resistance to chemotherapy and disease development in some cancers. In this study, we investigated the relation of APAF1 methylation status with its expression and clinicopathological factors in myelodysplastic syndrome (MDS) patients. Materials and Methods: Methylation Sensitive-High Resolution Melting Curve Analysis (MS-HRM) was employed in studying the methylation of CpG islands in the APAF1promoter region in MDS. Gene expression was analyzed by using real time RT-PCR. Results: 42.6% of patient samples were methylated in promoter region of APAF1analyzed, while methylation of the gene was not seen in controls (P<0.05). Methylation of APAF1was significantly associated with the suppression of its mRNA expression (P=0.00). The methylation status of APAF1in advanced-stage MDS patients (80%) was significantly higher than that of the early-stage MDS patients (28.2%) (P=0.001). The difference in frequency of hypermethylatedAPAF1 gene was significant between good (37.5%) and poor (85.71%) cytogenetic risk groups (P=0.043). In addition, a higher frequency of APAF1hypermethylation was observed in higher-risk MDS group (69.2%) compared to lower-risk MDS group (34.14%) (P=0.026). Conclusion: Our study indicated that APAF1hypermethylation in MDS was associated to high-risk disease classified according to the IPSS, WHO and cytogenetic risk.

  7. Methylation status and protein expression of RASSF1A in breast cancer patients.

    Science.gov (United States)

    Hagrass, Hoda A; Pasha, Heba F; Shaheen, Mohamed A; Abdel Bary, Eman H; Kassem, Rasha

    2014-01-01

    Recently genetics and epigenetics alterations have been found to be characteristic of malignancy and hence can be used as targets for detection of neoplasia. RAS association domain family protein 1A (RASSF1A) gene hypermethylation has been a subject of interest in recent researches on cancer breast patients. The aim of the present study was to evaluate whether RASSF1A methylation status and RASSF1A protein expression are associated with the major clinico-pathological parameters. One hundred and twenty breast cancer Egyptian patients and 100-control subjects diagnosed with benign lesions of the breast were enrolled in this study. We evaluated RASSF1A methylation status in tissue and serum samples using Methyl specific PCR together with RASSF1A protein expression in tissues by immunohistochemistry. Results were studied in relation to known prognostic clinicopathological features in breast cancer. Frequency of RASSF1A methylation in tissues and serum were 70 and 63.3 % respectively and RASSF1A protein expression showed frequency of 46.7 %. There was an association between RASSF1A methylation in tissues, serum and loss of protein expression in tissues with invasive carcinoma, advanced stage breast cancer, L.N. metastasis, ER/PR and HER2 negativity. RASSF1A methylation in serum showed high degree of concordance with methylation in tissues (Kappa = 0.851, P < 0.001). RASSF1A hypermethylation in tissues and serum and its protein expression may be a valid, reliable and sensitive tool for detection and follow up of breast cancer patients.

  8. Methylation of the SPARC gene promoter and its clinical implication in pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Lv Shunli

    2010-03-01

    Full Text Available Abstract Background The secreted protein acidic and rich in cysteine (SPARC plays a pivotal role in regulating cell-matrix interactions and tumor angiogenesis, proliferation, and migration. Detection of SPARC gene methylation may be useful as a tumorigenesis marker for early detection of pancreatic cancer. Methods Methylation of the SPARC gene transcriptional regulation region (TRR was detected using bisulfite-specific (BSP PCR-based sequencing analysis in 40 cases of pancreatic cancer and the adjacent normal tissues, 6 chronic pancreatitis tissues, and 6 normal pancreatic tissues. BSP cloning-based sequencing analysis was also performed in selected cases. Clinicopathological data from the cancer patients were collected and analyzed. Results Analysis of SPARC gene TRR methylation showed two hypermethylation wave peak regions: CpG Region 1 (CpG site 1-7 and CpG Region 2 (CpG site 8-12. Pancreatic tissues have shown methylation in both regions with gradual increases from normal, chronic pancreatitis, and adjacent normal tissues to cancerous tissues. However, Methylation of CpG Region 2 was more sensitive than CpG Region 1 in pancreatic tumorigenesis. Furthermore, the methylation level of CpG Region 2 was associated with increased tumor size and exposure to the risk factors (tobacco smoke and alcohol consumption for developing pancreatic cancer. Conclusion Methylation of the SPARC gene, specifically CpG Region 2, may be an early event during pancreatic tumorigenesis and should be further evaluated as a tumorigenesis marker for early detection of pancreatic cancer.

  9. A high-throughput and sensitive method to measure Global DNA Methylation: Application in Lung Cancer

    Directory of Open Access Journals (Sweden)

    Mamaev Sergey

    2008-08-01

    Full Text Available Abstract Background Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets. Methods We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment. CpGlobal utilizes methyl-sensitive restriction enzymes, HRP Neutravidin to detect the biotinylated nucleotides incorporated in an end-fill reaction and a luminometer to measure the chemiluminescence. The assay shows high accuracy and reproducibility in measuring global DNA methylation. Furthermore, CpGlobal correlates significantly with High Performance Capillary Electrophoresis (HPCE, a gold standard technology. We have applied the technology to understand the role of global DNA methylation in the natural history of lung cancer. World-wide, it is the leading cause of death attributed to any cancer. The survival rate is 15% over 5 years due to the lack of any clinical symptoms until the disease has progressed to a stage where cure is limited. Results Through the use of cell lines and paired normal/tumor samples from patients with non-small cell lung cancer (NSCLC we show that global DNA hypomethylation is highly associated with the progression of the tumor. In addition, the results provide the first indication that the normal part of the lung from a cancer patient has already experienced a loss of methylation compared to a normal individual. Conclusion By detecting these changes in global DNA methylation, CpGlobal may have a role as a barometer for the onset and development of lung cancer.

  10. 2-Methyl-1H-benzimidazol-3-ium hydrogen phthalate

    Directory of Open Access Journals (Sweden)

    YuanQi Yu

    2011-10-01

    Full Text Available The asymmetric unit of the title compound, C8H9N2+·C8H5O4−, contains two independent ion pairs. In each 2-methyl-1H-benzimidazolium ion, an intramolecular O—H...O bond forms an S(7 graph-set motif. In the crystal, the components are linked by N—H...O hydrogen bonds, forming chains along [210]. Further stabilization is provided by weak C—H...O hydrogen bonds.

  11. Developmental immunotoxicity testing of 4-methyl anisole.

    Science.gov (United States)

    Tonk, Elisa C M; Verhoef, Aart; Gremmer, Eric R; van Loveren, Henk; Piersma, Aldert H

    2015-07-01

    The developmental immunotoxicity of 4-methyl anisole (4MA) was investigated in the rat. Four study designs were used, with either premating or post-weaning onset of exposure, continued to postnatal day 50, and with or without additional oral gavage of pups from postnatal day 10 onward. Reduced litter size (benchmark dose lower confidence limit (BMDL) 80mg/kg bw/day) was the most sensitive developmental parameter, with pup relative organ weight effects observed at similar BMDLs, in the absence of maternal toxicity. Eosinophil numbers were reduced at lower doses (BMDL 16mg/kg bw/day). KLH challenge resulted in increased IL-13 and TNF-α responses, and variably reduced IgG production (BMDL 27mg/kg bw/day). T4 levels were reduced by 11% at maximum with a BMDL of 73mg/kg bw/day. Differences between exposure cohorts were limited and were considered to be without biological significance. This study shows that 4MA induces developmental immunotoxicity at doses below those inducing developmental and general toxicity. These observations being independent of the study designs applied suggest that the post-weaning period, included in all designs, is the most relevant sensitive period for inducing 4MA mediated developmental immunotoxicity. Moreover, this study stresses the importance of including developmental immunotoxicity testing by default in regulatory toxicology. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Methylation patterns in marginal zone lymphoma.

    Science.gov (United States)

    Arribas, Alberto J; Bertoni, Francesco

    Promoter DNA methylation is a major regulator of gene expression and transcription. The identification of methylation changes is important for understanding disease pathogenesis, for identifying prognostic markers and can drive novel therapeutic approaches. In this review we summarize the current knowledge regarding DNA methylation in MALT lymphoma, splenic marginal zone lymphoma, nodal marginal zone lymphoma. Despite important differences in the study design for different publications and the existence of a sole large and genome-wide methylation study for splenic marginal zone lymphoma, it is clear that DNA methylation plays an important role in marginal zone lymphomas, in which it contributes to the inactivation of tumor suppressors but also to the expression of genes sustaining tumor cell survival and proliferation. Existing preclinical data provide the rationale to target the methylation machinery in these disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Wp specific methylation of highly proliferated LCLs

    International Nuclear Information System (INIS)

    Park, Jung-Hoon; Jeon, Jae-Pil; Shim, Sung-Mi; Nam, Hye-Young; Kim, Joon-Woo; Han, Bok-Ghee; Lee, Suman

    2007-01-01

    The epigenetic regulation of viral genes may be important for the life cycle of EBV. We determined the methylation status of three viral promoters (Wp, Cp, Qp) from EBV B-lymphoblastoid cell lines (LCLs) by pyrosequencing. Our pyrosequencing data showed that the CpG region of Wp was methylated, but the others were not. Interestingly, Wp methylation was increased with proliferation of LCLs. Wp methylation was as high as 74.9% in late-passage LCLs, but 25.6% in early-passage LCLs. From two Burkitt's lymphoma cell lines, Wp specific hypermethylation was also found (>80%). Interestingly, the expression of EBNA2 gene which located directly next to Wp was associated with its methylation. Our data suggested that Wp specific methylation may be important for the indicator of the proliferation status of LCLs, and the epigenetic viral gene regulation of EBNA2 gene by Wp should be further defined possibly with other biological processes

  14. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

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    Eric J Chater-Diehl

    Full Text Available The molecular basis of Fetal Alcohol Spectrum Disorders (FASD is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

  15. Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

    Science.gov (United States)

    Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

    2016-01-01

    The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

  16. The importance of board independence

    NARCIS (Netherlands)

    Zijl, N.J.M.

    2012-01-01

    Although the attributed importance of board independence is high, a clear definition of independence does not exist. Furthermore, the aim and consequences of independence are the subject of discussion and empirical evidence about the impact of independence is weak and disputable. Despite this lack

  17. From the 'PCR' function to the 'PCR' profession; de la fonction 'PCR' au metier 'PCR'

    Energy Technology Data Exchange (ETDEWEB)

    Perrin, L. [CERAP, 91 - Gif sur Yvette (France)

    2008-07-01

    After having recalled the legal context concerning the appointment and training of a radiation protection expert (PCR for 'personne competente en radioprotection'), the author outlines that the PCR's role has notably evolved: his function is now of primary importance in the company and his activity does not correspond to the legal framework any longer. Moreover, with the application of a European directive, some small establishments possessing ionizing radiation sources are disadvantaged, and the PCR is now facing an increasing number of missions and tasks. The author gives a list of them and assesses a needed time of 146 days per year: this means PCRs cannot have an other activity within their company

  18. Copper induces expression and methylation changes of early development genes in Crassostrea gigas embryos.

    Science.gov (United States)

    Sussarellu, Rossana; Lebreton, Morgane; Rouxel, Julien; Akcha, Farida; Rivière, Guillaume

    2018-03-01

    Copper contamination is widespread along coastal areas and exerts adverse effects on marine organisms such as mollusks. In the Pacific oyster, copper induces severe developmental abnormalities during early life stages; however, the underlying molecular mechanisms are largely unknown. This study aims to better understand whether the embryotoxic effects of copper in Crassostrea gigas could be mediated by alterations in gene expression, and the putative role of DNA methylation, which is known to contribute to gene regulation in early embryo development. For that purpose, oyster embryos were exposed to 4 nominal copper concentrations (0.1, 1, 10 and 20 μg L -1 Cu 2+ ) during early development assays. Embryotoxicity was monitored through the oyster embryo-larval bioassay at the D-larva stage 24 h post fertilization (hpf) and genotoxicity at gastrulation 7 hpf. In parallel, the relative expression of 15 genes encoding putative homeotic, biomineralization and DNA methylation proteins was measured at three developmental stages (3 hpf morula stage, 7 hpf gastrula stage, 24 hpf D-larvae stage) using RT-qPCR. Global DNA content in methylcytosine and hydroxymethylcytosine were measured by HPLC and gene-specific DNA methylation levels were monitored using MeDIP-qPCR. A significant increase in larval abnormalities was observed from copper concentrations of 10 μg L -1 , while significant genotoxic effects were detected at 1 μg L -1 and above. All the selected genes presented a stage-dependent expression pattern, which was impaired for some homeobox and DNA methylation genes (Notochord, HOXA1, HOX2, Lox5, DNMT3b and CXXC-1) after copper exposure. While global DNA methylation (5-methylcytosine) at gastrula stage didn't show significant changes between experimental conditions, 5-hydroxymethylcytosine, its degradation product, decreased upon copper treatment. The DNA methylation of exons and the transcript levels were correlated in control samples for HOXA1 but such

  19. PCR melting profile (PCR MP - a new tool for differentiation of Candida albicans strains

    Directory of Open Access Journals (Sweden)

    Nowak Magdalena

    2009-11-01

    Full Text Available Abstract Background We have previously reported the use of PCR Melting Profile (PCR MP technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains. Methods In total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE and RAPD techniques. Results The genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD. Conclusion Data presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

  20. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    Science.gov (United States)

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  1. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    Science.gov (United States)

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  2. Autonomy, Independence, Inclusion

    Directory of Open Access Journals (Sweden)

    Filippo Angelucci

    2015-04-01

    Full Text Available The living environment must not only meet the primary needs of living, but also the expectations of improvement of life and social relations and people’s work. The need for a living environment that responds to the needs of users with their different abilities, outside of standardizations, is increasingly felt as autonomy, independence and well-being are the result of real usability and adaptability of the spaces. The project to improve the inclusivity of living space and to promote the rehabilitation of fragile users need to be characterized as an interdisciplinary process in which the integration of specialized contributions leads to adaptive customization of space solutions and technological that evolve with the changing needs, functional capacities and abilities of individuals.

  3. Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M Heiat

    2010-07-01

    Full Text Available Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.

  4. DNA methylation analysis in rat kidney epithelial cells exposed to 3-MCPD and glycidol.

    Science.gov (United States)

    Senyildiz, Mine; Alpertunga, Buket; Ozden, Sibel

    2017-10-01

    3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been regarded as a rat carcinogen, which is known to induce Leydig-cell and mammary gland tumors in males, as well as kidney tumors in both genders. 3-MCPD is highly suspected to be a non-genotoxic carcinogen. 2,3-Epoxy-1-propanol (glycidol) can be formed via dehalogenation from 3-MCPD. We aimed to investigate the cytotoxic effects of 3-MCPD and glycidol, then to demonstrate the possible epigenetic mechanisms with global and gene-specific DNA methylation in rat kidney epithelial cells (NRK-52E). IC 50 value of 3-MCPD was determined as 48 mM and 41.39 mM, whereas IC 50 value of glycidol was 1.67 mM and 1.13 mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100 μM and 1000 μM for 3-MCPD and 100 μM and 500 μM for glycidol were observed after 48 h exposure by using 5-methylcytosine (5-mC) ELISA kit. Methylation changes were detected in promoter regions of c-myc and Rassf1a in 3-MCPD and glycidol treated NRK-52E cells by using methylation-specific PCR (MSP), whereas changes on gene expression of c-myc and Rassf1a were observed by using real-time PCR. However, e-cadherin, p16, VHL and p15 genes were unmethylated in their CpG promoter regions in response to treatment with 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol.

  5. Reduction of heteroduplex formation in PCR amplification

    Czech Academy of Sciences Publication Activity Database

    Michu, Elleni; Mráčková, Martina; Vyskot, Boris; Žlůvová, Jitka

    2010-01-01

    Roč. 54, č. 1 (2010), s. 173-176 ISSN 0006-3134 R&D Projects: GA AV ČR(CZ) KJB600040801; GA ČR(CZ) GD204/09/H002; GA AV ČR(CZ) IAA600040801; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : polymerase chain reaction * reconditioning PCR * mixed-template PCR Subject RIV: BO - Biophysics Impact factor: 1.582, year: 2010

  6. A naked-eye colorimetric "PCR developer"

    Science.gov (United States)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  7. Structure, bioactivity, and synthesis of methylated flavonoids.

    Science.gov (United States)

    Wen, Lingrong; Jiang, Yueming; Yang, Jiali; Zhao, Yupeng; Tian, Miaomiao; Yang, Bao

    2017-06-01

    Methylated flavonoids are an important type of natural flavonoid derivative with potentially multiple health benefits; among other things, they have improved bioavailability compared with flavonoid precursors. Flavonoids have been documented to have broad bioactivities, such as anticancer, immunomodulation, and antioxidant activities, that can be elevated, to a certain extent, by methylation. Understanding the structure, bioactivity, and bioavailability of methylated flavonoids, therefore, is an interesting topic with broad potential applications. Though methylated flavonoids are widely present in plants, their levels are usually low. Because developing efficient techniques to produce these chemicals would likely be beneficial, we provide an overview of their chemical and biological synthesis. © 2017 New York Academy of Sciences.

  8. Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC.

    Science.gov (United States)

    Scarpa, Marco; Scarpa, Melania; Castagliuolo, Ignazio; Erroi, Francesca; Kotsafti, Andromachi; Basato, Silvia; Brun, Paola; D'Incà, Renata; Rugge, Massimo; Angriman, Imerio; Castoro, Carlo

    2016-03-01

    BACKGROUND PROMOTER: hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC. The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC. Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR. Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance.

  9. Association of diminished expression of RASSF1A with promoter methylation in primary gastric cancer from patients of central China

    Directory of Open Access Journals (Sweden)

    Zhou Feng

    2007-07-01

    Full Text Available Abstract Background Although methylation-mediated inactivation of expression of RASSF1A, a candidate tumor suppressor gene, has been observed in several human cancers, the data concerning alteration of RASSF1A expression and methylation in Chinese primary gastric cancer are scarce. Moreover, direct evidence showing the association between protein expression of RASSF1A and primary human cancers is lacking. The aim of this study was to investigate RASSF1A expression in tissue of primary gastric cancer (GC at mRNA and protein levels, and to establish the possible relationship between DNA methylation status and protein expression of RASSF1A in Chinese. Methods Fifty-four patients with primary gastric cancers were included in the study of RASSF1A mRNA expression and methylation status between the cancer tissue and the corresponding adjacent normal tissue. 20 out of 54 patients were included for study of RASSF1A protein expression. The expression of RASSF1A at mRNA and protein levels was determined by RT-PCR and Western-blotting, respectively. The RASSF1A promoter methylation was detected by methylation-specific PCR. Results RASSF1A mRNA and protein expressions in GC were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2589 ± 0.2407 vs 0.5448 ± 0.2971, P P P P Conclusion Expression of RASSF1A was reduced in tissue of GC at mRNA and protein levels. Diminished expression of RASSF1A was associated with the promoter methylation.

  10. PCR detection of Burkholderia multivorans in water and soil samples.

    Science.gov (United States)

    Peeters, Charlotte; Daenekindt, Stijn; Vandamme, Peter

    2016-08-12

    Although semi-selective growth media have been developed for the isolation of Burkholderia cepacia complex bacteria from the environment, thus far Burkholderia multivorans has rarely been isolated from such samples. Because environmental B. multivorans isolates mainly originate from water samples, we hypothesized that water rather than soil is its most likely environmental niche. The aim of the present study was to assess the occurrence of B. multivorans in water samples from Flanders (Belgium) using a fast, culture-independent PCR assay. A nested PCR approach was used to achieve high sensitivity, and specificity was confirmed by sequencing the resulting amplicons. B. multivorans was detected in 11 % of the water samples (n = 112) and 92 % of the soil samples (n = 25) tested. The percentage of false positives was higher for water samples compared to soil samples, showing that the presently available B. multivorans recA primers lack specificity when applied to the analysis of water samples. The results of the present study demonstrate that B. multivorans DNA is commonly present in soil samples and to a lesser extent in water samples in Flanders (Belgium).

  11. A genome-wide methylation study on obesity Differential variability and differential methylation

    NARCIS (Netherlands)

    Xu, Xiaojing; Su, Shaoyong; Barnes, Vernon A.; De Miguel, Carmen; Pollock, Jennifer; Ownby, Dennis; Shi, Huidong; Zhu, Haidong; Snieder, Harold; Wang, Xiaoling

    2013-01-01

    Besides differential methylation, DNA methylation variation has recently been proposed and demonstrated to be a potential contributing factor to cancer risk. Here we aim to examine whether differential variability in methylation is also an important feature of obesity, a typical non-malignant common

  12. 21 CFR 177.2000 - Vinylidene chloride/methyl acrylate/methyl methacrylate polymers.

    Science.gov (United States)

    2010-04-01

    ... methacrylate polymers. 177.2000 Section 177.2000 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF...: POLYMERS Substances for Use as Basic Components of Single and Repeated Use Food Contact Surfaces § 177.2000 Vinylidene chloride/methyl acrylate/methyl methacrylate polymers. The vinylidene chloride/methyl acrylate...

  13. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    International Nuclear Information System (INIS)

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-01-01

    Research highlights: → Incubating PCR products at a high temperature causes smears in gel electrophoresis. → Smears interfere with the interpretation of methylation analysis using COBRA. → Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. → The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 o C or 65 o C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  14. A novel method for identification and quantification of consistently differentially methylated regions.

    Directory of Open Access Journals (Sweden)

    Ching-Lin Hsiao

    Full Text Available Advances in biotechnology have resulted in large-scale studies of DNA methylation. A differentially methylated region (DMR is a genomic region with multiple adjacent CpG sites that exhibit different methylation statuses among multiple samples. Many so-called "supervised" methods have been established to identify DMRs between two or more comparison groups. Methods for the identification of DMRs without reference to phenotypic information are, however, less well studied. An alternative "unsupervised" approach was proposed, in which DMRs in studied samples were identified with consideration of nature dependence structure of methylation measurements between neighboring probes from tiling arrays. Through simulation study, we investigated effects of dependencies between neighboring probes on determining DMRs where a lot of spurious signals would be produced if the methylation data were analyzed independently of the probe. In contrast, our newly proposed method could successfully correct for this effect with a well-controlled false positive rate and a comparable sensitivity. By applying to two real datasets, we demonstrated that our method could provide a global picture of methylation variation in studied samples. R source codes to implement the proposed method were freely available at http://www.csjfann.ibms.sinica.edu.tw/eag/programlist/ICDMR/ICDMR.html.

  15. High frequency of p 16 promoter methylation in non-small cell lung carcinomas from Chile

    Directory of Open Access Journals (Sweden)

    LEDA M GUZMAN

    2007-01-01

    Full Text Available The inactivation of tumour suppressor genes by aberrant methylation of promoter regions has been described as a frequent event in neoplasia development, including lung cancer. The p16 gene is a tumour suppressor gene involved in the regulation of cell cycle progression that has been reported to be inactivated by promoter methylation in lung carcinomas at variable frequencies around the world in a smoking habit dependent manner. The purpose of this study was to investigate the methylation status of the promoter region of the p16 gene in 74 non-small cell lung carcinomas from Chile. The frequency of p16 gene inactivation by promoter methylation was determined as 79.7% (59/74. When we considered histological type, we observed that p16 promoter methylation was significantly higher in squamous cell carcinomas (30/33, 91% compared with adenocarcinomas (21/30, 70% (p=0.029. In addition, no association between p16 promoter methylation and gender, age or smoking habit was found (p=0.202, 0.202 and 0.147 respectively. Our results suggest that p16 promoter hypermethylation is a very frequent event in non-small cell lung carcinomas from Chile and could be smoking habit-independent

  16. Prognostic value of MLH1 promoter methylation in male patients with esophageal squamous cell carcinoma.

    Science.gov (United States)

    Wu, Dongping; Chen, Xiaoying; Xu, Yan; Wang, Haiyong; Yu, Guangmao; Jiang, Luping; Hong, Qingxiao; Duan, Shiwei

    2017-04-01

    The DNA mismatch repair (MMR) gene MutL homolog 1 ( MLH1 ) is critical for the maintenance of genomic integrity. Methylation of the MLH1 gene promoter was identified as a prognostic marker for numerous types of cancer including glioblastoma, colorectal, ovarian and gastric cancer. The present study aimed to determine whether MLH1 promoter methylation was associated with survival in male patients with esophageal squamous cell carcinoma (ESCC). Formalin-fixed, paraffin-embedded ESCC tissues were collected from 87 male patients. MLH1 promoter methylation was assessed using the methylation-specific polymerase chain reaction approach. Kaplan-Meier survival curves and log-rank tests were used to evaluate the association between MLH1 promoter methylation and overall survival (OS) in patients with ESCC. Cox regression analysis was used to obtain crude and multivariate hazard ratios (HR), and 95% confidence intervals (CI). The present study revealed that MLH1 promoter methylation was observed in 53/87 (60.9%) of male patients with ESCC. Kaplan-Meier survival analysis demonstrated that MLH1 promoter hypermethylation was significantly associated with poorer prognosis in patients with ESCC (P=0.048). Multivariate survival analysis revealed that MLH1 promoter hypermethylation was an independent predictor of poor OS in male patients with ESCC (HR=1.716; 95% CI=1.008-2.921). Therefore, MLH1 promoter hypermethylation may be a predictor of prognosis in male patients with ESCC.

  17. Pros and cons of methylation-based enrichment methods for ancient DNA

    Science.gov (United States)

    Seguin-Orlando, Andaine; Gamba, Cristina; Sarkissian, Clio Der; Ermini, Luca; Louvel, Guillaume; Boulygina, Eugenia; Sokolov, Alexey; Nedoluzhko, Artem; Lorenzen, Eline D.; Lopez, Patricio; McDonald, H. Gregory; Scott, Eric; Tikhonov, Alexei; Stafford,, Thomas W.; Alfarhan, Ahmed H.; Alquraishi, Saleh A.; Al-Rasheid, Khaled A. S.; Shapiro, Beth; Willerslev, Eske; Prokhortchouk, Egor; Orlando, Ludovic

    2015-01-01

    The recent discovery that DNA methylation survives in fossil material provides an opportunity for novel molecular approaches in palaeogenomics. Here, we apply to ancient DNA extracts the probe-independent Methylated Binding Domains (MBD)-based enrichment method, which targets DNA molecules containing methylated CpGs. Using remains of a Palaeo-Eskimo Saqqaq individual, woolly mammoths, polar bears and two equine species, we confirm that DNA methylation survives in a variety of tissues, environmental contexts and over a large temporal range (4,000 to over 45,000 years before present). MBD enrichment, however, appears principally biased towards the recovery of CpG-rich and long DNA templates and is limited by the fast post-mortem cytosine deamination rates of methylated epialleles. This method, thus, appears only appropriate for the analysis of ancient methylomes from very well preserved samples, where both DNA fragmentation and deamination have been limited. This work represents an essential step toward the characterization of ancient methylation signatures, which will help understanding the role of epigenetic changes in past environmental and cultural transitions. PMID:26134828

  18. Factors Affecting Arsenic Methylation in Arsenic-Exposed Humans: A Systematic Review and Meta-Analysis.

    Science.gov (United States)

    Shen, Hui; Niu, Qiang; Xu, Mengchuan; Rui, Dongsheng; Xu, Shangzhi; Feng, Gangling; Ding, Yusong; Li, Shugang; Jing, Mingxia

    2016-02-06

    Chronic arsenic exposure is a critical public health issue in many countries. The metabolism of arsenic in vivo is complicated because it can be influenced by many factors. In the present meta-analysis, two researchers independently searched electronic databases, including the Cochrane Library, PubMed, Springer, Embase, and China National Knowledge Infrastructure, to analyze factors influencing arsenic methylation. The concentrations of the following arsenic metabolites increase (piAs), monomethyl arsenic (MMA), dimethyl arsenic (DMA), and total arsenic. Additionally, the percentages of iAs (standard mean difference (SMD): 1.00; 95% confidence interval (CI): 0.60-1.40; p< 0.00001) and MMA (SMD: 0.49; 95% CI: 0.21-0.77; p = 0.0006) also increase, while the percentage of DMA (SMD: -0.57; 95% CI: -0.80--0.31; p< 0.0001), primary methylation index (SMD: -0.57; 95% CI: -0.94--0.20; p = 0.002), and secondary methylation index (SMD: -0.27; 95% CI: -0.46--0.90; p = 0.004) decrease. Smoking, drinking, and older age can reduce arsenic methylation, and arsenic methylation is more efficient in women than in men. The results of this analysis may provide information regarding the role of arsenic oxidative methylation in the arsenic poisoning process.

  19. Detection of Mycobacterium Tuberculosis by using PCR

    International Nuclear Information System (INIS)

    Suhadi, F; Dadang-Sudrajat; Maria-Lina, R.

    1996-01-01

    Polymerase Chain Reaction (PCR) procedure using three primary set derived from repetitive DNA sequence specific to mycobacteria was used to diagnose pathogenic Mycobacterium tuberculosis. The assay was specific for M. tuberculosis and could be used to detect the amount DNA less than 10 -9 g

  20. PCR detection of potato cyst nematode.

    Science.gov (United States)

    Reid, Alex

    2009-01-01

    Potato cyst nematode (PCN) is responsible for losses in potato production totalling millions of euros every year in the EC. It is important for growers to know which species is present in their land as this determines its subsequent use. The two species Globodera pallida and Globodera rostochiensis can be differentiated using an allele-specific PCR.

  1. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Kubista, Mikael; Sjögreen, B.; Forootan, A.; Šindelka, Radek; Jonák, Jiří; Andrade, J.M.

    2007-01-01

    Roč. 1, - (2007), s. 56-60 ISSN 1360-8606 R&D Projects: GA AV ČR KJB500520601 Institutional research plan: CEZ:AV0Z50520514 Keywords : real - time PCR, * expression profiling * statistical analysis Subject RIV: EB - Genetics ; Molecular Biology

  2. Lessons from independence

    International Nuclear Information System (INIS)

    Hauptfuhrer, R.R.

    1990-01-01

    The recent history of Oryx provides invaluable lessons for those who plan future energy strategies, relates the author of this paper. When Oryx became an independent oil and gas company, its reserves were declining, its stock was selling below asset values, and the price of oil seemed stuck below $15 per barrel. The message from Oryx management to Oryx employees was: We are in charge of our own destiny. We are about to create our own future. Oryx had developed a new, positive corporate culture and the corporate credit required for growth. This paper points to two basic principles that have guided the metamorphosis in Oryx's performance. The first objective was to improve operational efficiency and to identify the right performance indicators to measure this improvement. It states that the most critical performance indicator for an exploration and production company must be replacement and expansion of reserves at a competitive replacement cost. Oryx has cut its finding costs from $12 to $5 per barrel, while the BP acquisition provided proven reserves at a cost of only $4 per barrel. Another performance indicator measures Oryx's standing in the financial markets

  3. Independents' group posts loss

    International Nuclear Information System (INIS)

    Sanders, V.; Price, R.B.

    1992-01-01

    Low oil gas prices and special charges caused the group of 50 U.S. independent producers Oil and Gas Journal tracks to post a combined loss in first half 1992. The group logged a net loss of $53 million in the first half compared with net earnings of $354 million in first half 1991, when higher oil prices during the Persian Gulf crisis buoyed earnings in spite of crude oil and natural gas production declines. The combined loss in the first half follows a 45% drop in the group's earnings in 1991 and compares with the OGJ group of integrated oil companies whose first half 1992 income fell 47% from the prior year. Special charges, generally related to asset writedowns, accounted for most of the almost $560 million in losses posted by about the third of the group. Nerco Oil and Gas Inc., Vancouver, Wash., alone accounted for almost half that total with charges related to an asset writedown of $238 million in the first quarter. Despite the poor first half performance, the outlook is bright for sharply improved group earnings in the second half, assuming reasonably healthy oil and gas prices and increased production resulting from acquisitions and in response to those prices

  4. PPARγ-Independent Mechanism

    Directory of Open Access Journals (Sweden)

    Christopher M. Hogan

    2011-01-01

    Full Text Available Acute and chronic lung inflammation is associated with numerous important disease pathologies including asthma, chronic obstructive pulmonary disease and silicosis. Lung fibroblasts are a novel and important target of anti-inflammatory therapy, as they orchestrate, respond to, and amplify inflammatory cascades and are the key cell in the pathogenesis of lung fibrosis. Peroxisome proliferator-activated receptor gamma (PPARγ ligands are small molecules that induce anti-inflammatory responses in a variety of tissues. Here, we report for the first time that PPARγ ligands have potent anti-inflammatory effects on human lung fibroblasts. 2-cyano-3, 12-dioxoolean-1, 9-dien-28-oic acid (CDDO and 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2 inhibit production of the inflammatory mediators interleukin-6 (IL-6, monocyte chemoattractant protein-1 (MCP-1, COX-2, and prostaglandin (PGE2 in primary human lung fibroblasts stimulated with either IL-1β or silica. The anti-inflammatory properties of these molecules are not blocked by the PPARγ antagonist GW9662 and thus are largely PPARγ independent. However, they are dependent on the presence of an electrophilic carbon. CDDO and 15d-PGJ2, but not rosiglitazone, inhibited NF-κB activity. These results demonstrate that CDDO and 15d-PGJ2 are potent attenuators of proinflammatory responses in lung fibroblasts and suggest that these molecules should be explored as the basis for novel, targeted anti-inflammatory therapies in the lung and other organs.

  5. Whole-genome DNA methylation status associated with clinical PTSD measures of OIF/OEF veterans

    Science.gov (United States)

    Hammamieh, R; Chakraborty, N; Gautam, A; Muhie, S; Yang, R; Donohue, D; Kumar, R; Daigle, B J; Zhang, Y; Amara, D A; Miller, S-A; Srinivasan, S; Flory, J; Yehuda, R; Petzold, L; Wolkowitz, O M; Mellon, S H; Hood, L; Doyle, F J; Marmar, C; Jett, M

    2017-01-01

    Emerging knowledge suggests that post-traumatic stress disorder (PTSD) pathophysiology is linked to the patients’ epigenetic changes, but comprehensive studies examining genome-wide methylation have not been performed. In this study, we examined genome-wide DNA methylation in peripheral whole blood in combat veterans with and without PTSD to ascertain differentially methylated probes. Discovery was initially made in a training sample comprising 48 male Operation Enduring Freedom (OEF)/Operation Iraqi Freedom (OIF) veterans with PTSD and 51 age/ethnicity/gender-matched combat-exposed PTSD-negative controls. Agilent whole-genome array detected ~5600 differentially methylated CpG islands (CpGI) annotated to ~2800 differently methylated genes (DMGs). The majority (84.5%) of these CpGIs were hypermethylated in the PTSD cases. Functional analysis was performed using the DMGs encoding the promoter-bound CpGIs to identify networks related to PTSD. The identified networks were further validated by an independent test set comprising 31 PTSD+/29 PTSD− veterans. Targeted bisulfite sequencing was also used to confirm the methylation status of 20 DMGs shown to be highly perturbed in the training set. To improve the statistical power and mitigate the assay bias and batch effects, a union set combining both training and test set was assayed using a different platform from Illumina. The pathways curated from this analysis confirmed 65% of the pool of pathways mined from training and test sets. The results highlight the importance of assay methodology and use of independent samples for discovery and validation of differentially methylated genes mined from whole blood. Nonetheless, the current study demonstrates that several important epigenetically altered networks may distinguish combat-exposed veterans with and without PTSD. PMID:28696412

  6. Distinct features between MLH1-methylated and unmethylated colorectal carcinomas with the CpG island methylator phenotype: implications in the serrated neoplasia pathway.

    Science.gov (United States)

    Kim, Jung Ho; Bae, Jeong Mo; Cho, Nam-Yun; Kang, Gyeong Hoon

    2016-03-22

    The presence or absence of MLH1 methylation may critically affect the heterogeneity of colorectal carcinoma (CRC) with the CpG island methylator phenotype (CIMP). Here, we investigated the differential characteristics of CIMP-high (CIMP-H) CRCs according to MLH1 methylation status. To further confirm the MLH1-dependent features in CIMP-H CRC, an independent analysis was performed using data from The Cancer Genome Atlas (TCGA). In our CIMP-H CRC samples, MLH1-methylated tumors were characterized by older patient age, proximal colonic location, mucinous histology, intense lymphoid reactions, RUNX3/SOCS1 promoter methylation, BRAF mutations, and microsatellite instability-high (MSI-H) status. By contrast, MLH1-unmethylated tumors were associated with earlier age of onset, increased distal colorectal localization, adverse pathologic features, and KRAS mutations. In the TCGA dataset, the MLH1-silenced CIMP-H CRC demonstrated proximal location, MSI-H status, hypermutated phenotype, and frequent BRAF mutations, but the MLH1-non-silenced CIMP-H CRC was significantly associated with high frequencies of KRAS and APC mutations. In conclusion, the differential nature of CIMP-H CRCs depends primarily on the MLH1 methylation status. Based on the current knowledge, the sessile serrated adenoma/polyp may be the major precursor of MLH1-methylated CIMP-H CRCs, whereas MLH1-unmethylated CIMP-H CRCs may develop predominantly from KRAS-mutated traditional serrated adenomas and less commonly from BRAF-mutated traditional serrated adenomas and/or sessile serrated adenomas/polyps.

  7. SNP rs16906252C>T is an expression and methylation quantitative trait locus associated with an increased risk of developing MGMT-methylated colorectal cancer

    Science.gov (United States)

    Kuroiwa-Trzmielina, Joice; Wang, Fan; Rapkins, Robert W.; Ward, Robyn L.; Buchanan, Daniel D.; Win, Aung Ko; Clendenning, Mark; Rosty, Christophe; Southey, Melissa C.; Winship, Ingrid M.; Hopper, John L.; Jenkins, Mark A.; Olivier, Jake; Hawkins, Nicholas J.; Hitchins, Megan P.

    2016-01-01

    Purpose Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter-enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer (CRC) featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues. Experimental design By applying a molecular pathological epidemiology case-control study design, associations between rs16906252C>T and risk for CRC overall, and CRC stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of CRC cases and controls. The test sample comprised 1054 CRC cases and 451 controls from Sydney, Australia. The validation sample comprised 612 CRC cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine if rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues. Results An association between rs16906252C>T and increased risk of developing MGMT-methylated CRC in the Sydney sample was observed (OR 3.3; 95%CI=2.0–5.3; PT represents an expression and methylation quantitative trait locus. Conclusions We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated CRC, likely mediated by constitutive epigenetic repression of the T allele. PMID:27267851

  8. CpG Island Methylator Phenotype Positive Tumors in the Absence of MLH1 Methylation Constitute a Distinct Subset of Duodenal Adenocarcinomas and Are Associated with Poor Prognosis

    Science.gov (United States)

    Fu, Tao; Pappou, Emmanouil P.; Guzzetta, Angela A.; Jeschke, Jana; Kwak, Ruby; Dave, Pujan; Hooker, Craig M.; Morgan, Richard; Baylin, Stephen B.; Iacobuzio-Donahue, Christine A.; Wolfgang, Christopher L.; Ahuja, Nita

    2012-01-01

    Purpose Little information is available on genetic and epigenetic changes in duodenal adenocarcinomas. The purpose was to identify possible subsets of duodenal adenocarcinomas based on microsatellite instability (MSI), DNA methylation, mutations in the KRAS and BRAF genes, clinicopathologic features, and prognosis. Experimental Design Demographics, tumor characteristics and survival were available for 99 duodenal adenocarcinoma patients. Testing for KRAS and BRAF mutations, MSI, MLH1 methylation and CpG island methylator phenotype (CIMP) status was performed. A Cox proportional hazard model was built to predict survival. Results CIMP+ was detected in 27 of 99 (27.3%) duodenal adenocarcinomas, and was associated with MSI (P = 0.011) and MLH1 methylation (P CIMP− tumors. No BRAF V600E mutation was detected. Among the CIMP+ tumors, 15 (55.6%) were CIMP+/MLH1-unmethylated (MLH1-U). Kaplan-Meier analysis showed tumors classified by CIMP, CIMP/MLH1 methylation status or CIMP/MSI status could predict overall survival (OS; P = 0.047, 0.002, and 0.002, respectively), while CIMP/MLH1 methylation status could also predict time-to-recurrence (TTR; P = 0.016). In multivariate analysis, CIMP/MLH1 methylation status showed a significant prognostic value regarding both OS (P CIMP+/MLH1-U tumors had the worst OS and TTR. Conclusions Our results demonstrate existence of CIMP in duodenal adenocarcinomas. The combination of CIMP+/MLH1-U appears to be independently associated with poor prognosis in patients with duodenal adenocarcinomas. This study also suggests that BRAF mutations are not involved in duodenal tumorigenesis, MSI or CIMP development. PMID:22825585

  9. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  10. Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

    Science.gov (United States)

    Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

    2017-01-01

    The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

  11. Real-time PCR (qPCR) primer design using free online software.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  12. Aberrant TET1 Methylation Closely Associated with CpG Island Methylator Phenotype in Colorectal Cancer.

    Science.gov (United States)

    Ichimura, Norihisa; Shinjo, Keiko; An, Byonggu; Shimizu, Yasuhiro; Yamao, Kenji; Ohka, Fumiharu; Katsushima, Keisuke; Hatanaka, Akira; Tojo, Masayuki; Yamamoto, Eiichiro; Suzuki, Hiromu; Ueda, Minoru; Kondo, Yutaka

    2015-08-01

    Inactivation of methylcytosine dioxygenase, ten-eleven translocation (TET) is known to be associated with aberrant DNA methylation in cancers. Tumors with a CpG island methylator phenotype (CIMP), a distinct subgroup with extensive DNA methylation, show characteristic features in the case of colorectal cancer. The relationship between TET inactivation and CIMP in colorectal cancers is not well understood. The expression level of TET family genes was compared between CIMP-positive (CIMP-P) and CIMP-negative (CIMP-N) colorectal cancers. Furthermore, DNA methylation profiling, including assessment of the TET1 gene, was assessed in colorectal cancers, as well as colon polyps. The TET1 was silenced by DNA methylation in a subset of colorectal cancers as well as cell lines, expression of which was reactivated by demethylating agent. TET1 methylation was more frequent in CIMP-P (23/55, 42%) than CIMP-N (2/113, 2%, P CIMP-P, 16/40, 40%; CIMP-N, 2/24, 8%; P = 0.002), suggesting that TET1 methylation is an early event in CIMP tumorigenesis. TET1 methylation was significantly associated with BRAF mutation but not with hMLH1 methylation in the CIMP-P colorectal cancers. Colorectal cancers with TET1 methylation have a significantly greater number of DNA methylated genes and less pathological metastasis compared to those without TET1 methylation (P = 0.007 and 0.045, respectively). Our data suggest that TET1 methylation may contribute to the establishment of a unique pathway in respect to CIMP-mediated tumorigenesis, which may be incidental to hMLH1 methylation. In addition, our findings provide evidence that TET1 methylation may be a good biomarker for the prediction of metastasis in colorectal cancer. ©2015 American Association for Cancer Research.

  13. [Mifepristone inhibites the migration of endometrial cancer cells through regulating H19 methylation].

    Science.gov (United States)

    Lu, Z Z; Yan, L; Zhang, H; Li, M J; Zhang, X H; Zhao, X X

    2016-06-23

    To investigate the effect and mechanism of mifepristone on the migration of human endometrial carcinoma cells. A human endometrial carcinoma cell line, Ishikawa cells, was cultured in vitro and treated with mifepristone at different concentrations. Wound healing assay was applied to detect the migration of Ishikawa cells. RT-PCR and methylation-specific PCR (MSP) were used to detect the levels of H19 mRNA and its DNA methylation. Western-blot was used to detect the expressions of HMGA2 and epithelial to mesenchymal transition (EMT) related proteins. When treated with different concentrations of mifepristone for 48 hours, the width of scratch of the the control group, the 5 mg/L and the 10 mg/L mifepristone treatment groups were (4.18±0.07)mm, (4.68±0.07)mm, and(4.99±0.07)mm, respectively (Pendometrial carcinoma cells partially through methylation-induced of transcriptional inhibition of H19, which results in the down-regulation of HMGA2 and vimentin and upregulation of E-cadherin.

  14. 5-Chloro-2-methyl-3-phenylsulfonyl-1-benzofuran

    Directory of Open Access Journals (Sweden)

    Hong Dae Choi

    2008-07-01

    Full Text Available The title compound, C15H11ClO3S, was prepared by the oxidation of 5-chloro-2-methyl-3-phenylsulfanyl-1-benzofuran with 3-chloroperoxybenzoic acid. There are two symmetry-independent molecules in the asymmetric unit. The dihedral angles formed by the phenyl ring and the plane of the benzofuran system are 77.80 (8 and 78.34 (8°. The crystal structure is stabilized by aromatic π–π stacking interactions between the furan ring and the benzene rings of neighbouring benzofuran fragments from two symmetry-independent molecules; the centroid–centroid distances within the stacks are 3.689 (4, 3.702 (4, 3.825 (4 and 3.826 (4 Å. Additionally, the stacked molecules exhibit inter- and intramolecular C—H...O interactions.

  15. Association of promoter methylation of VGF and PGP9.5 with ovarian cancer progression.

    Directory of Open Access Journals (Sweden)

    Mariana Brait

    Full Text Available To elucidate the role of biological and clinical impact of aberrant promoter hypermethylation (PH in ovarian cancer (OC.PH of PGP9.5, HIC1, AIM1, APC, PAK3, MGMT, KIF1A, CCNA1, ESR1, SSBP2, GSTP1, FKBP4 and VGF were assessed by quantitative methylation specific PCR (QMSP in a training set. We selected two genes (VGF and PGP9.5 for further QMSP analysis in a larger independent validation (IV set with available clinical data. Biologic relevance of VGF gene was also evaluated.PH frequency for PGP9.5 and VGF were 85% (316/372 and 43% (158/366 respectively in the IV set of samples while no PH was observed in controls. In 372 OC cases with available follow up, PGP9.5 and VGF PH were correlated with better patient survival [Hazard Ratios (HR for overall survival (OS were 0.59 (95% Confidence Intervals (CI  = 0.42-0.84, p = 0.004, and 0.73 (95%CI = 0.55-0.97, p = 0.028 respectively, and for disease specific survival (DSS were 0.57 (95%CI 0.39-0.82, p = 0.003 and 0.72 (95%CI 0.54-0.96, p = 0.027. In multivariate analysis, VGF PH remained an independent prognostic factor for OS (HR 0.61, 95%CI 0.43-0.86, p<0.005 and DSS (HR 0.58, 95%CI 0.41-0.83, p<0.003. Furthermore, PGP9.5 PH was significantly correlated with lower grade, early stage tumors, and with absence of residual disease. Forced expression of VGF in OC cell lines inhibited cell growth.Our results indicate that VGF and PGP9.5 PH are potential biomarkers for ovarian carcinoma. Confirmatory cohorts with longitudinal follow-up are required in future studies to define the clinical impact of VGF and PGP9.5 PH before clinical application.

  16. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  17. The Synthesis of Methyl Salicylate: Amine Diazotization.

    Science.gov (United States)

    Zanger, Murray; McKee, James R.

    1988-01-01

    Notes that this experiment takes safety and noncarcinogenic reactants into account. Demonstrates the use of diazonium salts for the replacement of an aromatic amine group by a phenolic hydroxyl. Involves two pleasant-smelling organic compounds, methyl anthranilate (grape) and methyl salicylate (oil of wintergreen). (MVL)

  18. Evidence Suggesting Absence of Mitochondrial DNA Methylation

    DEFF Research Database (Denmark)

    Mechta, Mie; Ingerslev, Lars R; Fabre, Odile

    2017-01-01

    , 16S, ND5 and CYTB, suggesting that mtDNA supercoiled structure blocks the access to bisulfite conversion. Here, we identified an artifact of mtDNA bisulfite sequencing that can lead to an overestimation of mtDNA methylation levels. Our study supports that cytosine methylation is virtually absent...

  19. DNA Methylation Modulates Nociceptive Sensitization after Incision.

    Directory of Open Access Journals (Sweden)

    Yuan Sun

    Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

  20. Methyl 3-(Quinolin-2-ylindolizine-1-carboxylate

    Directory of Open Access Journals (Sweden)

    Roumaissa Belguedj

    2015-12-01

    Full Text Available A novel compound, methyl 3-(quinolin-2-ylindolizine-1-carboxylate (2 has been synthesized by cycloaddition reaction of 1-(quinolin-2-ylmethylpyridinium ylide (1 with methyl propiolate in presence of sodium hydride in THF. The structure of this compound was established by IR, 1H-NMR, 13C-NMR and MS data

  1. Adenine N6-methylation in diverse fungi

    NARCIS (Netherlands)

    Seidl, Michael F.

    2017-01-01

    A DNA modification - methylation of cytosines and adenines - has important roles in diverse processes such as regulation of gene expression and genome stability, yet until recently adenine methylation had been considered to be only a hallmark of prokaryotes. A new study identifies abundant

  2. Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection.

    Science.gov (United States)

    Harrus, Shimon; Kenny, Martin; Miara, Limor; Aizenberg, Itzhak; Waner, Trevor; Shaw, Susan

    2004-11-01

    This report presents evidence that dogs recover from acute canine monocytic ehrlichiosis (CME) after 16 days of doxycycline treatment (10 mg/kg of body weight every 24 h). Blood PCR was as valuable as splenic aspirate PCR for early diagnosis of acute CME. Splenic aspirate PCR was, however, superior to blood PCR for the evaluation of ehrlichial elimination.

  3. Commonwealth of (Independent States

    Directory of Open Access Journals (Sweden)

    Vrućinić Dušan

    2013-01-01

    Full Text Available Following the stages from the establishment itself to the present day of the functioning of such a specific regional organization as the Commonwealth of Independent States (CIS, the article seeks to further explain the meaning of its existence, efficiency and functioning. The CIS was created in order to make the dissolution of a major world super-power, which throughout the 20th century together with the USA defined the bipolar world, as painless as possible, especially for the new countries and its nationally and ethnically diverse population. During the early years after the dissolution of the USSR, the CIS played a major role in a more flexible and less severe dissolution of the Soviet empire, alleviating the consequences for its people. A more efficient functioning among the republics in all fields was also one of the tasks of the Commonwealth, to which it was devoted to the extent which was permitted by the then, not too favourable circumstances. Difficult years of economic crisis did not allow the CIS to mutually integrate its members as much as possible on the economy level. Thanks to the economic recovery of the post-Soviet states in the early 21st century, the Commonwealth has also been transformed, reformed, and renewed, and all this in order to achieve better and more fruitful cooperation between the members. The CIS may serve as a proper example of how the former Soviet Union states are inextricably linked by social, security-political, economic, cultural, communication-transport, and other ties, thanks to the centuries-long existence of the peoples of these states in this area, despite both internal and external factors which occasionally, but temporarily halt the post-Soviet integration. Mathematically expressed, the CIS members are naturally predisposed, to be reciprocally depended on each other, just as they also have the capacity for successful cooperation in the future times and epochs brought on by the modern world.

  4. DNA methylation in inflammatory genes among children with obstructive sleep apnea.

    Science.gov (United States)

    Kim, Jinkwan; Bhattacharjee, Rakesh; Khalyfa, Abdelnaby; Kheirandish-Gozal, Leila; Capdevila, Oscar Sans; Wang, Yang; Gozal, David

    2012-02-01

    Pediatric obstructive sleep apnea (OSA) leads to multiple end-organ morbidities that are mediated by the cumulative burden of oxidative stress and inflammation. Because not all children with OSA exhibit increased systemic inflammation, genetic and environmental factors may be affecting patterns of DNA methylation in genes subserving inflammatory functions. DNA from matched children with OSA with and without high levels of high-sensitivity C-reactive protein (hsCRP) were assessed for DNA methylation levels of 24 inflammatory-related genes. Primer-based polymerase chain reaction assays in a case-control setting involving 47 OSA cases and 31 control subjects were conducted to confirm the findings; hsCRP and myeloid-related protein (MRP) 8/14 levels were also assayed. Forkhead box P3 (FOXP3) and interferon regulatory factor 1 (IRF1) showed higher methylation in six children with OSA and high hsCRP levels compared with matched children with OSA and low hsCRP levels (P DNA methylation levels compared with children with OSA and low CRP levels and control subjects. IRF1 did not exhibit significant differences. FOXP3 DNA methylation levels correlated with hsCRP and MRP 8/14 levels and with apnea-hypopnea index (AHI), BMI z score, and apolipoprotein B levels. A stepwise multiple regression model showed that AHI was independently associated with FOXP3 DNA methylation levels (P gene, which regulates expression of T regulatory lymphocytes, is more likely to display increased methylation among children with OSA who exhibit increased systemic inflammatory responses. Thus, epigenetic modifications may constitute an important determinant of inflammatory phenotype in OSA, and FOXP3 DNA methylation levels may provide a potential biomarker for end-organ vulnerability.

  5. A DNA methylation-based definition of biologically distinct breast cancer subtypes.

    Science.gov (United States)

    Stefansson, Olafur A; Moran, Sebastian; Gomez, Antonio; Sayols, Sergi; Arribas-Jorba, Carlos; Sandoval, Juan; Hilmarsdottir, Holmfridur; Olafsdottir, Elinborg; Tryggvadottir, Laufey; Jonasson, Jon G; Eyfjord, Jorunn; Esteller, Manel

    2015-03-01

    In cancer, epigenetic states are deregulated and thought to be of significance in cancer development and progression. We explored DNA methylation-based signatures in association with breast cancer subtypes to assess their impact on clinical presentation and patient prognosis. DNA methylation was analyzed using Infinium 450K arrays in 40 tumors and 17 normal breast samples, together with DNA copy number changes and subtype-specific markers by tissue microarrays. The identified methylation signatures were validated against a cohort of 212 tumors annotated for breast cancer subtypes by the PAM50 method (The Cancer Genome Atlas). Selected markers were pyrosequenced in an independent validation cohort of 310 tumors and analyzed with respect to survival, clinical stage and grade. The results demonstrate that DNA methylation patterns linked to the luminal-B subtype are characterized by CpG island promoter methylation events. In contrast, a large fraction of basal-like tumors are characterized by hypomethylation events occurring within the gene body. Based on these hallmark signatures, we defined two DNA methylation-based subtypes, Epi-LumB and Epi-Basal, and show that they are associated with unfavorable clinical parameters and reduced survival. Our data show that distinct mechanisms leading to changes in CpG methylation states are operative in different breast cancer subtypes. Importantly, we show that a few selected proxy markers can be used to detect the distinct DNA methylation-based subtypes thereby providing valuable information on disease prognosis. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  6. CpG methylation differences between neurons and glia are highly conserved from mouse to human.

    Science.gov (United States)

    Kessler, Noah J; Van Baak, Timothy E; Baker, Maria S; Laritsky, Eleonora; Coarfa, Cristian; Waterland, Robert A

    2016-01-15

    Understanding epigenetic differences that distinguish neurons and glia is of fundamental importance to the nascent field of neuroepigenetics. A recent study used genome-wide bisulfite sequencing to survey differences in DNA methylation between these two cell types, in both humans and mice. That study minimized the importance of cell type-specific differences in CpG methylation, claiming these are restricted to localized genomic regions, and instead emphasized that widespread and highly conserved differences in non-CpG methylation distinguish neurons and glia. We reanalyzed the data from that study and came to markedly different conclusions. In particular, we found widespread cell type-specific differences in CpG methylation, with a genome-wide tendency for neuronal CpG-hypermethylation punctuated by regions of glia-specific hypermethylation. Alarmingly, our analysis indicated that the majority of genes identified by the primary study as exhibiting cell type-specific CpG methylation differences were misclassified. To verify the accuracy of our analysis, we isolated neuronal and glial DNA from mouse cortex and performed quantitative bisulfite pyrosequencing at nine loci. The pyrosequencing results corroborated our analysis, without exception. Most interestingly, we found that gene-associated neuron vs. glia CpG methylation differences are highly conserved across human and mouse, and are very likely to be functional. In addition to underscoring the importance of independent verification to confirm the conclusions of genome-wide epigenetic analyses, our data indicate that CpG methylation plays a major role in neuroepigenetics, and that the mouse is likely an excellent model in which to study the role of DNA methylation in human neurodevelopment and disease. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  7. Identifying DNA Methylation Biomarkers for Non-Endoscopic Detection of Barrett’s Esophagus

    Science.gov (United States)

    Moinova, Helen R.; LaFramboise, Thomas; Lutterbaugh, James D.; Chandar, Apoorva Krishna; Dumot, John; Faulx, Ashley; Brock, Wendy; De la Cruz Cabrera, Omar; Guda, Kishore; Barnholtz-Sloan, Jill S.; Iyer, Prasad G.; Canto, Marcia I.; Wang, Jean S.; Shaheen, Nicholas J.; Thota, Prashanti N.; Willis, Joseph E.; Chak, Amitabh; Markowitz, Sanford D.

    2018-01-01

    We report a biomarker-based non-endoscopic method for detecting Barrett’s esophagus (BE), based on detecting methylated DNAs retrieved via a swallowable balloon-based esophageal sampling device. BE is the precursor of, and a major recognized risk factor for, developing esophageal adenocarcinoma (EAC). Endoscopy, the current standard for BE detection, is not cost-effective for population screening. We performed genome-wide screening to ascertain regions targeted for recurrent aberrant cytosine methylation in BE, identifying high-frequency methylation within the CCNA1 locus. We tested CCNA1 DNA methylation as a BE biomarker in cytology brushings of the distal esophagus from 173 individuals with or without BE. CCNA1 DNA methylation demonstrated an area under the curve (AUC)=0.95 for discriminating BE-related metaplasia and neoplasia cases versus normal individuals, performing identically to methylation of VIM DNA, an established BE biomarker. When combined, the resulting two biomarker panel was 95% sensitive and 91% specific. These results were replicated in an independent validation cohort of 149 individuals, who were assayed using the same cutoff values for test positivity established in the training population. To progress toward non-endoscopic esophageal screening, we engineered a well-tolerated, swallowable, encapsulated balloon device able to selectively sample the distal esophagus within 5 minutes. In balloon samples from 86 individuals, tests of CCNA1 plus VIM DNA methylation detected BE metaplasia with 90.3% sensitivity and 91.7% specificity. Combining the balloon sampling device with molecular assays of CCNA1 plus VIM DNA methylation enables an efficient, well-tolerated, sensitive, and specific method of screening at-risk populations for BE. PMID:29343623

  8. Gene methylation profiles of normal mucosa, and benign and malignant colorectal tumors identify early onset markers

    Directory of Open Access Journals (Sweden)

    Vatn Morten

    2008-12-01

    Full Text Available Abstract Background Multiple epigenetic and genetic changes have been reported in colorectal tumors, but few of these have clinical impact. This study aims to pinpoint epigenetic markers that can discriminate between non-malignant and malignant tissue from the large bowel, i.e. markers with diagnostic potential. The methylation status of eleven genes (ADAMTS1, CDKN2A, CRABP1, HOXA9, MAL, MGMT, MLH1, NR3C1, PTEN, RUNX3, and SCGB3A1 was determined in 154 tissue samples including normal mucosa, adenomas, and carcinomas of the colorectum. The gene-specific and widespread methylation status among the carcinomas was related to patient gender and age, and microsatellite instability status. Possible CIMP tumors were identified by comparing the methylation profile with microsatellite instability (MSI, BRAF-, KRAS-, and TP53 mutation status. Results The mean number of methylated genes per sample was 0.4 in normal colon mucosa from tumor-free individuals, 1.2 in mucosa from cancerous bowels, 2.2 in adenomas, and 3.9 in carcinomas. Widespread methylation was found in both adenomas and carcinomas. The promoters of ADAMTS1, MAL, and MGMT were frequently methylated in benign samples as well as in malignant tumors, independent of microsatellite instability. In contrast, normal mucosa samples taken from bowels without tumor were rarely methylated for the same genes. Hypermethylated CRABP1, MLH1, NR3C1, RUNX3, and SCGB3A1 were shown to be identifiers of carcinomas with microsatellite instability. In agreement with the CIMP concept, MSI and mutated BRAF were associated with samples harboring hypermethylation of several target genes. Conclusion Methylated ADAMTS1, MGMT, and MAL are suitable as markers for early tumor detection.

  9. [Association between serum aluminium level and methylation of amyloid precursor protein gene in workers engaged in aluminium electrolysis].

    Science.gov (United States)

    Yang, X J; Yuan, Y Z; Niu, Q

    2016-04-20

    To investigate the association between serum aluminium level and methylation of the promoter region of amyloid precursor protein (APP)gene in workers engaged in aluminium electrolysis. In 2012, 366 electrolysis workers in an aluminium factory were enrolled as exposure group (working years >10 and age >40 years)and divided into low-exposure group and high-exposure group based on the median serum aluminium level. Meanwhile, 102 workers in a cement plant not exposed to aluminium were enrolled as control group. Graphite furnace atomic absorption spectrometry was used to measure serum aluminium level, methylation specific PCR was used to measure the methylation rate of the promoter region of APP gene, and ELI-SA was used to measure the protein expression of APP in lymphocytes in peripheral blood. The exposure group had a significantly higher serum aluminium level than the control group (45.07 μg/L vs 30.51 μg/L, P0.05). The multivariate logistic regression analysis showed that with reference to the control group, low aluminium exposure (OR=1.86, 95% CI 1.67~3.52)and high aluminium exposure (OR=2.98, 95% CI 1.97~4.15)were risk factors for a reduced methylation rate of the promoter region of APP gene. Reduced methylation of the promoter region of APP gene may be associated with increased serum aluminium level, and downregulated methylation of the promoter region of APP gene may accelerate APP gene transcription.

  10. Effects of γ-radiation on cell growth, cell cycle and promoter methylation of 22 cell cycle genes in the 1321NI astrocytoma cell line.

    Science.gov (United States)

    Alghamian, Yaman; Abou Alchamat, Ghalia; Murad, Hossam; Madania, Ammar

    2017-09-01

    DNA damage caused by radiation initiates biological responses affecting cell fate. DNA methylation regulates gene expression and modulates DNA damage pathways. Alterations in the methylation profiles of cell cycle regulating genes may control cell response to radiation. In this study we investigated the effect of ionizing radiation on the methylation levels of 22 cell cycle regulating genes in correlation with gene expression in 1321NI astrocytoma cell line. 1321NI cells were irradiated with 2, 5 or 10Gy doses then analyzed after 24, 48 and 72h for cell viability using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu bromide) assay. Flow cytometry were used to study the effect of 10Gy irradiation on cell cycle. EpiTect Methyl II PCR Array was used to identify differentially methylated genes in irradiated cells. Changes in gene expression was determined by qPCR. Azacytidine treatment was used to determine whether DNA methylation affectes gene expression. Our results showed that irradiation decreased cell viability and caused cell cycle arrest at G2/M. Out of 22 genes tested, only CCNF and RAD9A showed some increase in DNA methylation (3.59% and 3.62%, respectively) after 10Gy irradiation, and this increase coincided with downregulation of both genes (by 4 and 2 fold, respectively). with azacytidine confirmed that expression of CCNF and RAD9A genes was regulated by methylation. 1321NI cell line is highly radioresistant and that irradiation of these cells with a 10Gy dose increases DNA methylation of CCNF and RAD9A genes. This dose down-regulates these genes, favoring G2/M arrest. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

  11. On the potential of models for location and scale for genome-wide DNA methylation data.

    Science.gov (United States)

    Wahl, Simone; Fenske, Nora; Zeilinger, Sonja; Suhre, Karsten; Gieger, Christian; Waldenberger, Melanie; Grallert, Harald; Schmid, Matthias

    2014-07-03

    With the help of epigenome-wide association studies (EWAS), increasing knowledge on the role of epigenetic mechanisms such as DNA methylation in disease processes is obtained. In addition, EWAS aid the understanding of behavioral and environmental effects on DNA methylation. In terms of statistical analysis, specific challenges arise from the characteristics of methylation data. First, methylation β-values represent proportions with skewed and heteroscedastic distributions. Thus, traditional modeling strategies assuming a normally distributed response might not be appropriate. Second, recent evidence suggests that not only mean differences but also variability in site-specific DNA methylation associates with diseases, including cancer. The purpose of this study was to compare different modeling strategies for methylation data in terms of model performance and performance of downstream hypothesis tests. Specifically, we used the generalized additive models for location, scale and shape (GAMLSS) framework to compare beta regression with Gaussian regression on raw, binary logit and arcsine square root transformed methylation data, with and without modeling a covariate effect on the scale parameter. Using simulated and real data from a large population-based study and an independent sample of cancer patients and healthy controls, we show that beta regression does not outperform competing strategies in terms of model performance. In addition, Gaussian models for location and scale showed an improved performance as compared to models for location only. The best performance was observed for the Gaussian model on binary logit transformed β-values, referred to as M-values. Our results further suggest that models for location and scale are specifically sensitive towards violations of the distribution assumption and towards outliers in the methylation data. Therefore, a resampling procedure is proposed as a mode of inference and shown to diminish type I error rate in

  12. Alteration in Methylation Pattern of Retinoblastoma 1 Gene Promotor Region in Intestinal Metaplasia with or without Helicobacter pylori and Gastric Cancer Patients.

    Science.gov (United States)

    Boyacioglu, Seda Orenay; Kasap, Elmas; Yuceyar, Hakan; Korkmaz, Mehmet

    2016-01-01

    Helicobacter pylori, intestinal metaplasia (IM), and gene methylation play important roles in gastric carcinogenesis. However, the association among H. pylori infection, IM, gastric cancer (GC), and gene methylation is not fully understood. Cell cycle control involving retinoblastoma 1 (RB1) gene is one of the main regulatory pathways reported to be altered in gastric carcinogenesis. The purpose of this research is to assess the methylation status of RB1 gene in GC and IM with or without H. pylori infection, and to discuss the possible role of H. pylori-induced RB1 gene methylation in the mechanism of gastric carcinogenesis. The methylation profile of RB1 gene was analyzed by sodium bisulfite modification and methylation-specific PCR in GC (n = 24), IM patients with H. pylori positive (n = 20) and negative (n = 20), and control subjects (n = 20). According to methylation levels in RB1 gene; the high correlation values were detected between H. pylori positive-IM group and GC group, and between H. pylori positive-IM and H. pylori negative-IM groups (p gene. High methylation levels in RB1 gene in H. pylori positive individuals may suggest an elevated risk of gastric cancer occurrence.

  13. The mGluR5 antagonist AFQ056 does not affect methylation and transcription of the mutant FMR1 gene in vitro

    Directory of Open Access Journals (Sweden)

    Tabolacci Elisabetta

    2012-03-01

    Full Text Available Abstract Background Fragile X syndrome (FXS, the leading cause of inherited mental retardation, is due to expansion and methylation of a CGG sequence in the FMR1 gene, which result in its silencing and consequent absence of FMRP protein. This absence causes loss of repression of metabotropic glutamate receptor 5 (mGluR5-mediated pathways resulting in the behavioral and cognitive impairments associated with FXS. In a randomized, double-blind trial it was recently demonstrated a beneficial effect of AFQ056, a selective inhibitor of metabotrobic glutamate receptor type 5 (mGluR5, on fully methylated FXS patients respect to partially methylated FXS ones. Methods To determine whether AFQ056 may have secondary effects on the methylation and transcription of FMR1, here we treated three FXS lymphoblastoid cell lines and one normal control male line. A quantitative RT-PCR was performed to assess transcriptional reactivation of the FMR1 gene. To assess the methylation status of the FMR1 gene promoter it was carried out a bisulphite sequencing analysis. Results Both FMR1-mRNA levels and DNA methylation were unmodified with respect to untreated controls. Conclusions These results demonstrate that the AFQ056 effect on fully methylated FXS patients is not due to a secondary effect on DNA methylation and consequent transcriptional activation of FMR1.

  14. Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

    DEFF Research Database (Denmark)

    Lyng, Maria B; Laenkholm, Anne-Vibeke; Pallisgaard, Niels

    2008-01-01

    BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various...... means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent...... of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER...

  15. Effects of cytosine methylation on transcription factor binding sites

    KAUST Repository

    Medvedeva, Yulia A; Khamis, Abdullah M.; Kulakovskiy, Ivan V; Ba Alawi, Wail; Bhuyan, Md Shariful I; Kawaji, Hideya; Lassmann, Timo; Harbers, Matthias; Forrest, Alistair RR; Bajic, Vladimir B.

    2014-01-01

    Background: DNA methylation in promoters is closely linked to downstream gene repression. However, whether DNA methylation is a cause or a consequence of gene repression remains an open question. If it is a cause, then DNA methylation may affect

  16. DMPD: TLR ignores methylated RNA? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 16111629 TLR ignores methylated RNA? Ishii KJ, Akira S. Immunity. 2005 Aug;23(2):11...1-3. (.png) (.svg) (.html) (.csml) Show TLR ignores methylated RNA? PubmedID 16111629 Title TLR ignores methylated

  17. Media independence and dividend policy

    DEFF Research Database (Denmark)

    Farooq, Omar; Dandoune, Salma

    2012-01-01

    independence and dividend policies in emerging markets. Using a dataset from twenty three emerging markets, we show a significantly negative relationship between dividend policies (payout ratio and decision to pay dividend) and media independence. We argue that independent media reduces information asymmetries...... for stock market participants. Consequently, stock market participants in emerging markets with more independent media do not demand as high and as much dividends as their counterparts in emerging markets with less independent media. We also show that press independence is more important in defining......Can media pressurize managers to disgorge excess cash to shareholders? Do firms in countries with more independent media follow different dividend policies than firms with less independent media? This paper seeks to answer these questions and aims to document the relationship between media...

  18. Modeling spatiotemporal dynamics of DNA methylation

    DEFF Research Database (Denmark)

    Lövkvist, Cecilia Elisabet

    into how epigenetic marks are distributed in the human genome. In the first part of the thesis, we investigate DNA methylation and maintenance of methylation patterns throughout cell division. We argue that collaborative models, those where the methylation of CpG sites depends on the methylation status...... into the game more explicitly in another type of model that speaks out the duality of the two aspects. Using statistical analysis of experimental data, this thesis further explores a link between DNA methylation and nucleosome occupancy. By comparing the patterns on promoters to regions with similar Cp...... division. The patterns of epigentic marks depend on enzymes that ensure their maintenance and introduction. Using theoretical models, this thesis proposes new mechanisms for how enzymes operate to maintain patterns of epigenetic marks. Through analysis of experimental data this work gives new insight...

  19. Protein methylation reactions in intact pea chloroplasts

    International Nuclear Information System (INIS)

    Niemi, K.J.

    1989-01-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ( 3 H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented

  20. Radiation effects on DNA methylation in mice

    International Nuclear Information System (INIS)

    Komura, J.; Kurishita, A.; Miyamura, Y.; Ono, T.; Tawa, R.; Sakurai, H.

    1992-01-01

    Effects of ionizing radiation on DNA methylation in liver, brain and spleen were examined by high performance liquid chromatography (HPLC). The total methylated cytosine level in the genome was reduced within 8 hours after 3.8 Gy of irradiation in liver of adult mice. But no appreciable effect was observed in brain and spleen. When mice were irradiated at newborn, liver DNA revealed no change in methylated cytosine level. Even though slight effects of radiation were detected in he methylation of the c-myc and c-fos genes, they were only temporary and no long-term effects were observed. These data suggest that the effect of radiation on DNA methylation in vivo is not prevailing a DNA damage, but rather influenced much through biological parameters. (author)

  1. Methylation analysis of polysaccharides: Technical advice.

    Science.gov (United States)

    Sims, Ian M; Carnachan, Susan M; Bell, Tracey J; Hinkley, Simon F R

    2018-05-15

    Glycosyl linkage (methylation) analysis is used widely for the structural determination of oligo- and poly-saccharides. The procedure involves derivatisation of the individual component sugars of a polysaccharide to partially methylated alditol acetates which are analysed and quantified by gas chromatography-mass spectrometry. The linkage positions for each component sugar can be determined by correctly identifying the partially methylated alditol acetates. Although the methods are well established, there are many technical aspects to this procedure and both careful attention to detail and considerable experience are required to achieve a successful methylation analysis and to correctly interpret the data generated. The aim of this article is to provide the technical details and critical procedural steps necessary for a successful methylation analysis and to assist researchers (a) with interpreting data correctly and (b) in providing the comprehensive data required for reviewers to fully assess the work. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Dimos Gaidatzis

    2014-02-01

    Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

  3. Detection of DNA methylation changes in micropropagated banana plants using methylation-sensitive amplification polymorphism (MSAP).

    Science.gov (United States)

    Peraza-Echeverria, S; Herrera-Valencia, V A.; Kay, A -J.

    2001-07-01

    The extent of DNA methylation polymorphisms was evaluated in micropropagated banana (Musa AAA cv. 'Grand Naine') derived from either the vegetative apex of the sucker or the floral apex of the male inflorescence using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 465 fragments, each representing a recognition site cleaved by either or both of the isoschizomers were amplified using eight combinations of primers. A total of 107 sites (23%) were found to be methylated at cytosine in the genome of micropropagated banana plants. In plants micropropagated from the male inflorescence explant 14 (3%) DNA methylation events were polymorphic, while plants micropropagated from the sucker explant produced 8 (1.7%) polymorphisms. No DNA methylation polymorphisms were detected in conventionally propagated banana plants. These results demonstrated the usefulness of MSAP to detect DNA methylation events in micropropagated banana plants and indicate that DNA methylation polymorphisms are associated with micropropagation.

  4. The origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

    Science.gov (United States)

    Wolff, George A.; Lamb, Neil A.; Maxwell, James R.

    1986-03-01

    Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalysed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4β-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4α-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4β-methyl steranes decrease gradually in abundance relative to their 4α-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

  5. Diffuse large B-cell lymphoma with combined TP53 mutation and MIR34A> methylation

    DEFF Research Database (Denmark)

    Asmar, Fazila; Hother, Christoffer; Kulosman, Gorjan

    2014-01-01

    and MIR34A methylation ("double hit") and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (Phit") influence on survival. The TP53/MIR34A "double-hit" is an independent...... negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A "double hit" characterizes a very aggressive subgroup of DLBCL, which may be treatable...

  6. A refined, rapid and reproducible high resolution melt (HRM-based method suitable for quantification of global LINE-1 repetitive element methylation

    Directory of Open Access Journals (Sweden)

    Tse M Yat

    2011-12-01

    Full Text Available Abstract Background The methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation. Findings Using high resolution melt (HRM curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986 with a high amplification efficiency (90-105%, capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach. Conclusions In summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies.

  7. Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR.

    OpenAIRE

    Gretch, D R; Wilson, J J; Carithers, R L; dela Rosa, C; Han, J H; Corey, L

    1993-01-01

    We evaluated a new hepatitis C virus RNA assay based on one-stage PCR followed by liquid hybridization with an oligonucleotide probe and compared it with nested-set PCR. The one-stage and nested-set PCR assays had identical sensitivities in analytical experiments and showed 100% concordance when clinical specimens were used. One-stage PCR may be less prone to contamination than nested-set PCR.

  8. Validations of SCT Methylation as a Hallmark Biomarker for Lung Cancers

    Science.gov (United States)

    Fujimoto, Junya; Wistuba, Ignacio; Lam, Stephen; Yatabe, Yasushi; Wang, Yi-Wei; Stastny, Victor; Gao, Boning; Larsen, Jill E; Girard, Luc; Liu, Xiaoyun; Song, Kai; Behrens, Carmen; Kalhor, Neda; Xie, Yang; Zhang, Michael Q; Minna, John D; Gazdar, Adi F

    2016-01-01

    Background The human secretin (SCT) gene encodes secretin, a hormone with limited tissue distribution. Analysis of The Cancer Genome Atlas (TCGA) 450K methylation array data indicated that the SCT promoter region is differentially hypermethylated in lung cancer. Our purpose was to validate SCT methylation as a potential cancer biomarker for lung cancer. Methods We analyzed TCGA data, and developed and applied SCT-specific bisulfite DNA sequencing and quantitative methylation specific PCR (qMSP) assays. Results The analyses of TCGA 450K data of 801 samples showed that SCT hypermethylation has an area under curve (AUC) value >0.98 to distinguish lung adenocarcinomas or squamous cell carcinomas from non-malignant lung. We confirmed the highly discriminative SCT methylation by bisulfite sequencing of lung cancer cell lines and normal blood cells. By applying qMSP, we found that SCT hypermethylation was frequently detected in all major subtypes of malignant NSCLC (AUC=0.92, n=108) and SCLC cancers (AUC=0.93, n=40) but less frequently present in lung carcinoids (AUC=0.54, n=20). SCT hypermethylation appeared in lung carcinoma in situ samples during multistage pathogenesis and increased in invasive samples. Further analyses of TCGA 450K data showed that SCT hypermethylation is highly discriminative in most types of other malignant tumors but less frequently present in low-grade malignant tumors. The only normal tissue with high methylation was the placenta. Conclusions Our findings demonstrated that SCT methylation is a highly discriminative biomarker for lung and other malignant tumors, and less frequently present in low-grade malignant tumors including lung carcinoids, and appears at the carcinoma in situ stage. PMID:26725182

  9. Methylation associated transcriptional repression of ELOVL5 in novel colorectal cancer cell lines.

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    Arnoud Boot

    Full Text Available Genetic and epigenetic alterations mark colorectal cancer (CRC. Global hypomethylation is observed in nearly all CRC, but a distinct subset of CRC show the CpG Island Methylator Phenotype (CIMP. These tumors show DNA hypermethylation of a specific subset of CpG islands, resulting in transcriptional downregulation of nearby genes. Recently we reported the establishment of novel CRC cell lines derived from primary and metastatic CRC tissues. In this study we describe the DNA methylation profiling of these low passage CRC cell lines. We generated global DNA methylation profiles with Infinium HumanMethylation450 BeadChips and analysed them in conjunction with matching gene expression profiles. Multidimensional scaling of the DNA methylation and gene expression datasets showed that BRAF mutated cell lines form a distinct group. In this group we investigated the 706 loci which we have previously identified to be hypermethylated in BRAF mutant CRC. We validated the significant findings in the The Cancer Genome Atlas colon adenocarcinoma dataset. Our analysis identified ELOVL5, FAM127B, MTERF1, ZNF606 to be subject to transcriptional downregulation through DNA hypermethylation in CRC. We further investigated ELOVL5 with qPCR and immunohistochemical staining, validating our results, but did not find a clear relation between ELOVL5 expression and tumor stage or relapse free survival. ELOVL5, FAM127B, MTERF1, ZNF606 are involved in important cellular processes such as apoptosis, lipogenesis and the downstream transcriptional effect of the MAPK-pathway. We have identified a DNA methylation profile regulating key cellular processes in CRC, resulting in a growth advantage to the tumor cells.

  10. Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

    Science.gov (United States)

    Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

    2013-08-01

    The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

  11. Genome-wide screen of DNA methylation changes induced by low dose X-ray radiation in mice.

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    Jingzi Wang

    Full Text Available Epigenetic mechanisms play a key role in non-targeted effects of radiation. The purpose of this study was to investigate global hypomethylation and promoter hypermethylation of particular genes induced by low dose radiation (LDR. Thirty male BALB/c mice were divided into 3 groups: control, acutely exposed (0.5 Gy X-rays, and chronic exposure for 10 days (0.05Gy/d×10d. High-performance liquid chromatography (HPLC and MeDIP-quantitative polymerase chain reaction (qPCR were used to study methylation profiles. DNMT1 and MBD2 expression was determined by qPCR and western blot assays. Methylation and expression of Rad23b and Ddit3 were determined by bisulfate sequencing primers (BSP and qPCR, respectively. The results show that LDR induced genomic hypomethylation in blood 2 h postirraditaion, but was not retained at 1-month. DNMT1 and MBD2 were downregulated in a tissue-specific manner but did not persist. Specific hypermethylation was observed for 811 regions in the group receiving chronic exposure, which covered almost all key biological processes as indicated by GO and KEGG pathway analysis. Eight hypermethylated genes (Rad23b, Tdg, Ccnd1, Ddit3, Llgl1, Rasl11a, Tbx2, Scl6a15 were verified by MeDIP-qPCR. Among them, Rad23b and Ddit3 gene displayed tissue-specific methylation and downregulation, which persisted for 1-month postirradiation. Thus, LDR induced global hypomethylation and tissue-specific promoter hypermethylation of particular genes. Promoter hypermethylation, rather than global hypomethylation, was relatively stable. Dysregulation of methylation might be correlated with down-regulation of DNMT1 and MBD2, but much better understanding the molecular mechanisms involved in this process will require further study.

  12. SMYD2 promoter DNA methylation is associated with abdominal aortic aneurysm (AAA) and SMYD2 expression in vascular smooth muscle cells.

    Science.gov (United States)

    Toghill, Bradley J; Saratzis, Athanasios; Freeman, Peter J; Sylvius, Nicolas; Bown, Matthew J

    2018-01-01

    Abdominal aortic aneurysm (AAA) is a deadly cardiovascular disease characterised by the gradual, irreversible dilation of the abdominal aorta. AAA is a complex genetic disease but little is known about the role of epigenetics. Our objective was to determine if global DNA methylation and CpG-specific methylation at known AAA risk loci is associated with AAA, and the functional effects of methylation changes. We assessed global methylation in peripheral blood mononuclear cell DNA from 92 individuals with AAA and 93 controls using enzyme-linked immunosorbent assays, identifying hyper-methylation in those with large AAA and a positive linear association with AAA diameter ( P  AAA risk loci identified in genome-wide association studies, using bisulphite next-generation sequencing (NGS) in vascular smooth muscle cells (VSMCs) taken from aortic tissues of 44 individuals (24 AAAs and 20 controls). In IL6R , 2 CpGs were hyper-methylated ( P  = 0.0145); in ERG , 13 CpGs were hyper-methylated ( P  = 0.0005); in SERPINB9 , 6 CpGs were hypo-methylated ( P  = 0.0037) and 1 CpG was hyper-methylated ( P  = 0.0098); and in SMYD2 , 4 CpGs were hypo-methylated ( P  = 0.0012).RT-qPCR was performed for each differentially methylated gene on mRNA from the same VSMCs and compared with methylation. This analysis revealed downregulation of SMYD2 and SERPINB9 in AAA, and a direct linear relationship between SMYD2 promoter methylation and SMYD2 expression ( P  = 0.038). Furthermore, downregulation of SMYD2 at the site of aneurysm in the aortic wall was further corroborated in 6 of the same samples used for methylation and gene expression analysis with immunohistochemistry. This study is the first to assess DNA methylation in VSMCs from individuals with AAA using NGS, and provides further evidence there is an epigenetic basis to AAA. Our study shows that methylation status of the SMYD2 promoter may be linked with decreased SMYD2 expression in disease pathobiology. In

  13. DNA methylation is an independent prognostic marker of survival in adrenocortical cancer

    NARCIS (Netherlands)

    Jouinot, Anne; Assie, Guillaume; Libe, Rossella; Fassnacht, Martin; Papathomas, Thomas; Barreau, Olivia; De La Villeon, Bruno; Faillot, Simon; Hamzaoui, Nadim; Neou, Mario; Perlemoine, Karine; Rene-Corail, Fernande; Rodriguez, Stephanie; Sibony, Mathilde; Tissier, Frederique; Dousset, Bertrand; Sbiera, Silviu; Ronchi, Cristina; Kroiss, Matthias; Korpershoek, Esther; De Krijger, Ronald; Waldmann, Jens; Bartsch, Detlef K.; Quinkler, Marcus; Haissaguerre, Magalie; Tabarin, Antoine; Chabre, Olivier; Sturm, Nathalie; Luconi, Michaela; Mantero, Franco; Mannelli, Massimo; Cohen, Regis; Kerlan, Veronique; Touraine, Philippe; Barrande, Gaelle; Groussin, Lionel; Bertagna, Xavier; Baudin, Eric; Amar, Laurence; Beuschlein, Felix; Clauser, Eric; Coste, Joel; Bertherat, Jerome

    2017-01-01

    Context: Adrenocortical cancer (ACC) is an aggressive tumor with a heterogeneous outcome. Prognostic stratification is difficult even based on tumor stage and Ki67. Recently integrated genomics studies have demonstrated that CpG islands hypermethylation is correlated with poor survival. Objective:

  14. DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

    NARCIS (Netherlands)

    Glasow, Annegret; Barrett, Angela; Petrie, Kevin; Gupta, Rajeev; Boix-Chornet, Manuel; Zhou, Da-Cheng; Grimwade, David; Gallagher, Robert; von Lindern, Marieke; Waxman, Samuel; Enver, Tariq; Hildebrandt, Guido; Zelent, Arthur

    2008-01-01

    The retinoic acid receptor (RAR) alpha gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARalpha2) isoform increases with myelomonocytic differentiation and appears to be

  15. DNA methylation-independent loss of RARA gene expression in acute myeloid leukemia

    NARCIS (Netherlands)

    A. Glasow (Annegret); A. Barrett (Angela); K. Petrie (Kevin); R. Gupta (Rajeev); M. Boix-Chornet (Manuel); D.C. Zhou; D. Grimwade (David); R. Gallagher (Robert); M.M. von Lindern (Marieke); S. Waxman (Sergio); T. Enver (Tariq); G. Hildebrandt (Guido); A. Zelent (Arthur)

    2008-01-01

    textabstractThe retinoic acid receptor (RAR) α gene (RARA) encodes 2 major isoforms and mediates positive effects of all-trans retinoic acid (ATRA) on myelomonocytic differentiation. Expression of the ATRA-inducible (RARα2) isoform increases with myelomonocytic differentiation and appears to be

  16. Genome-Wide DNA Methylation Indicates Silencing of Tumor Suppressor Genes in Uterine Leiomyoma

    Science.gov (United States)

    Navarro, Antonia; Yin, Ping; Monsivais, Diana; Lin, Simon M.; Du, Pan; Wei, Jian-Jun; Bulun, Serdar E.

    2012-01-01

    Background Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown. Principal Findings We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels. Conclusions These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women. PMID:22428009

  17. Promoter Methylation of RASSF1A Associates to Adult Secondary Glioblastomas and Pediatric Glioblastomas.

    Science.gov (United States)

    Muñoz, Jorge; Inda, María Del Mar; Lázcoz, Paula; Zazpe, Idoya; Fan, Xing; Alfaro, Jorge; Tuñón, Teresa; Rey, Juan A; Castresana, Javier S

    2012-01-01

    While allelic losses and mutations of tumor suppressor genes implicated in the etiology of astrocytoma have been widely assessed, the role of epigenetics is still a matter of study. We analyzed the frequency of promoter hypermethylation by methylation-specific PCR (MSP) in five tumor suppressor genes (PTEN, MGMT, RASSF1A, p14(ARF), and p16(INK4A)), in astrocytoma samples and cell lines. RASSF1A was the most frequently hypermethylated gene in all grades of astrocytoma samples, in cell lines, and in adult secondary GBM. It was followed by MGMT. PTEN showed a slight methylation signal in only one GBM and one pilocytic astrocytoma, and in two cell lines; while p14(ARF) and p16(INK4A) did not show any evidence of methylation in primary tumors or cell lines. In pediatric GBM, RASSF1A was again the most frequently altered gene, followed by MGMT; PTEN, p14 and p16 showed no alterations. Lack or reduced expression of RASSF1A in cell lines was correlated with the presence of methylation. RASSF1A promoter hypermethylation might be used as a diagnostic marker for secondary GBM and pediatric GBM. Promoter hypermethylation might not be an important inactivation mechanism in other genes like PTEN, p14(ARF) and p16(INK4A), in which other alterations (mutations, homozygous deletions) are prevalent.

  18. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

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    Yogita Maheshwari

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  19. Clostridium perfringens isolate typing by multiplex PCR

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    MR Ahsani

    2010-01-01

    Full Text Available Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

  20. DNA methylation in the human cerebral cortex is dynamically regulated throughout the life span and involves differentiated neurons.

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    Kimberly D Siegmund

    Full Text Available The role of DNA cytosine methylation, an epigenetic regulator of chromatin structure and function, during normal and pathological brain development and aging remains unclear. Here, we examined by MethyLight PCR the DNA methylation status at 50 loci, encompassing primarily 5' CpG islands of genes related to CNS growth and development, in temporal neocortex of 125 subjects ranging in age from 17 weeks of gestation to 104 years old. Two psychiatric disease cohorts--defined by chronic neurodegeneration (Alzheimer's or lack thereof (schizophrenia--were included. A robust and progressive rise in DNA methylation levels across the lifespan was observed for 8/50 loci (GABRA2, GAD1, HOXA1, NEUROD1, NEUROD2, PGR, STK11, SYK typically in conjunction with declining levels of the corresponding mRNAs. Another 16 loci were defined by a sharp rise in DNA methylation levels within the first few months or years after birth. Disease-associated changes were limited to 2/50 loci in the Alzheimer's cohort, which appeared to reflect an acceleration of the age-related change in normal brain. Additionally, methylation studies on sorted nuclei provided evidence for bidirectional methylation events in cortical neurons during the transition from childhood to advanced age, as reflected by significant increases at 3, and a decrease at 1 of 10 loci. Furthermore, the DNMT3a de novo DNA methyl-transferase was expressed across all ages, including a subset of neurons residing in layers III and V of the mature cortex. Therefore, DNA methylation is dynamically regulated in the human cerebral cortex throughout the lifespan, involves differentiated neurons, and affects a substantial portion of genes predominantly by an age-related increase.

  1. Association of promoter methylation and 32-bp deletion of the PTEN gene with susceptibility to metabolic syndrome.

    Science.gov (United States)

    Hashemi, Mohammad; Rezaei, Hamzeh; Eskandari-Nasab, Ebrahim; Kaykhaei, Mahmoud-Ali; Taheri, Mohsen

    2013-01-01

    Metabolic syndrome (MeS), a cluster of several metabolic disorders, is increasingly being recognized as a risk factor for type II diabetes (T2D) and cardiovascular disease. Genetic and epigenetic alteration of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) has been associated with components of MeS. The aim of the present study was to investigate the possible association of a 32-bp deletion polymorphism and promoter methylation of the PTEN gene with MeS. DNA was extracted from the peripheral blood of 151 subjects with and 149 subjects without MeS. The 32-bp deletion variant of PTEN was detected by polymerase chain reaction (PCR) and PTEN promoter methylation was defined by a nested methylation‑specific PCR (MSP) method. No significant differences were found in the allelic and genotypic frequencies of the 32-bp deletion variant of PTEN between the groups [odds ratio (OR), 0.77; 95% confidence interval (CI), 0.41-1.45; P=0.431]. However, patients with MeS were identified to have lower levels of PTEN promoter hypermethylation than subjects without MeS. Promoter methylation may be a protective factor against susceptibility to MeS (OR, 0.52; 95% CI, 0.29-0.92; P=0.029). Our findings suggest that PTEN promoter methylation may be a mechanism for PTEN downregulation or silencing in MeS, which remains to be fully clarified.

  2. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis.

    Science.gov (United States)

    Devonshire, Alison S; O'Sullivan, Denise M; Honeyborne, Isobella; Jones, Gerwyn; Karczmarczyk, Maria; Pavšič, Jernej; Gutteridge, Alice; Milavec, Mojca; Mendoza, Pablo; Schimmel, Heinz; Van Heuverswyn, Fran; Gorton, Rebecca; Cirillo, Daniela Maria; Borroni, Emanuele; Harris, Kathryn; Barnard, Marinus; Heydenrych, Anthenette; Ndusilo, Norah; Wallis, Carole L; Pillay, Keshree; Barry, Thomas; Reddington, Kate; Richter, Elvira; Mozioğlu, Erkan; Akyürek, Sema; Yalçınkaya, Burhanettin; Akgoz, Muslum; Žel, Jana; Foy, Carole A; McHugh, Timothy D; Huggett, Jim F

    2016-08-03

    Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification

  3. DNA methylation analysis reveals distinct methylation signatures in pediatric germ cell tumors

    International Nuclear Information System (INIS)

    Amatruda, James F; Frazier, A Lindsay; Poynter, Jenny N; Ross, Julie A; Christensen, Brock; Fustino, Nicholas J; Chen, Kenneth S; Hooten, Anthony J; Nelson, Heather; Kuriger, Jacquelyn K; Rakheja, Dinesh

    2013-01-01

    Aberrant DNA methylation is a prominent feature of many cancers, and may be especially relevant in germ cell tumors (GCTs) due to the extensive epigenetic reprogramming that occurs in the germ line during normal development. We used the Illumina GoldenGate Cancer Methylation Panel to compare DNA methylation in the three main histologic subtypes of pediatric GCTs (germinoma, teratoma and yolk sac tumor (YST); N = 51) and used recursively partitioned mixture models (RPMM) to test associations between methylation pattern and tumor and demographic characteristics. We identified genes and pathways that were differentially methylated using generalized linear models and Ingenuity Pathway Analysis. We also measured global DNA methylation at LINE1 elements and evaluated methylation at selected imprinted loci using pyrosequencing. Methylation patterns differed by tumor histology, with 18/19 YSTs forming a distinct methylation class. Four pathways showed significant enrichment for YSTs, including a human embryonic stem cell pluripotency pathway. We identified 190 CpG loci with significant methylation differences in mature and immature teratomas (q < 0.05), including a number of CpGs in stem cell and pluripotency-related pathways. Both YST and germinoma showed significantly lower methylation at LINE1 elements compared with normal adjacent tissue while there was no difference between teratoma (mature and immature) and normal tissue. DNA methylation at imprinted loci differed significantly by tumor histology and location. Understanding methylation patterns may identify the developmental stage at which the GCT arose and the at-risk period when environmental exposures could be most harmful. Further, identification of relevant genetic pathways could lead to the development of new targets for therapy

  4. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Koji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Munetsuna, Eiji [Department of Biochemistry, Fujita Health University School of Medicine, Toyoake (Japan); Yamada, Hiroya, E-mail: hyamada@fujita-hu.ac.jp [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan); Ando, Yoshitaka [Department of Joint Research Laboratory of Clinical Medicine, Fujita Health University Hospital, Toyoake (Japan); Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Suzuki, Koji [Department of Public Health, Fujita Health University School of Health Sciences, Toyoake (Japan); Teradaira, Ryoji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Hashimoto, Shuji [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan)

    2015-12-04

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  5. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    International Nuclear Information System (INIS)

    Ohashi, Koji; Munetsuna, Eiji; Yamada, Hiroya; Ando, Yoshitaka; Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki; Suzuki, Koji; Teradaira, Ryoji; Hashimoto, Shuji

    2015-01-01

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  6. Gene mdpC plays a regulatory role in the methyl-tert-butyl ether degradation pathway of Methylibium petroleiphilum strain PM1.

    Science.gov (United States)

    Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

    2015-04-01

    Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Investigating the Connection between hgcA and Mercury Methylation Rates in the Environment

    Science.gov (United States)

    King, A. J.; Christensen, G. A.; Wymore, A. M.; Podar, M.; Hurt, R. A., Jr.; Brown, S. D.; Palumbo, A. V.; Bender, K. S.; Fields, M. W.; Gilmour, C. C.; Santillan, E. F. U.; Brandt, C. C.; Elias, D. A.

    2015-12-01

    Methylmercury (MeHg) is a common contaminant in many natural environments and is known to be a neurotoxin that impacts human health through bioaccumulation in food webs. The anaerobic conversion of mercury (Hg) to MeHg by microorganisms requires the presence of both HgcA and HgcB. In an effort to link hgcAB abundance and diversity with MeHg generation rates, we performed metagenomic and 16S rRNA sequencing as well as qualitative polymerase chain reaction (qPCR) of hgcA on samples from eight mercury-contaminated sites ranging from tidal marshes to Arctic permafrost. Custom algorithms were developed to filter hgcA sequences from the metagenomes, and to then select for those lineages that also contained hgcB. In the metagenomes, the Deltaproteobacteria dominated the pool of hgcAB from all eight sites; however, Firmicutes and methanogenic Archaea were each 50% less abundant. In parallel to the metagenomics studies, clone libraries of hgcAB were constructed for each site. This more cost-effective approach allowed us to verify the identity of the hgcAB+ organism, and yielded similar results to the metagenomes. Additionally, to determine the accuracy of our new degenerate qPCR primer sets (three sets specific to the three major clades of mercury methylators) in the environment, qPCR hgcA abundance values were compared to those derived from the metagenomes. Finally, we present evidence that hgcA abundance can correlate with MeHg concentrations but that the relationship is influenced by local environmental conditions. Our work demonstrates the relative efficacy of genetic methods for assessing the presence of mercury-methylators in eight different environments contaminated with mercury as well as the strength of association between abundance of hgcA and the rate of mercury methylation.

  8. Culture -independent Pathogenic Bacterial Communities in Bottled Mineral Water

    Directory of Open Access Journals (Sweden)

    Hamdy A. Hassan

    2015-08-01

    Full Text Available Bottled mineral water (BMW is an alternative to mains water and consider it to be better and safer. Access to safe BMW from the bacteria involving potential health hazard is essential to health. Cultivation-independent technique PCR-based single-strand conformation polymorphism (SSCP for genetic profiling of PCR-amplified 16S rRNA genes was performed using Com primer set targeting the 16S rRNA genes for detection of pathogenic bacteria in bottled mineral water from the final product of six factories for bottled mineral drinking water in Wadi El-natron region- Egypt. These factories use often ozone technology to treat large quantities of water because of its effectiveness in purifying and conditioning water. A total of 27 single products were isolated from the profiles by PCR re-amplification and cloning. Sequence analysis of 27 SSCP bands revealed that the 16S rRNA sequences were clustered into seven operational taxonomic units (OTUs and the compositions of the communities of the six samples were all common. The results showed that most communities from phyla Alphaproteobacteria and certainly in the Sphingomonas sp. Culture-independent approaches produced complementary information, thus generating a more accurate view for the bacterial community in the BMW, particularly in the disinfection step, as it constitutes the final barrier before BMW distribution to the consumer

  9. CD133 expression is not an independent prognostic factor in stage II and III colorectal cancer but may predict the better outcome in patients with adjuvant therapy

    International Nuclear Information System (INIS)

    Mia-Jan, Khalilullah; Jung, So Young; Kim, Ik-Yong; Oh, Sung Soo; Choi, EunHee; Chang, Sei Jin; Kang, Tae Young; Cho, Mee-Yon

    2013-01-01

    Cancer stem cells (CSCs) are notorious for their capacity of tumor progression, metastasis or resistance to chemo-radiotherapy. However, the undisputed role of cancer stem marker, CD133, in colorectal cancers (CRCs) is not clear yet. We assessed 271 surgically-resected stage II and III primary CRCs with (171) and without (100) adjuvant therapy after surgery. CD133 expression was analyzed by immunohistochemical (IHC) staining and real-time RT-PCR. CD133 promoter methylation was quantified by pyrosequencing. The CD133 IHC expression was significantly correlated with mRNA expression (p=0.0257) and inversely correlated with the promoter methylation (p=0.0001). CD133 was expressed more frequently in rectal cancer (p=0.0035), and in moderately differentiated tumors (p=0.0378). In survival analysis, CD133 expression was not significantly correlated with overall survival (OS) (p=0.9689) as well as disease-free survival (DFS) (p=0.2103). However, CD133+ tumors were significantly associated with better OS in patients with adjuvant therapy compared to those without adjuvant therapy (p<0.0001, HR 0.125, 95% CI 0.052-0.299). But the patients with CD133- tumors did not show any significant difference of survival according to adjuvant therapy (p=0.055, HR 0.500, 95% CI 0.247-1.015). In stage II and III CRCs, CD133 IHC expression may signify the benefit for adjuvant therapy although it is not an independent prognostic factor

  10. Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

    Directory of Open Access Journals (Sweden)

    Xiang Gao

    Full Text Available Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3 and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

  11. Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

    Science.gov (United States)

    Gao, Xiang; Cao, Donghui; Liu, Jie; Wang, Xiaoping; Geng, Shujuan; Liu, Bao; Shi, Decheng

    2013-01-01

    Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3) and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

  12. Boards: Independent and Committed Directors?

    OpenAIRE

    Christophe Volonté

    2011-01-01

    Regulators, proxy advisors and shareholders are regularly calling for independent directors. However, at the same time, independent directors commonly engage in numerous outside activities potentially reducing their time and commitment with the particular firm. Using Tobin's Q as an approximation of market valuation and controlling for endogeneity, our empirical analysis reveals that neither is independence positively related to firm performance nor are outside activities negatively related t...

  13. Independent component analysis: recent advances

    OpenAIRE

    Hyv?rinen, Aapo

    2013-01-01

    Independent component analysis is a probabilistic method for learning a linear transform of a random vector. The goal is to find components that are maximally independent and non-Gaussian (non-normal). Its fundamental difference to classical multi-variate statistical methods is in the assumption of non-Gaussianity, which enables the identification of original, underlying components, in contrast to classical methods. The basic theory of independent component analysis was mainly developed in th...

  14. Avaliação das técnicas de RT-PCR e heminested RT-PCR em cérebros de cães com sinais neurológicos compatíveis com cinomose

    Directory of Open Access Journals (Sweden)

    Adriana Cortez

    2015-12-01

    Full Text Available The diagnostic value of RT-PCR and hemi-nested RT-PCR (hnRT-PCR was compared in brain samples of dogs presenting neurological signs compatible with canine distemper. Samples of central nervous system (CNS were collected from 68 dogs and tested by direct immunofluorescence test (RFID and, independent of the results, they were stored at -20°C for at least three years. They were submitted to the RT-PCR and hnRT-PCR techniques aiming to determine the gene responsible for the viral nucleoprotein decoding. Fifty-nine samples were positive for RIFD, 40 for RT-PCR (Kappa = 0.358 and 54 for hnRT-PCR (Kappa = 0.740. All nine RIFD negative samples were also negative for RT-PCR and hnRT-PCR. In spite of the storage duration and proper sample conditions, the estimated accordance between hnRT-PCR and RIFD demonstrated that hnRT-PCR technique can be applied in retrospective studies.

  15. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    Science.gov (United States)

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Age-associated sperm DNA methylation alterations: possible implications in offspring disease susceptibility.

    Science.gov (United States)

    Jenkins, Timothy G; Aston, Kenneth I; Pflueger, Christian; Cairns, Bradley R; Carrell, Douglas T

    2014-07-01

    Recent evidence demonstrates a role for paternal aging on offspring disease susceptibility. It is well established that various neuropsychiatric disorders (schizophrenia, autism, etc.), trinucleotide expansion associated diseases (myotonic dystrophy, Huntington's, etc.) and even some forms of cancer have increased incidence in the offspring of older fathers. Despite strong epidemiological evidence that these alterations are more common in offspring sired by older fathers, in most cases the mechanisms that drive these processes are unclear. However, it is commonly believed that epigenetics, and specifically DNA methylation alterations, likely play a role. In this study we have investigated the impact of aging on DNA methylation in mature human sperm. Using a methylation array approach we evaluated changes to sperm DNA methylation patterns in 17 fertile donors by comparing the sperm methylome of 2 samples collected from each individual 9-19 years apart. With this design we have identified 139 regions that are significantly and consistently hypomethylated with age and 8 regions that are significantly hypermethylated with age. A representative subset of these alterations have been confirmed in an independent cohort. A total of 117 genes are associated with these regions of methylation alterations (promoter or gene body). Intriguingly, a portion of the age-related changes in sperm DNA methylation are located at genes previously associated with schizophrenia and bipolar disorder. While our data does not establish a causative relationship, it does raise the possibility that the age-associated methylation of the candidate genes that we observe in sperm might contribute to the increased incidence of neuropsychiatric and other disorders in the offspring of older males. However, further study is required to determine whether, and to what extent, a causative relationship exists.

  17. Regulation of homocysteine metabolism and methylation in human and mouse tissues

    Science.gov (United States)

    Chen, Natalie C.; Yang, Fan; Capecci, Louis M.; Gu, Ziyu; Schafer, Andrew I.; Durante, William; Yang, Xiao-Feng; Wang, Hong

    2010-01-01

    Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine (Hcy) metabolism involves multiple enzymes; however, tissue Hcy metabolism and its relevance to methylation remain unknown. Here, we established gene expression profiles of 8 Hcy metabolic and 12 methylation enzymes in 20 human and 19 mouse tissues through bioinformatic analysis using expression sequence tag clone counts in tissue cDNA libraries. We analyzed correlations between gene expression, Hcy, S-adenosylhomocysteine (SAH), and S-adenosylmethionine (SAM) levels, and SAM/SAH ratios in mouse tissues. Hcy metabolic and methylation enzymes were classified into two types. The expression of Type 1 enzymes positively correlated with tissue Hcy and SAH levels. These include cystathionine β-synthase, cystathionine-γ-lyase, paraxonase 1, 5,10-methylenetetrahydrofolate reductase, betaine:homocysteine methyltransferase, methionine adenosyltransferase, phosphatidylethanolamine N-methyltransferases and glycine N-methyltransferase. Type 2 enzyme expressions correlate with neither tissue Hcy nor SAH levels. These include SAH hydrolase, methionyl-tRNA synthase, 5-methyltetrahydrofolate:Hcy methyltransferase, S-adenosylmethionine decarboxylase, DNA methyltransferase 1/3a, isoprenylcysteine carboxyl methyltransferases, and histone-lysine N-methyltransferase. SAH is the only Hcy metabolite significantly correlated with Hcy levels and methylation enzyme expression. We established equations expressing combined effects of methylation enzymes on tissue SAH, SAM, and SAM/SAH ratios. Our study is the first to provide panoramic tissue gene expression profiles and mathematical models of tissue methylation regulation.—Chen, N. C., Yang, F., Capecci, L. M., Gu, Z., Schafer, A. I., Durante, W., Yang, X.-F., Wang, H. Regulation of homocysteine metabolism and methylation in human and mouse tissues. PMID:20305127

  18. Differential DNA Methylation in Relation to Age and Health Risks of Obesity

    Directory of Open Access Journals (Sweden)

    María Luisa