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Sample records for methylation independent pcr

  1. Primer design versus PCR bias in methylation independent PCR amplifications.

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    Wojdacz, Tomasz K; Borgbo, Tanni; Hansen, Lise Lotte

    2009-05-16

    Many protocols in methylation studies utilize one primer set to generate a PCR product from bisulfite modified template regardless of its methylation status (methylation independent amplification MIP). However, proportional amplification of methylated and unmethylated alleles is hard to achieve due to PCR bias favoring amplification of unmethylated relatively GC poor sequence. Two primer design systems have been proposed to overcome PCR bias in methylation independent amplifications. The first advises against including any CpG dinucleoteides into the primer sequence (CpG-free primers) and the second, recently published by us, is based on inclusion of a limited number of CpG sites into the primer sequence. Here we used the Methylation Sensitive High Resolution Melting (MS-HRM) technology to investigate the ability of primers designed according to both of the above mentioned primer design systems to proportionally amplify methylated and unmethylated templates. Ten "CpG-free" primer pairs and twenty primers containing limited number of CpGs were tested. In reconstruction experiments the "CpG-free" primers showed primer specific sensitivity and allowed us to detect methylation levels in the range from 5 to 50%. Whereas while using primers containing limited number of CpG sites we were able to consistently detect 1-0.1% methylation levels and effectively control PCR amplification bias. In conclusion, the primers with limited number of CpG sites are able to effectively reverse PCR bias and therefore detect methylated templates with significantly higher sensitivity than CpG free primers.

  2. 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.

  3. MGMT promoter methylation in gliomas-assessment by pyrosequencing and quantitative methylation-specific PCR

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    Håvik Annette

    2012-03-01

    Full Text Available Abstract Background Methylation of the O6-methylguanine-DNA methyltransferase (MGMT gene promoter is a favorable prognostic factor in glioblastoma patients. However, reported methylation frequencies vary significantly partly due to lack of consensus in the choice of analytical method. Method We examined 35 low- and 99 high-grade gliomas using quantitative methylation specific PCR (qMSP and pyrosequencing. Gene expression level of MGMT was analyzed by RT-PCR. Results When examined by qMSP, 26% of low-grade and 37% of high-grade gliomas were found to be methylated, whereas 97% of low-grade and 55% of high-grade gliomas were found methylated by pyrosequencing. The average MGMT gene expression level was significantly lower in the group of patients with a methylated promoter independent of method used for methylation detection. Primary glioblastoma patients with a methylated MGMT promoter (as evaluated by both methylation detection methods had approximately 5 months longer median survival compared to patients with an unmethylated promoter (log-rank test; pyrosequencing P = .02, qMSP P = .06. One third of the analyzed samples had conflicting methylation results when comparing the data from the qMSP and pyrosequencing. The overall survival analysis shows that these patients have an intermediate prognosis between the groups with concordant MGMT promoter methylation results when comparing the two methods. Conclusion In our opinion, MGMT promoter methylation analysis gives sufficient prognostic information to merit its inclusion in the standard management of patients with high-grade gliomas, and in this study pyrosequencing came across as the better analytical method.

  4. A novel methylation PCR that offers standardized determination of FMR1 methylation and CGG repeat length without southern blot analysis.

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    Grasso, Marina; Boon, Elles M J; Filipovic-Sadic, Stela; van Bunderen, Patrick A; Gennaro, Elena; Cao, Ru; Latham, Gary J; Hadd, Andrew G; Coviello, Domenico A

    2014-01-01

    Fragile X syndrome and associated disorders are characterized by the number of CGG repeats and methylation status of the FMR1 gene for which Southern blot (SB) historically has been required for analysis. This study describes a simple PCR-only workflow (mPCR) to replace SB analysis, that incorporates novel procedural controls, treatment of the DNA in separate control and methylation-sensitive restriction endonuclease reactions, amplification with labeled primers, and two-color amplicon sizing by capillary electrophoresis. mPCR was evaluated in two independent laboratories with 76 residual clinical samples that represented typical and challenging fragile X alleles in both males and females. mPCR enabled superior size resolution and analytical sensitivity for size and methylation mosaicism compared to SB. Full mutation mosaicism was detected down to 1% in a background of 99% normal allele with 50- to 100-fold less DNA than required for SB. A low level of full mutation mosaicism in one sample was detected using mPCR but not observed using SB. Overall, the sensitivity for detection of full mutation alleles was 100% (95% CI: 89%-100%) with an accuracy of 99% (95% CI: 93%-100%). mPCR analysis of DNA from individuals with Klinefelter and Turner syndromes, and DNA from sperm and blood, were consistent with SB. As such, mPCR enables accurate, sensitive, and standardized methods of FMR1 analysis that can harmonize results across different laboratories.

  5. A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

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    Claus, Rainer; Wilop, Stefan; Hielscher, Thomas; Sonnet, Miriam; Dahl, Edgar; Galm, Oliver; Jost, Edgar; Plass, Christoph

    2012-01-01

    Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R2 = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly. PMID:22647397

  6. 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.

  7. 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.

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    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

  8. A new approach to primer design for the control of PCR bias in methylation studies

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    Hansen Lise

    2008-07-01

    Full Text Available Abstract Primer design for PCR-based methylation analysis following bisulfite conversion of DNA is considerably more complex than primer design for regular PCR. The choice of the optimal primer set is critical to the performance and correct interpretation of the results. Most methodologies in methylation analysis utilize primers that theoretically amplify methylated and unmethylated templates at the same time. The proportional amplification of all templates is critical but difficult to achieve due to PCR bias favouring the amplification of the unmethylated template. The focus of this brief communication is to point out the important criteria needed for the successful choice of primers that will enable the control of PCR bias in bisulfite based methylation-screening protocols.

  9. Methylation-sensitive restriction enzyme nested real time PCR, a potential approach for sperm DNA identification.

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    Bai, Lijuan; Yan, Peng; Cao, Ximei; Jia, Linna; Zhang, Ce; Guo, Dawei

    2015-08-01

    Mammal H19 gene is an imprinting gene in which the paternal allele is silenced. On H19 imprinting control region (ICR), one of the mechanisms regulating the paternal allelic specific silence is DNA methylation in somatic cells throughout the individual's whole life. Nevertheless, this pattern of DNA methylation is erased and re-established in germline. As results, in mature sperm H19 ICR shows biallelic methylation instead of paternal specific methylation in somatic cells. Although the data were mainly from experiments on mice the same mechanisms are believed existing in human germline. We designed an experiment to probe the sperm DNA by methylation sensitive restriction enzyme based nested qPCR (MSRE-nested-qPCR). The genomic DNA digested/undigested by HhaI was amplified by outer primers encompassing four HhaI sites on H19 ICR. These PCR products were used as templates for second round real-time PCR to quantify the DNA methylation level. The results showed that DNA methylation level at H19 ICR were 55.27 ± 8.36% in 32 blood samples and 101.94 ± 11.66% in 31 semen samples. Based on our data sperm DNA could be identified if H19 ICR methylation level is over 78.62%.

  10. Trisomy 21 Alters DNA Methylation in Parent-of-Origin-Dependent and -Independent Manners.

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    Alves da Silva, Antônio Francisco; Machado, Filipe Brum; Pavarino, Érika Cristina; Biselli-Périco, Joice Matos; Zampieri, Bruna Lancia; da Silva Francisco Junior, Ronaldo; Mozer Rodrigues, Pedro Thyago; Terra Machado, Douglas; Santos-Rebouças, Cíntia Barros; Gomes Fernandes, Maria; Chuva de Sousa Lopes, Susana Marina; Lopes Rios, Álvaro Fabricio; Medina-Acosta, Enrique

    2016-01-01

    The supernumerary chromosome 21 in Down syndrome differentially affects the methylation statuses at CpG dinucleotide sites and creates genome-wide transcriptional dysregulation of parental alleles, ultimately causing diverse pathologies. At present, it is unknown whether those effects are dependent or independent of the parental origin of the nondisjoined chromosome 21. Linkage analysis is a standard method for the determination of the parental origin of this aneuploidy, although it is inadequate in cases with deficiency of samples from the progenitors. Here, we assessed the reliability of the epigenetic 5mCpG imprints resulting in the maternally (oocyte)-derived allele methylation at a differentially methylated region (DMR) of the candidate imprinted WRB gene for asserting the parental origin of chromosome 21. We developed a methylation-sensitive restriction enzyme-specific PCR assay, based on the WRB DMR, across single nucleotide polymorphisms (SNPs) to examine the methylation statuses in the parental alleles. In genomic DNA from blood cells of either disomic or trisomic subjects, the maternal alleles were consistently methylated, while the paternal alleles were unmethylated. However, the supernumerary chromosome 21 did alter the methylation patterns at the RUNX1 (chromosome 21) and TMEM131 (chromosome 2) CpG sites in a parent-of-origin-independent manner. To evaluate the 5mCpG imprints, we conducted a computational comparative epigenomic analysis of transcriptome RNA sequencing (RNA-Seq) and histone modification expression patterns. We found allele fractions consistent with the transcriptional biallelic expression of WRB and ten neighboring genes, despite the similarities in the confluence of both a 17-histone modification activation backbone module and a 5-histone modification repressive module between the WRB DMR and the DMRs of six imprinted genes. We concluded that the maternally inherited 5mCpG imprints at the WRB DMR are uncoupled from the parental allele

  11. Novel identification of biofluids using a multiplex methylation sensitive restriction enzyme-PCR system.

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    Lin, Yu-Chih; Tsai, Li-Chin; Lee, James Chun-I; Su, Chih-Wen; Tzen, Jason Tze-Cheng; Linacre, Adrian; Hsieh, Hsing-Mei

    2016-11-01

    The identification of a specific body fluid encountered in a forensic investigation can give crucial information. This identification can be aided by methylation profiles based on selected markers specific to a range of biofluids. In this study, the open database of Infinium HumanMethylation450 BeadChip was searched for markers specific for semen, vaginal fluids, saliva, venous blood and menstrual blood. A total of 8 biofluid-specific methylated markers and 2 control markers were combined into a 10-plex methylation sensitive restriction enzyme-PCR (MSRE-PCR) system. Based upon the analysis of 100 DNA samples from these 5 biofluid types, unambiguous results were obtained to identify the body fluid from which it originated. Validation studies of the developed 10-plex MSRE-PCR included sensitivity, reproducibility and mixed body fluids. Co-amplification of the established MSRE-PCR system and the microsatellite loci in AmpFlSTR(®) MiniFiler™ PCR Amplification Kit was performed to generate both the methylation profile for biofluid type and the miniSTR profile. This allowed human identification and the identification of the body fluid type to be performed in a single reaction. The results of this study displayed the applicability of this 10-plex MSRE-PCR system in forensic science.

  12. A novel three primers PCR (TP-PCR) method to obtain recombinant DNA molecule independent of restriction enzyme

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In this note, we report a novel and efficient three primers PCR (TP-PCR) method to rapidly generate recombinant DNA molecule at precise junction between two arbitrary DNA fragments. TP-PCR method is characterized by its reaction system with two templates and three primers, which can produce a recombinant DNA molecule in one PCR reaction. The main advantages of this method are the independence of sequences at the recombination site, the rapidness, and the easy establishment of adequate conditions. This method has been successfully applied to constructing a fusion protein gene, sck gene.

  13. 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.

  14. Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP.

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    Bian, Y S; Yan, P; Osterheld, M C; Fontolliet, C; Benhattar, J

    2001-01-01

    Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes. The combination of bisulfite modification and PCR results in the conversion of unmethylated cytosines to thymines, whereas methylated cytosines remain unchanged. This sequence conversion can lead to methylation-dependent alterations of single-strand conformation, which can be detected by SSCA. An analysis of mixtures of methylated and unmethylated DNA at known ratios revealed that the relative intensities of the corresponding bands following MS-SSCA were maintained. MS-SSCA was applied for methylation analysis of human p16 promoter region using genomic DNA obtained from either frozen, fixed, or microdissected fixed tissue sections. MS-SSCA is a rapid, specific, and semiquantitative approach that allows the detection of methylation of the p16 gene promoter. In reconstruction experiments, the method permits the detection of 10% or less of cells harboring a methylated p16 promoter. We have been successful in analyzing by MS-SSCA almost all (96%) tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results. In addition, when microdissection was performed, the clonality of this genetic alteration could be identified.

  15. Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR

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    Lázcoz Paula

    2008-02-01

    Full Text Available Abstract Background We present two melting curve analysis (MCA-based semiquantitative real time PCR techniques to detect the promoter methylation status of genes. The first, MCA-MSP, follows the same principle as standard MSP but it is performed in a real time thermalcycler with results being visualized in a melting curve. The second, MCA-Meth, uses a single pair of primers designed with no CpGs in its sequence. These primers amplify both unmethylated and methylated sequences. In clinical applications the MSP technique has revolutionized methylation detection by simplifying the analysis to a PCR-based protocol. MCA-analysis based techniques may be able to further improve and simplify methylation analyses by reducing starting DNA amounts, by introducing an all-in-one tube reaction and by eliminating a final gel stage for visualization of the result. The current study aimed at investigating the feasibility of both MCA-MSP and MCA-Meth in the analysis of promoter methylation, and at defining potential advantages and shortcomings in comparison to currently implemented techniques, i.e. bisulfite sequencing and standard MSP. Methods The promoters of the RASSF1A (3p21.3, BLU (3p21.3 and MGMT (10q26 genes were analyzed by MCA-MSP and MCA-Meth in 13 astrocytoma samples, 6 high grade glioma cell lines and 4 neuroblastoma cell lines. The data were compared with standard MSP and validated by bisulfite sequencing. Results Both, MCA-MSP and MCA-Meth, successfully determined promoter methylation. MCA-MSP provided information similar to standard MSP analyses. However the analysis was possible in a single tube and avoided the gel stage. MCA-Meth proved to be useful in samples with intermediate methylation status, reflected by a melting curve position shift in dependence on methylation extent. Conclusion We propose MCA-MSP and MCA-Meth as alternative or supplementary techniques to MSP or bisulfite sequencing.

  16. 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.

  17. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood.

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    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-10-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

  18. A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

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    Wiencke, John K; Bracci, Paige M; Hsuang, George; Zheng, Shichun; Hansen, Helen; Wrensch, Margaret R; Rice, Terri; Eliot, Melissa; Kelsey, Karl T

    2014-01-01

    Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells. PMID:25437051

  19. Detection of Serum DNA in Cancer Patients by Modified Methylation Specific PCR

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    LIYUongchang; SUNJunning; 等

    2002-01-01

    Ojbective To search for circulating DNA as a biomarker for the cancer burden of patients[1].Methods Methylation specific PCR(MSP),according to Herman et al,was used to identify this epigenetic modification of DNA from serum and tissue of 111 patients with different cancers.In parallel the mutant K-ras gene was analyzed in 55 cases for comparison.Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference Results Methylation of the p16 gene was found in serum or cancer tissue in about 50% of patients with a statistically significant difference to healthy controls(P<0.05).There was no difference between the DNA from the tumor and from the neighboring normal tissue or from sereum in early cancer.The positivity rate increased with progression of the tumor.20 days after the operation methylated DNA nearly disappeared.The positivity rate for mutant K-ras was only 30% and thus much lower than that of methlated DNA.The combination of the two markers increased the positivity rate of tumors by 13%.Conclusion The detection of methylation of the p16 gene by MSP combined with mutant K-ras is serum of cancer patients would be a very effective method to estimate the cancer burden.

  20. 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.

  1. Methylation of serum SST gene is an independent prognostic marker in colorectal cancer

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    Liu, Yanqun; Chew, Min Hoe; Tham, Chee Kian; Tang, Choong Leong; Ong, Simon YK; Zhao, Yi

    2016-01-01

    There is an increasing demand for accurate prognostication for colorectal cancer (CRC). This study sought to assess prognostic potentials of methylation targets in the serum of CRC patients. A total of 165 CRC patients were enrolled in this prospective study. Promoter methylation levels of seven genes in pre-operative sera and matched tumor tissues were evaluated by quantitative methylation-specific PCR. Kaplan-Meier test, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. After a median follow-up of 56 months, 43 patients (28.7%) experienced tumor recurrence. In univariate survival analyses, serum methylation levels of SST and MAL were significantly predictive of cancer-specific death (Pcancer death and recurrence, respectively). When focusing on stage II and III patients, prognostication with serum methylated SST remained significant. Methylated SST detected in all serum samples can be traced back to the matched primary tumor tissues. We believe that methylated SST detected in the pre-operative sera of CRC patients appear to be a novel promising prognostic marker and probably can be auxiliary to tumor staging system and serum carcinoembryonic antigen towards better risk stratification.

  2. Novel methylation specific real-time PCR test for the diagnosis of Klinefelter syndrome

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    Akanksha Mehta

    2014-10-01

    Full Text Available The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome (KS, based on the detection of supernumerary X-chromosomes (X-chs. DNA was extracted from peripheral blood samples of twenty-six 47,XXY males; two 46,XY/47,XXY males; twenty-two 46,XY males; and 15 females; and deaminated. Methylation-specific quantitative polymerase chain reaction (MS-qPCR was performed using primers for unmethylated and methylated copies of the X-ch inactive-specific transcript (XIST-U and XIST-M gene. X-ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and fluorescent in situ hybridization (FISH results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points (CPs by fit-point analysis and melting curve analysis. X-ch disomy was detected in all female controls and KS patients; male controls expressed XIST-M only. CPs ranged from 29.5 to 32.5 (standard deviation (s.d. 0.8 for XIST-U and from 29 to 31 (s.d. 0.6 for XIST-M. Limit of detection of mosaicism was 1%. Based on XIST-U/XIST-M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism (1.8% and 17.8% was comparable to FISH results (2.3% and 15%, respectively. Turnaround time from DNA deamination to final data analysis was under 9 h. We conclude that MS-qPCR is a sensitive, specific and rapid test for the detection of X-ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade 47,XXY/46,XY mosaicism.

  3. Long-term survival in glioblastoma: methyl guanine methyl transferase (MGMT) promoter methylation as independent favourable prognostic factor

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    Smrdel, Uros; Zwitter, Matjaz; Bostjancic, Emanuela; Zupan, Andrej; Kovac, Viljem; Glavac, Damjan; Bokal, Drago; Jerebic, Janja

    2016-01-01

    Abstract Background In spite of significant improvement after multi-modality treatment, prognosis of most patients with glioblastoma remains poor. Standard clinical prognostic factors (age, gender, extent of surgery and performance status) do not clearly predict long-term survival. The aim of this case-control study was to evaluate immuno-histochemical and genetic characteristics of the tumour as additional prognostic factors in glioblastoma. Patients and methods Long-term survivor group were 40 patients with glioblastoma with survival longer than 30 months. Control group were 40 patients with shorter survival and matched to the long-term survivor group according to the clinical prognostic factors. All patients underwent multimodality treatment with surgery, postoperative conformal radiotherapy and temozolomide during and after radiotherapy. Biopsy samples were tested for the methylation of MGMT promoter (with methylation specific polymerase chain reaction), IDH1 (with immunohistochemistry), IDH2, CDKN2A and CDKN2B (with multiplex ligation-dependent probe amplification), and 1p and 19q mutations (with fluorescent in situ hybridization). Results Methylation of MGMT promoter was found in 95% and in 36% in the long-term survivor and control groups, respectively (p < 0.001). IDH1 R132H mutated patients had a non-significant lower risk of dying from glioblastoma (p = 0.437), in comparison to patients without this mutation. Other mutations were rare, with no significant difference between the two groups. Conclusions Molecular and genetic testing offers additional prognostic and predictive information for patients with glioblastoma. The most important finding of our analysis is that in the absence of MGMT promoter methylation, longterm survival is very rare. For patients without this mutation, alternative treatments should be explored. PMID:27904447

  4. COLD-PCR amplification of bisulfite-converted DNA allows the enrichment and sequencing of rare un-methylated genomic regions.

    Science.gov (United States)

    Castellanos-Rizaldos, Elena; Milbury, Coren A; Karatza, Elli; Chen, Clark C; Makrigiorgos, G Mike; Merewood, Anne

    2014-01-01

    Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.

  5. Novel methylation speciifc real-time PCR test for the diagnosis of Klinefelter syndrome

    Institute of Scientific and Technical Information of China (English)

    Akanksha Mehta; Anna Mielnik; Peter N Schlegel; Darius A Paduch

    2014-01-01

    The aim of this study was to design a molecular assay for the diagnosis of Klinefelter syndrome(KS), based on the detection of supernumerary X‑chromosomes(X‑chs). DNA was extracted from peripheral blood samples of twenty‑six47,XXY males; two 46,XY/47,XXY males; twenty‑two46,XY males; and 15females; and deaminated. Methylation‑speciifc quantitative polymerase chain reaction(MS‑qPCR) was performed using primers for unmethylated and methylated copies of the X‑ch inactive‑speciifc transcript(XIST‑U and XIST‑M) gene. X‑ch disomy was determined on the basis of XIST methylation status. Degree of mosaicism in the 46,XY/47,XXY males was compared with karyotype and lfuorescentinsituhybridization(FISH) results. Data analysis was performed using the Roche® LightCycler software V. 3.5.3., including determination of crossing points(CPs) by ift‑point analysis and melting curve analysis. X‑ch disomy was detected in all female controls and KS patients; male controls expressed XIST‑M only. CPs ranged from 29.5 to 32.5(standard deviation(s.d.) 0.8) for XIST‑U and from 29 to 31(s.d. 0.6) for XIST‑M. Limit of detection of mosaicism was 1%. Based on XIST‑U/XIST‑M ratios for the two 47,XXY/46,XY patients, the calculated degree of mosaicism(1.8%and 17.8%) was comparable to FISH results(2.3% and 15%, respectively). Turnaround time from DNA deamination to ifnal data analysis was under 9h. We conclude that MS‑qPCR is a sensitive, speciifc and rapid test for the detection of X‑ch disomy, with applicability for the screening and diagnosis of KS, even in the setting of low grade47,XXY/46,XY mosaicism.

  6. Evaluating the methylation status of CXCR4 promoter: A cost-effective and sensitive two-step PCR method.

    Science.gov (United States)

    Bianchessi, Valentina; Lauri, Andrea; Vigorelli, Vera; Toia, Martina; Vinci, Maria Cristina

    2017-02-15

    The chemokine receptor CXCR4 plays a key role in the bone marrow microenvironment maintenance and in the hematopoietic stem and progenitor cells migration. In addition, CXCR4 is expressed in a broad spectrum of solid tumors where its methylation state has been recently proposed as a biomarker for cancer prognosis. To evaluate methylation status of CXCR4 promoter we developed a sensitive, accurate, specific and cost-effective two-step PCR method that does not require any specific equipment other than a conventional real-time PCR instrument. The principle of the technique relies on a novel normalization strategy which allows the detection and quantification of small methylation differences among pre-amplified DNA samples deriving from low amount of starting material. In addition, the analysis of melting curve profiles of PCR products provides additional information about the methylation status of CpG sites in between the primers. Finally, the principle of this technique can potentially be adapted for the investigation of the methylation status of any other DNA region.

  7. Identification of a DNA methylation-independent imprinting control region at the Arabidopsis MEDEA locus.

    Science.gov (United States)

    Wöhrmann, Heike J P; Gagliardini, Valeria; Raissig, Michael T; Wehrle, Wendelin; Arand, Julia; Schmidt, Anja; Tierling, Sascha; Page, Damian R; Schöb, Hanspeter; Walter, Jörn; Grossniklaus, Ueli

    2012-08-15

    Genomic imprinting is exclusive to mammals and seed plants and refers to parent-of-origin-dependent, differential transcription. As previously shown in mammals, studies in Arabidopsis have implicated DNA methylation as an important hallmark of imprinting. The current model suggests that maternally expressed imprinted genes, such as MEDEA (MEA), are activated by the DNA glycosylase DEMETER (DME), which removes DNA methylation established by the DNA methyltransferase MET1. We report the systematic functional dissection of the MEA cis-regulatory region, resulting in the identification of a 200-bp fragment that is necessary and sufficient to mediate MEA activation and imprinted expression, thus containing the imprinting control region (ICR). Notably, imprinted MEA expression mediated by this ICR is independent of DME and MET1, consistent with the lack of any significant DNA methylation in this region. This is the first example of an ICR without differential DNA methylation, suggesting that factors other than DME and MET1 are required for imprinting at the MEA locus.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Potent Methyl Oxidation of 5-Methyl-2′-deoxycytidine by Halogenated Quinoid Carcinogens and Hydrogen Peroxide via a Metal-independent Mechanism

    OpenAIRE

    Shao, Jie; Huang, Chun-Hua; Kalyanaraman, Balaraman; Zhu, Ben-Zhan

    2013-01-01

    Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical (•OH) can be produced by halogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2′-deoxycytidine (5mdC) to its methyl oxidation prod...

  10. DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells.

    Directory of Open Access Journals (Sweden)

    Myrna Candelaria

    Full Text Available BACKGROUND: Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.

  11. Development of a caseinase assay for PCR independent detection of esp gene carriage among enterococci

    Science.gov (United States)

    Dada, Ayokunle Christopher; Asmat, Ahmad; Lee, Yook Heng; Usup, Gires

    2013-11-01

    Currently, there is no known relationship between caseinase and carriage of esp gene. Also, no breakpoints exist for phenotypic assays that are used to infer virulence characteristics among Enterococci. In the present study, caseinase activity was measured by a radial diffusion assay for 113 enterococci isolates. A standard curve with predictive r2 value of 0.939 was produced by dispensing several doubling dilutions of proteinase K into 3% skimmed milk agar wells. Caseinase activity for all tested enterococci was subsequently converted into proteinase K activity, using the obtained chart. Caseinase activity ranged from 1.74 × 10-8 to 4.47 × 10-7ug/ml and 6.37 × 10-8 to 8.82 × 10-8 ug/ml per colony of environmental and clinical enterocococci tested, proportionate to proteinase K activity. Caseinase activity among environmental strains was five-fold higher than was observed among clinical strains. Fishers exact test revealed significant associations between esp gene carriage and caseinase activity (diameter on skimmed milk, z=8 to 13mm) at p<0.1. However, the probability of association was strongest at z=13 mm (p=0.033) suggesting a range of diameter cut-offs that was exclusive to and may be used to predict the presence of environmental enterococci strains harbouring esp gene. Results obtained from sensitivity analysis showed increasing assay sensitivity from cut-off of 9 mm (61.54%) up to 84.62% (13 mm). Specificity of the caseinase assay slightly decreased from 50% to 42.86% as cut-off increased from 9 to 13 mm. The caseinase assay described here potentially proves useful in preliminary PCR independent screening of environmental enterococci isolates for the detection of strains which carry the esp gene known to increase the severity of enterococcal infections.

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

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje;

    2010-01-01

    , for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant....... The method allowed accurate quantification from 4.4×102 to at least 2.2×107 CFU Salmonella per swab sample using qPCR (without preceding DNA extraction) or selective plating on xylose lysine deoxycholate agar. Samples with 50 CFU could be detected occasionally but fell outside the linear range...... of the standard curve. The swab samples showed a broad biological diversity; for seven samples not inoculated with Salmonella, the microbial background flora (BGF) was determined to 5.0 ± 2.2 log CFU/ml sample withdrawn after flotation. It was determined that the proceeding PCR step was inhibited by BGF...

  13. Seed response to strigolactone is controlled by abscisic acid-independent DNA methylation in the obligate root parasitic plant, Phelipanche ramosa L. Pomel.

    Science.gov (United States)

    Lechat, Marc-Marie; Brun, Guillaume; Montiel, Grégory; Véronési, Christophe; Simier, Philippe; Thoiron, Séverine; Pouvreau, Jean-Bernard; Delavault, Philippe

    2015-06-01

    Seed dormancy release of the obligate root parasitic plant, Phelipanche ramosa, requires a minimum 4-day conditioning period followed by stimulation by host-derived germination stimulants, such as strigolactones. Germination is then mediated by germination stimulant-dependent activation of PrCYP707A1, an abscisic acid catabolic gene. The molecular mechanisms occurring during the conditioning period that silence PrCYP707A1 expression and regulate germination stimulant response are almost unknown. Here, global DNA methylation quantification associated with pharmacological approaches and cytosine methylation analysis of the PrCYP707A1 promoter were used to investigate the modulation and possible role of DNA methylation during the conditioning period and in the PrCYP707A1 response to GR24, a synthetic strigolactone analogue. Active global DNA demethylation occurs during the conditioning period and is required for PrCYP707A1 activation by GR24 and for subsequent seed germination. Treatment with 5-azacytidine, a DNA-hypomethylating molecule, reduces the length of the conditioning period. Conversely, hydroxyurea, a hypermethylating agent, inhibits PrCYP707A1 expression and seed germination. Methylated DNA immunoprecipitation followed by PCR experiments and bisulfite sequencing revealed that DNA demethylation particularly impacts a 78-nucleotide sequence in the PrCYP707A1 promoter. The results here demonstrate that the DNA methylation status during the conditioning period plays a crucial role independently of abscisic acid in the regulation of P. ramosa seed germination by controlling the strigolactone-dependent expression of PrCYP707A1.

  14. 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......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... concentrations of ≥ 6.1 × 108 CFU/swab sample, but not by concentrations ≤ 6.1 × 106 CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture independent...

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

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje

    2010-01-01

    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......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... concentrations of ≥ 6.1×108 CFU/swab sample, but not by concentrations ≤ 6.1×106 CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture independent quantification...

  16. 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......, for culture-independent quantification of intact Salmonella in pig carcass gauze swabs (100 cm2) prior to quantitative PCR (qPCR). A novel approach was investigated, excluding the homogenization step prior to flotation, to improve the detection limit and speed up the quantification procedure. The buoyant...... concentrations of ≥ 6.1 × 108 CFU/swab sample, but not by concentrations ≤ 6.1 × 106 CFU/swab sample. By using the gauze swabs directly in the flotation procedure, the homogenization step normally used for preparation of food-related samples could be excluded, which simplified the culture independent...

  17. Potent methyl oxidation of 5-methyl-2'-deoxycytidine by halogenated quinoid carcinogens and hydrogen peroxide via a metal-independent mechanism.

    Science.gov (United States)

    Shao, Jie; Huang, Chun-Hua; Kalyanaraman, Balaraman; Zhu, Ben-Zhan

    2013-07-01

    Halogenated quinones are a class of carcinogenic intermediates and are newly identified chlorination disinfection by-products in drinking water. We found recently that the highly reactive and biologically important hydroxyl radical ((•)OH) can be produced by halogenated quinones and H2O2 independent of transition metal ions. However, it is not clear whether these quinoid carcinogens and H2O2 can oxidize the nucleoside 5-methyl-2'-deoxycytidine (5mdC) to its methyl oxidation products and, if so, what the underlying molecular mechanism is. Here we show that three methyl oxidation products, 5-(hydroperoxymethyl)-, 5-(hydroxymethyl)-, and 5-formyl-2'-deoxycytidine, could be produced when 5mdC was treated with tetrachloro-1,4-benzoquinone (TCBQ) and H2O2. The formation of the oxidation products was markedly inhibited by typical (•)OH scavengers and under anaerobic conditions. Analogous effects were observed with other halogenated quinones and the classic Fenton system. Based on these data, we propose that the oxidation of 5mdC by TCBQ/H2O2 might be through the following mechanism: (•)OH produced by TCBQ/H2O2 may first abstract hydrogen from the methyl group of 5mdC, leading to the formation of 5-(2'-deoxycytidylyl)methyl radical, which may combine with O2 to form the peroxyl radical. The unstable peroxyl radical transforms into the corresponding hydroperoxide 5-(hydroperoxymethyl)-2'-deoxycytidine, which reacts with TCBQ and results in the formation of 5-(hydroxymethyl)-2'-deoxycytidine and 5-formyl-2'-deoxycytidine. This is the first report that halogenated quinoid carcinogens and H2O2 can induce potent methyl oxidation of 5mdC via a metal-independent mechanism, which may partly explain their potential carcinogenicity. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Development and validation of a multiplex methylation specific PCR-coupled liquid bead array for liquid biopsy analysis.

    Science.gov (United States)

    Parisi, C; Mastoraki, S; Markou, A; Strati, A; Chimonidou, M; Georgoulias, V; Lianidou, E S

    2016-10-01

    Liquid biopsy is based on minimally invasive blood tests and has the potential to characterize the evolution of a solid tumor in real time, by extracting molecular information from circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Epigenetic silencing of tumor and metastasis suppressor genes plays a key role in survival and metastatic potential of cancer cells. Our group was the first to show the presence of epigenetic alterations in CTCs. We present the development and analytical validation of a highly specific and sensitive Multiplex Methylation Specific PCR-coupled liquid bead array (MMSPA) for the simultaneous detection of the methylation status of three tumor and metastasis suppressor genes (CST6, SOX17 and BRMS1) in liquid biopsy material (CTCs, corresponding ctDNA) and paired primary breast tumors. In the EpCAM-positive CTCs fraction we observed methylation of: a) CST6, in 11/30(37%) and 11/30(37%), b) BRMS1 in 8/30(27%) and 11/30(37%) c) SOX17 in 8/30(27%) and 13/30(43%) early breast cancer patients and patients with verified metastasis respectively. In ctDNA we observed methylation of: a) CST6, in 5/30(17%) and 10/31(32%), b) BRMS1 in 8/30 (27%) and 8/31 (26%) c) SOX17 in 5/30(17%) and 13/31(42%) early breast cancer patients and patients with verified metastasis respectively. Our results indicate a high cancerous load at the epigenetic level in EpCAM-positive CTCs fractions and corresponding ctDNA in breast cancer. The main principle of the developed methodology has the potential to be extended in a large number of gene-targets and be applied in many types of cancer. Copyright © 2016. Published by Elsevier B.V.

  19. 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.

  20. Accurate and easy-to-use assessment of contiguous DNA methylation sites based on proportion competitive quantitative-PCR and lateral flow nucleic acid biosensor.

    Science.gov (United States)

    Xu, Wentao; Cheng, Nan; Huang, Kunlun; Lin, Yuehe; Wang, Chenguang; Xu, Yuancong; Zhu, Longjiao; Du, Dan; Luo, Yunbo

    2016-06-15

    Many types of diagnostic technologies have been reported for DNA methylation, but they require a standard curve for quantification or only show moderate accuracy. Moreover, most technologies have difficulty providing information on the level of methylation at specific contiguous multi-sites, not to mention easy-to-use detection to eliminate labor-intensive procedures. We have addressed these limitations and report here a cascade strategy that combines proportion competitive quantitative PCR (PCQ-PCR) and lateral flow nucleic acid biosensor (LFNAB), resulting in accurate and easy-to-use assessment. The P16 gene with specific multi-methylated sites, a well-studied tumor suppressor gene, was used as the target DNA sequence model. First, PCQ-PCR provided amplification products with an accurate proportion of multi-methylated sites following the principle of proportionality, and double-labeled duplex DNA was synthesized. Then, a LFNAB strategy was further employed for amplified signal detection via immune affinity recognition, and the exact level of site-specific methylation could be determined by the relative intensity of the test line and internal reference line. This combination resulted in all recoveries being greater than 94%, which are pretty satisfactory recoveries in DNA methylation assessment. Moreover, the developed cascades show significantly high usability as a simple, sensitive, and low-cost tool. Therefore, as a universal platform for sensing systems for the detection of contiguous multi-sites of DNA methylation without external standards and expensive instrumentation, this PCQ-PCR-LFNAB cascade method shows great promise for the point-of-care diagnosis of cancer risk and therapeutics.

  1. 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.

  2. 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.; Falcomata, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; Braud, F. De; Ruda, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; Hamer, P. de Witt; Pietrantonio, F.; Siena, S. Di; Esteller, M.; Sartore-Bianchi, A.; Nicolantonio, F. Di

    2015-01-01

    BACKGROUND: O(6)-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 a

  3. 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.; Falcomata, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; Braud, F. De; Ruda, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; Hamer, P. de Witt; Pietrantonio, F.; Siena, S. Di; Esteller, M.; Sartore-Bianchi, A.; Nicolantonio, F. Di

    2015-01-01

    BACKGROUND: O(6)-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 a

  4. 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.; Falcomata, C.; Fiano, V.; Cassingena, A.; Siravegna, G.; Milione, M.; Cassoni, P.; Braud, F. De; Ruda, R.; Soffietti, R.; Venesio, T.; Bardelli, A.; Wesseling, P.; Hamer, P. de Witt; Pietrantonio, F.; Siena, S. Di; Esteller, M.; Sartore-Bianchi, A.; Nicolantonio, F. Di

    2015-01-01

    BACKGROUND: O(6)-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

  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, 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

  6. Rep-PCR--a variant to RAPD or an independent technique of bacteria genotyping? A comparison of the typing properties of rep-PCR with other recognised methods of genotyping of microorganisms.

    Science.gov (United States)

    Baldy-Chudzik, K

    2001-01-01

    The paper presents technical aspects of rep-PCR fingerprinting technique and compares its typing abilities, differentiation power and reproducibility with other recognised and recommended genotyping methods. Although rep-PCR fingerprinting is similar to MAAP techniques, it demonstrates some essentially different elements. The data presented in this review, indicate a rep-PCR genomic fingerprinting technique as a highly discriminating, independent screening method for determining the taxonomical diversity of bacterial population.

  7. Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing.

    Directory of Open Access Journals (Sweden)

    Hitomi Miyazaki

    2013-11-01

    Full Text Available Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII, thereby manifesting intermediate steps for the establishment of transcriptional activation. Here, using genetics and DRB, we show that mammalian Absent, small, or homeotic discs 1-like (Ash1l, a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, we found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. We also found that Brg1 is another key player involved in the response. Our results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing.

  8. HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster.

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    Margarida L A Figueiredo

    Full Text Available Heterochromatin protein 1 (HP1 proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me, are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs are associated with the methylation of H3K9: Su(var3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2 and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.

  9. A Pol V-mediated silencing, independent of RNA-directed DNA methylation, applies to 5S rDNA.

    Directory of Open Access Journals (Sweden)

    Julien Douet

    2009-10-01

    Full Text Available The plant-specific RNA polymerases Pol IV and Pol V are essential to RNA-directed DNA methylation (RdDM, which also requires activities from RDR2 (RNA-Dependent RNA Polymerase 2, DCL3 (Dicer-Like 3, AGO4 (Argonaute, and DRM2 (Domains Rearranged Methyltransferase 2. RdDM is dedicated to the methylation of target sequences which include transposable elements, regulatory regions of several protein-coding genes, and 5S rRNA-encoding DNA (rDNA arrays. In this paper, we have studied the expression of the 5S-210 transcript, a marker of silencing release at 5S RNA genes, to show a differential impact of RNA polymerases IV and V on 5S rDNA arrays during early development of the plant. Using a combination of molecular and cytological assays, we show that Pol IV, RDR2, DRM2, and Pol V, actors of the RdDM, are required to maintain a transcriptional silencing of 5S RNA genes at chromosomes 4 and 5. Moreover, we have shown a derepression associated to chromatin decondensation specific to the 5S array from chromosome 4 and restricted to the Pol V-loss of function. In conclusion, our results highlight a new role for Pol V on 5S rDNA, which is RdDM-independent and comes specifically at chromosome 4, in addition to the RdDM pathway.

  10. Cholesterol-independent effects of methyl-β-cyclodextrin on chemical synapses.

    Directory of Open Access Journals (Sweden)

    Kiel G Ormerod

    Full Text Available The cholesterol chelating agent, methyl-β-cyclodextrin (MβCD, alters synaptic function in many systems. At crayfish neuromuscular junctions, MβCD is reported to reduce excitatory junctional potentials (EJPs by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MβCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MβCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05 in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05. MβCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05. MβCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05. This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MβCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MβCD can affect both presynaptic and postsynaptic properties, and that some effects of MβCD are unrelated to cholesterol chelation.

  11. A/T Run Geometry of B-form DNA Is Independent of Bound Methyl-CpG Binding Domain, Cytosine Methylation and Flanking Sequence.

    Science.gov (United States)

    Chia, Jyh Yea; Tan, Wen Siang; Ng, Chyan Leong; Hu, Nien-Jen; Foo, Hooi Ling; Ho, Kok Lian

    2016-08-09

    DNA methylation in a CpG context can be recognised by methyl-CpG binding protein 2 (MeCP2) via its methyl-CpG binding domain (MBD). An A/T run next to a methyl-CpG maximises the binding of MeCP2 to the methylated DNA. The A/T run characteristics are reported here with an X-ray structure of MBD A140V in complex with methylated DNA. The A/T run geometry was found to be strongly stabilised by a string of conserved water molecules regardless of its flanking nucleotide sequences, DNA methylation and bound MBD. New water molecules were found to stabilise the Rett syndrome-related E137, whose carboxylate group is salt bridged to R133. A structural comparison showed no difference between the wild type and MBD A140V. However, differential scanning calorimetry showed that the melting temperature of A140V constructs in complex with methylated DNA was reduced by ~7 °C, although circular dichroism showed no changes in the secondary structure content for A140V. A band shift analysis demonstrated that the larger fragment of MeCP2 (A140V) containing the transcriptional repression domain (TRD) destabilises the DNA binding. These results suggest that the solution structure of MBD A140V may differ from the wild-type MBD although no changes in the biochemical properties of X-ray A140V were observed.

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

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    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.

  13. A convenient method to generate methylated and un-methylated control DNA in methylation studies

    Directory of Open Access Journals (Sweden)

    Mehdi Manoochehri

    2013-09-01

    Full Text Available Methylated and un-methylated control DNA is an important part of DNA methylation studies. Although human and mouse DNA methylation control sets are commercially available, in case of methylation studies on other species such as animals, plants, and bacteria, control sets need to be prepared. In this paper a simple method of generating methylated and un-methylated control DNA is described. Whole genome amplification and enzymatic methylation were performed to generate un-methylated and methylated DNA. The generated DNA were confirmed using methylation sensitive/dependant enzymes, and methylation specific PCR. Control reaction assays confirmed the generated methylated and un-methylated DNA.

  14. The SET-domain protein SUVR5 mediates H3K9me2 deposition and silencing at stimulus response genes in a DNA methylation-independent manner.

    Directory of Open Access Journals (Sweden)

    Elena Caro

    Full Text Available In eukaryotic cells, environmental and developmental signals alter chromatin structure and modulate gene expression. Heterochromatin constitutes the transcriptionally inactive state of the genome and in plants and mammals is generally characterized by DNA methylation and histone modifications such as histone H3 lysine 9 (H3K9 methylation. In Arabidopsis thaliana, DNA methylation and H3K9 methylation are usually colocated and set up a mutually self-reinforcing and stable state. Here, in contrast, we found that SUVR5, a plant Su(var3-9 homolog with a SET histone methyltransferase domain, mediates H3K9me2 deposition and regulates gene expression in a DNA methylation-independent manner. SUVR5 binds DNA through its zinc fingers and represses the expression of a subset of stimulus response genes. This represents a novel mechanism for plants to regulate their chromatin and transcriptional state, which may allow for the adaptability and modulation necessary to rapidly respond to extracellular cues.

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

    DEFF Research Database (Denmark)

    Löfström, Charlotta; Schelin, Jenny; Norling, Börje

    2010-01-01

    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, for...

  16. Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes.

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    Frank Thieme

    Full Text Available We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning, for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.

  17. Methylation-independent repression of Dnmt3b contributes to oncogenic activity of Dnmt3a in mouse MYC-induced T-cell lymphomagenesis.

    Science.gov (United States)

    Haney, S L; Hlady, R A; Opavska, J; Klinkebiel, D; Pirruccello, S J; Dutta, S; Datta, K; Simpson, M A; Wu, L; Opavsky, R

    2015-10-01

    DNA methyltransferase 3A (DNMT3A) catalyzes cytosine methylation of mammalian genomic DNA. In addition to myeloid malignancies, mutations in DNMT3A have been recently reported in T-cell lymphoma and leukemia, implying a possible involvement in the pathogenesis of human diseases. However, the role of Dnmt3a in T-cell transformation in vivo is poorly understood. Here we analyzed the functional consequences of Dnmt3a inactivation in a mouse model of MYC-induced T-cell lymphomagenesis (MTCL). Loss of Dnmt3a delayed tumorigenesis by suppressing cellular proliferation during disease progression. Gene expression profiling and pathway analysis identified upregulation of 17 putative tumor suppressor genes, including DNA methyltransferase Dnmt3b, in Dnmt3a-deficient lymphomas as molecular events potentially responsible for the delayed lymphomagenesis in Dnmt3a(Δ/Δ) mice. Interestingly, promoter and gene body methylation of these genes was not substantially changed between control and Dnmt3a-deficient lymphomas, suggesting that Dnmt3a may inhibit their expression in a methylation-independent manner. Re-expression of both wild type and catalytically inactive Dnmt3a in Dnmt3a(Δ/Δ) lymphoma cells in vitro inhibited Dnmt3b expression, indicating that Dnmt3b upregulation may be directly repressed by Dnmt3a. Importantly, genetic inactivation of Dnmt3b accelerated lymphomagenesis in Dnmt3a(Δ/Δ) mice, demonstrating that upregulation of Dnmt3b is a relevant molecular change in Dnmt3a-deficient lymphomas that inhibits disease progression. Collectively, our data demonstrate an unexpected oncogenic role for Dnmt3a in MTCL through methylation-independent repression of Dnmt3b and possibly other tumor suppressor genes.

  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

    2017-01-31

    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.Leukemia advance online publication, 31 January 2017; doi:10.1038/leu.2017.12.

  19. 甲基化特异性PCR检测Prader-Willi综合征%Detection of Prader-Willi syndrome by methylation-specific PCR

    Institute of Scientific and Technical Information of China (English)

    宋明浩; 李麓芸; 傅俊江; 李秀蓉; 卢光琇

    2000-01-01

    目的:探讨一种高效、快速检测Prader-Willi综合征(Prader-Willi syndrome,PWS)的方法.方法:采用甲基化特异性聚合酶链反应(methylation-specific PCR,MSPCR),其作用原理基于DNA经亚硫酸氢钠处理后,来自父方非甲基化序列的胞嘧啶转变为尿嘧啶,而来自母方甲基化序列则保持不变,随后用具有高度特异性的引物进行扩增.结果:PWS患者的DNA经亚硫酸氢钠处理后,扩增仅能得到来自母方甲基化序列174 bp的产物,与用PW71B印迹杂交甲基化分析结果一致,与临床诊断相符,确诊为PWS.结论:MSPCR可检测由目前已知3种机理导致的PWS,是一种有效的首选筛查方法.

  20. 应用甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR检测Prader-Willi综合征%Detecting Prader - Willi syndrome with methylation specific multiplex ligation -dependent probe amplification and methylation - specific PCR

    Institute of Scientific and Technical Information of China (English)

    欧展辉; 梁雄; 孙筱放

    2012-01-01

    目的 比较甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR两种方法检测Prader - Willi综合征.方法 应用细胞遗传学、甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR方法检测1个Prader - Willi综合征家系.结果 甲基化特异性多重连接依赖性探针扩增和甲基化特异性PCR方法均能对患者进行检测,为缺失型致病,而其父母未见异常.结论 甲基化特异性多重连接依赖性探针扩增方法检测Prader - Willi综合征比甲基化特异性PCR方法提供更多的致病信息.%Objective; Comparing methylation specific multiplex ligation -dependent probe amplification lo methylation - specific PCR methods of detecting Prader - Willi syndrome. Methods; Using cytogenelic analysis, methylation specific multiplex ligation-dependent probe amplification and methylaiion - specific PCR methods lo detect a Prader - Willi syndrome family. Results; methylation specific multiplex ligalion - dependent probe amplification and methylation - specific PCR methods could be used to detect Prader -Willi syndrome, and the patient was caused by deletion of relative gene in the 15q11 - q13 region, but his parents were normal. Conclusion ; Methylation specific multiplex ligation - dependent probe amplification was able to give more messages than methylation - specific PCR method.

  1. 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

    2017-01-01

    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. PMID:28139759

  2. Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids.

    Science.gov (United States)

    Koponen, S; Keinänen, R; Roivainen, R; Hirvonen, T; Närhi, M; Chan, P H; Koistinaho, J

    1999-01-01

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and

  3. Methylation Linear Discriminant Analysis (MLDA for identifying differentially methylated CpG islands

    Directory of Open Access Journals (Sweden)

    Vass J Keith

    2008-08-01

    Full Text Available Abstract Background Hypermethylation of promoter CpG islands is strongly correlated to transcriptional gene silencing and epigenetic maintenance of the silenced state. As well as its role in tumor development, CpG island methylation contributes to the acquisition of resistance to chemotherapy. Differential Methylation Hybridisation (DMH is one technique used for genome-wide DNA methylation analysis. The study of such microarray data sets should ideally account for the specific biological features of DNA methylation and the non-symmetrical distribution of the ratios of unmethylated and methylated sequences hybridised on the array. We have therefore developed a novel algorithm tailored to this type of data, Methylation Linear Discriminant Analysis (MLDA. Results MLDA was programmed in R (version 2.7.0 and the package is available at CRAN 1. This approach utilizes linear regression models of non-normalised hybridisation data to define methylation status. Log-transformed signal intensities of unmethylated controls on the microarray are used as a reference. The signal intensities of DNA samples digested with methylation sensitive restriction enzymes and mock digested are then transformed to the likelihood of a locus being methylated using this reference. We tested the ability of MLDA to identify loci differentially methylated as analysed by DMH between cisplatin sensitive and resistant ovarian cancer cell lines. MLDA identified 115 differentially methylated loci and 23 out of 26 of these loci have been independently validated by Methylation Specific PCR and/or bisulphite pyrosequencing. Conclusion MLDA has advantages for analyzing methylation data from CpG island microarrays, since there is a clear rational for the definition of methylation status, it uses DMH data without between-group normalisation and is less influenced by cross-hybridisation of loci. The MLDA algorithm successfully identified differentially methylated loci between two classes of

  4. MBD5 regulates iron metabolism via methylation-independent genomic targeting of Fth1 through KAT2A in mice.

    Science.gov (United States)

    Tao, Yunlong; Wu, Qian; Guo, Xin; Zhang, Zhuzhen; Shen, Yuanyuan; Wang, Fudi

    2014-07-01

    Ferritin plays important roles in iron metabolism and controls iron absorption in the intestine. The ferritin subunits ferritin heavy chain (Fth1) and ferritin light chain (Ftl1) are tightly regulated at both the transcriptional and post-transcriptional levels. However, mechanisms of maintaining stable, basal expression of Fth1 are poorly understood. Here, we show that global deletion of Mbd5 in mice induces an iron overload phenotype. Liver and serum iron levels in Mbd5(-/-) mice were 3·2-fold and 1·5-fold higher respectively, than wild-type littermates; moreover, serum ferritin was increased >5-fold in the Mbd5(-/-) mice. Mbd5 encodes a member of the methyl-CpG binding domain family; however, the precise function of this gene is poorly understood. Here, we found that intestinal Fth1 mRNA levels were decreased in Mbd5(-/-) mice. Loss of Fth1 expression in the intestine could lead to iron over-absorption. Furthermore, deleting Mbd5 specifically in the intestine resulted in a phenotype similar to that of conditional deletion of Fth1 mice. An Fth1 promoter-report luciferase assay indicated that overexpression of Mbd5 enhanced Fth1 transcription in a dose-dependent manner. Histone H4 acetylation of the Fth1 promoter was reduced in the intestine of Mbd5(-/-) mice and further analysis showed that histone acetyltransferase KAT2A was essential for MBD5-induced Fth1 transcription.

  5. Lysine Methylation of Progesterone Receptor at Activation Function 1 Regulates both Ligand-independent Activity and Ligand Sensitivity of the Receptor*

    Science.gov (United States)

    Chung, Hwa Hwa; Sze, Siu Kwan; Woo, Amanda Rui En; Sun, Yang; Sim, Kae Hwan; Dong, Xue Ming; Lin, Valerie C-L.

    2014-01-01

    Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity. PMID:24415758

  6. Lysine methylation of progesterone receptor at activation function 1 regulates both ligand-independent activity and ligand sensitivity of the receptor.

    Science.gov (United States)

    Chung, Hwa Hwa; Sze, Siu Kwan; Woo, Amanda Rui En; Sun, Yang; Sim, Kae Hwan; Dong, Xue Ming; Lin, Valerie C-L

    2014-02-28

    Progesterone receptor (PR) exists in two isoforms, PRA and PRB, and both contain activation functions AF-1 and AF-2. It is believed that AF-1 is primarily responsible for the ligand-independent activity, whereas AF-2 mediates ligand-dependent PR activation. Although more than a dozen post-translational modifications of PR have been reported, no post-translational modification on AF-1 or AF-2 has been reported. Using LC-MS/MS-based proteomic analysis, this study revealed AF-1 monomethylation at Lys-464. Mutational analysis revealed the remarkable importance of Lys-464 in regulating PR activity. Single point mutation K464Q or K464A led to ligand-independent PR gel upshift similar to the ligand-induced gel upshift. This upshift was associated with increases in both ligand-dependent and ligand-independent PR phosphorylation and PR activity due to the hyperactivation of AF-1. In contrast, mutation of Lys-464 to the bulkier phenylalanine to mimic the effect of methylation caused a drastic decrease in PR activity. Importantly, PR-K464Q also showed heightened ligand sensitivity, and this was associated with increases in its functional interaction with transcription co-regulators NCoR1 and SRC-1. These results suggest that monomethylation of PR at Lys-464 probably has a repressive effect on AF-1 activity and ligand sensitivity.

  7. RNA Polymerase V Functions in Arabidopsis Interphase Heterochromatin Organization Independently of the 24-nt siRNA-Directed DNA Methylation Pathway

    Institute of Scientific and Technical Information of China (English)

    Olga Pontes; Pedro Costa-Nunes; Paul Vithayathil; Craig S.Pikaard

    2009-01-01

    In Arabidopsis,pericentromeric repeats,retroelements,and silenced rRNA genes are assembled into heterochromatin within nuclear structures known as chromocenters.The mechanisms governing higher-order heterochromatin organization are poorly understood but 24-nt small interfering RNAs (siRNAs) are known to play key roles in heterochromatin formation.Nuclear RNA polymerase IV (Pol IV),RNA-DEPENDENT RNA POLYMERASE 2 (RDR2),and DICER-LIKE 3 (DCL3) are required for biogenesis of 24-nt siRNAs that associate with ARGONAUTE 4 (AGO4).Nuclear RNA polymerase V (Pol V) collaborates with DRD1 (DEFICIENT IN RNA-DEPENDENT DNA METHYLATION 1) to generate transcripts at heterochromatic loci that are hypothesized to bind to siRNA-AGO4 complexes and subsequently recruit the de-novo DNA methylation and/or histone modifying machinery.Here,we report that decondensation of the major pericentromeric repeats and depletion of the heterochromatic mark histone H3 lysine 9 dimethylation at chromocenters occurs specifically in pol V and drd1 mutants.Disruption of pericentromeric repeats condensation is coincident with transcriptional reactivation of specific classes of pericentromeric 180-bp repeats.We further demonstrate that Pol V functions independently of Pol IV,RDR2,and DCL3-mediated siRNA production to affect interphase heterochromatin organization,possibly by involving RNAs that recruit structural or chromatin-modifying proteins.

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

    Energy Technology Data Exchange (ETDEWEB)

    Liang, Ya-Chen; Hsu, Chiao-Yu [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China); Yao, Ya-Li [Department of Biotechnology, Asia University, Taichung 41354, Taiwan (China); Yang, Wen-Ming, E-mail: yangwm@nchu.edu.tw [Institute of Molecular Biology, National Chung Hsing University, Taichung 40227, Taiwan (China)

    2013-02-01

    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.

  9. 实时荧光定量PCR和甲基化特异性PCR检测结直肠癌APC、MLH1基因甲基化状态%Detection of APC and MLH1 Gene Hypermethylation with Real-time Fluorescent Quantitative PCR and Methylation-specific PCR in Colorectal Cancer

    Institute of Scientific and Technical Information of China (English)

    潘程宇; 曹俊; 季兴; 张丽莉; 郭慧敏; 邹晓平

    2011-01-01

    背景:启动子区甲基化致肿瘤抑制基因失活在结直肠癌的发生、发展中起重要作用,检测肿瘤相关基因的甲基化状态,可能为寻找新的结直肠癌诊断、预后相关标记物提供依据.目的:比较实时荧光定量PCR(FO-PCR)与甲基化特异性PCR(MSP)检测基因甲基化状态的差异.方法:以FQ-PCR检测66例结直肠癌组织和20例癌旁组织中与结直肠癌的发生、发展相关的抑癌基因APC和错配修复基因MLH1的甲基化状态,同时以MSP检测结直肠癌组织中APC基因的甲基化状态.结果:根据FQ-PCR结果,结直肠癌组织中APC、MLH1基因甲基化阳性率显著高于癌旁组织(48.5%和54.5%对0%和0%,P=0.000).FQ-PCR和MSP可检出的甲基化阳性对照DNA最低浓度分别为0.015 ng/μl和1.5 ng/μl,两者对APC基因甲基化状态的检测结果差异有统计学意义(P<0.05).结论:在DNA甲基化的检测手段中,FQ-PCR的敏感度优于MSP.%Background: Inactivation of tumor suppressor genes by promotor methylation plays an important role in the development and progression of colorectal cancer. Detection of hypermethylation of tumorigenesis-related genes might provide promising biomarkers for the diagnosis and prognosis of colorectal cancer. Aims: To compare the value of realtime fluorescent quantitative PCR (FQ-PCR) and methylation-specific PCR (MSP) for detecting the gene methylation.Methods: Hypermethylations of tumor suppressor gene APC and mismatch repair gene MLH1, which were related with the development and progression of colorectal cancer, were detected in 66 colorectal cancer specimens and 20 adjacent tissue specimens by FQ-PCR. Meanwhile, hypermethylation of APC in colorectal cancer specimens was also detected by MSP. Results: APC hypermethylation and MLH1 hypermethylation were detected in 32 and 36 cases of colorectal cancer tissues respectively by FQ-PCR, with the positivity rates significantly higher than those in adjacent tissues (48.5% and 54.5

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

    Science.gov (United States)

    Adewumi, Moses Olubusuyi; Coker, Bamidele Atinuke; Nudamajo, Felix Yasha; Adeniji, Johnson Adekunle

    2016-01-01

    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. PMID:27087810

  11. Culture-independent nested PCR method reveals high diversity of actinobacteria associated with the marine sponges Hymeniacidon perleve and Sponge sp.

    Science.gov (United States)

    Xin, Yanjuan; Huang, Jianyu; Deng, Maicun; Zhang, Wei

    2008-11-01

    A culture-independent nested polymerase chain reaction (PCR) technique was used to investigate the diversity of actinobacteria communities associated with the sponges Hymeniacidon perleve and Sponge sp. The phylogenetic affiliation of sponge-derived actinobacteria was then assessed by 16S rRNA sequencing of cloned DNA fragments. A total of 196 positive clones were screened by restriction fragment length polymorphism (RFLP) analysis; 48 unique operational taxonomic units (OTUs) were selected for sequencing. Rarefaction analysis indicated that the clone libraries represented 93% and 94% of the total estimated diversity for the two species, respectively. Phylogenetic analysis of sequence data revealed representatives of various phylogenetic divisions, which were related to the following ten actinobacterial genera: Acidimicrobium, Corynebacterium, Propionibacterium, Actinomyces, Micrococcus, Microbacterium, Streptomyces, Mycobacterium, Cellulosimicrobium, Sporichthya, and unidentified actinobacterial clones. A sponge-specific, previously uncultured actinobacteria community grouped within the subclass Acidimicrobidae was discovered from both H. perleve and Sponge sp. Sequences belonging to Acidimicrobium in the H. perleve and the Sponge sp. clone libraries represented 33% and 24% of the clones, respectively. In the Sponge sp. clone library Mycobacterium dominated, accounting for 70% of all clones. The presence of Acidimicrobium and mycobacteria within two sponges can lay the groundwork for attempts to culture these interesting bacteria for industrial applications.

  12. 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.

  13. Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

    Science.gov (United States)

    Decock, Anneleen; Ongenaert, Maté; Cannoodt, Robrecht; Verniers, Kimberly; De Wilde, Bram; Laureys, Geneviève; Van Roy, Nadine; Berbegall, Ana P.; Bienertova-Vasku, Julie; Bown, Nick; Clément, Nathalie; Combaret, Valérie; Haber, Michelle; Hoyoux, Claire; Murray, Jayne; Noguera, Rosa; Pierron, Gaelle; Schleiermacher, Gudrun; Schulte, Johannes H.; Stallings, Ray L.; Tweddle, Deborah A.; De Preter, Katleen; Speleman, Frank; Vandesompele, Jo

    2016-01-01

    Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylation-specific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and event-free survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature. PMID:26646589

  14. 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.

  15. Direct, real-time PCR (MethyLight) assay for methylation of O6-methylguanine-DNA methyltransferase promoter in glioma

    Institute of Scientific and Technical Information of China (English)

    CHEN Gong; WU Xing; YAO Yu; ZHOU Liang-fu; MAO Ying

    2009-01-01

    @@ O6-Methylguanine-DNA methyltransferase (MGMT) is a cellular DNA repair protein that rapidly reverses alkylation (e.g. methylation) at the O6 position of guanine,thereby neutralizing the cytotoxic effects of alkylating agent therapy such as temozolomide (TMZ) and carmustine.1It has been shown that epigenetic silencing of the MGMT gene by promoter methylation shuts down gene transcription2 and reflects a common alteration in primary human tumors leading to MGMT deficiency)Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival in several studies of glioma" patients treated with the alkylating agent. 56therapy4 and has been substantiated in two clinical trials.5.6

  16. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    Directory of Open Access Journals (Sweden)

    Miyuki Uno

    2011-01-01

    Full Text Available OBJECTIVES: 1 To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT promoter to its gene and protein expression levels in glioblastoma and 2 to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001. However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297. The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing, and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue.

  17. 甲基化特异性PCR方法诊断Prader-Willi综合征%Diagnosis of Prader-Willi syndrome by methylation-specific PCR

    Institute of Scientific and Technical Information of China (English)

    王薇; 吴晓燕; 宋红梅; 邱正庆; 魏珉

    2008-01-01

    目的 Prader-Willi综合征(PWS)是多系统异常的复杂临床综合征,仅根据临床症状很难诊断,国外已建立快速、高效、特异性、敏感性均佳的甲基化特异性PCR(MS-PCR)方法用于临床诊断,但我国还未有系统的对照研究.该研究目的 便是建立PWS的MS-PCR诊断方法,并对临床疑似患者进行筛查.方法 将44例受试者,分为正常对照组(16例)、临床确诊患者组(7例)及临床疑似患者组(21例).采用盐析法提取基因组DNA;应用CpGemoneTM Fast Modification试剂盒行亚硫酸盐修饰;以正常人为阴性对照,未修饰的基因组DNA为系统对照,以M(母源)、P(父源)两对引物同时对修饰产物行PCR;扩增产物以琼脂糖凝胶电泳分离.结果 ①16例正常对照的PCR结果同时显示M,P两条带,7例临床确诊的PWS患者均只显示一条M带;②经MS-PCR筛查的21例临床疑似患者,2例确诊为PWS,其他19例排除PWS.结论 该研究成功建立MS-PCR,并对疑似患者进行了筛查确诊.MS-PCR为特异高效的PWS确诊方法且方便易行.

  18. 乳腺癌MCF-7细胞Alu甲基化水平的MSRE-qPCR法检测%Detection of Alu methylation in MCF-7 breast cancer cells by MSRE-qPCR

    Institute of Scientific and Technical Information of China (English)

    徐酩; 吕京澴; 孙玉洁

    2010-01-01

    目的:寻找简单、可靠、经济并适合大批量检测肿瘤组织标本和肿瘤细胞系中Alu甲基化水平的实验方法.方法:用对甲基化敏感的限制性内切酶联合定量PCB(methylation-sensitive restriction endonuclease digestion and quantitative PCR,MSREqPCR)法检测乳腺癌细胞系MCF-7中Alu序列的甲基化水平,并与常用的亚硫酸氢盐修饰结合测序法(bisulfite-sequencing PCR,BSP)进行比较.结果:MSRE-qPCR显示Alu甲基化水平在BstU I酶切位点约为33.7%,在HpaⅡ酶切位点约为56.1%,与BSP法比较,两种方法都显示BstU I位点甲基化水平较低,而HpaⅡ位点甲基化水平较高,趋势一致,并提示MCF-7细胞的Alu甲基化水平明显低于正常细胞(84.6%或者更高).结论:MSRE-qPCR法简单、可靠、经济,适合用于大批量检测Alu甲基化水平.

  19. 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.

  20. 甲基化特异性多重连接探针扩增及甲基化特异性PCR诊断Prader-Willi综合征方法比较%Comparison between Methylation-specific Multiplex Ligation-dependent Probe Amplification and Methylation-specific PCR in Diagnosis of Prader-Willi Syndrome

    Institute of Scientific and Technical Information of China (English)

    王薇; 魏珉; 宋红梅; 邱正庆; 施惠萍; 赵时敏

    2014-01-01

    目的:应用甲基化特异性 PCRmethylation-specific PCR, MS-PCR )和甲基化特异性多重连接探针扩增(methylation-specific multiplex ligation-dependent probe amplification, MS-MLPA)方法对Prader-Willi综合征(Prader-Willi syn-drome, PWS)进行诊断并比较两种方法的差异。方法回顾性研究2005年10月至2014年2月到北京协和医院就诊的患儿及正常对照共102例,男57例,女45例。其中正常对照16例;荧光原位杂交或高分辨染色体确诊PWS阳性对照2例;临床疑似PWS患儿84例。提取受试者外周血基因组DNA分别应用MS-PCR及MS-MLPA方法进行基因分析,对疑似患儿进行确诊和遗传类型分型,并计算两种方法的特异性、敏感性及准确度,应用卡方检验对两种方法进行比较。结果 MS-PCR结果示正常对照及阳性对照与其表型全部相符,84例疑似患儿中有39例提示为PWS,45例提示正常。 MS-MLPA结果示除正常对照外的86例受试者中,29例提示为父源缺失型PWS;9例提示为母源二体型PWS;47例提示为正常;1例因DNA过于陈旧未检出有效结果。对比两种方法检测结果,有2例MS-PCR结果显示为PWS,但MS-MLPA结果显示为正常,通过增加DNA用量重新进行MS-PCR检测后,除外PWS。结合临床表现,受试者中明确诊断PWS的患儿39例,非PWS者63例。 MS-PCR 方法共出现2例假阳性,假阳性率为3.17%(2/63)。 MS-PCR 方法敏感性100%,特异性96.83%,准确度98.03%; MS-MLPA方法敏感性97.43%,特异性100%,准确度99.02%。两种方法的敏感性、特异性及准确度差异无统计学意义(P均>0.05)。结论 MS-PCR及MS-MLPA敏感性、特异性及准确度皆佳,均可用于PWS诊断, MS-MLPA可区分父源缺失型及母源二体型。 MS-PCR应保证DNA用量充足以避免假阳性出现。 MS-MLPA应使用新鲜提取的DNA,且实验条件要求更为严格。建议

  1. Detection of DNA methylation in eucaryotic cells.

    Directory of Open Access Journals (Sweden)

    Lech Chyczewski

    2008-01-01

    Full Text Available The methods of molecular biology allow for analyzing the methylation pattern in the whole genome and in particular genes. We differentiate methylated sequences from unmethylated ones by means of cutting the genomic template with methylation-sensitive restriction enzymes or by sodium bisulfite DNA modification. Chemical modification precedes most quantitative and qualitative PCR techniques: MS-PCR, MS-nested PCR, Real-Time PCR, QAMA, HeavyMethyl, MSHRM. Restriction enzymes, on the other hand, may be used together with PCR or hybridisation methods (Southern blot and microarrays. PCRs are conducted with primers specific for methylated and unmethylated sequences and sometimes, similarly to hybridisation techniques, with specifically labeled probes or dyes intercalating to double-stranded nucleic acids. The most advanced methylation detection techniques (MALDI-TOF MS and HPLC significantly reduce the amount of biological material used for tests, but they require specialist equipment.

  2. ID4基因甲基化定量PCR检测在急性白血病中的临床意义%Clinical Significance of ID4 Methylation Detection by Quantitative Methylation-Specific PCR in Acute Leukemia

    Institute of Scientific and Technical Information of China (English)

    刘洋; 钟文雯; 康慧媛; 王莉莉; 卢学春; 于力; 朱宏丽

    2014-01-01

    尽管治疗的进步极大地改善了急性白血病患者的预后和生存,但时至今日多数类型的急性白血病没有特异性的生物标记,因此对于多数白血病患者,影响生存的复发这一重要因素缺少有效的顸警机制.ID4基因启动子区甲基化广泛发生于各类型急性白血病.本研究在前期建立的甲基化定量PCR体系的基础上,用该方法检测患者骨髓样本,探讨ID4甲基化定量指标(percentage of methylated reference,PMR)的临床意义.采集我院门诊及住院确诊的初治、完全缓解、复发3个阶段的急性白血病患者骨髓样本及正常对照者骨髓样本.应用ID4甲基化定量PCR体系对样本进行检测.按初治、完全缓解、复发分组比较PMR值.比较相同病例不同疾病状态的PMR的动态变化.结果表明,初治组PMR最高,其次为复发组,而完全缓解组最低.初治组PMR与完全缓解组比较存在统计学差异.4例随访病例的PMR值波动与病情变化一致.在1例复发病例中,PMR升高早于骨髓细胞学检查确认复发1.7个月.结论:本研究通过ID4基因启动子区甲基定量检测的方法初步验证:甲基化水平的量化指标PMR值与急性白血病患者肿瘤细胞负荷关系密切.PMR动态监测波动与疾病变化一致,可能具有预测复发的作用,但ID4甲基化定量指标的临床价值还有待进一步研究证据的支持.

  3. Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

    Science.gov (United States)

    Cairns, M D; Preston, M D; Hall, C L; Gerding, D N; Hawkey, P M; Kato, H; Kim, H; Kuijper, E J; Lawley, T D; Pituch, H; Reid, S; Kullin, B; Riley, T V; Solomon, K; Tsai, P J; Weese, J S; Stabler, R A; Wren, B W

    2017-03-01

    The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents. Copyright © 2017 Cairns et al.

  4. 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.

  5. Quantification of PKC family genes in sporadic breast cancer by qRT-PCR: evidence that PKCι/λ overexpression is an independent prognostic factor.

    Science.gov (United States)

    Awadelkarim, Khalid Dafaallah; Callens, Céline; Rossé, Carine; Susini, Aurélie; Vacher, Sophie; Rouleau, Etienne; Lidereau, Rosette; Bièche, Ivan

    2012-12-15

    Drugs targeting protein kinase C (PKC) show promising therapeutic activity. However, little is known about the expression patterns of the 11 PKC genes in human tumors, and the clinical significance of most PKC genes is unknown. We used qRT-PCR assays to quantify mRNA levels of the 11 PKC genes in 458 breast tumors from patients with known clinical/pathological status and long-term outcome. The proportion of tumors in which the expression of the different genes was altered varied widely, from 9.6% for PKN2 to 40.2% for PKCι/λ. In breast tumors, overexpression was the main alteration observed for PKCι/λ (33.4%), PKCδ (29.5%) and PKCζ (9.6%), whereas underexpression was the main alteration observed for PKCα (27.3%), PKCε (11.6%), PKCη (8.7%) and PKN2 (8.1%). Both overexpression and underexpression were observed for PKCβ (underexpression 15.5%, overexpression 13.8%), PKCθ (underexpression 14.8%, overexpression 10.0%) and PKN1 (underexpression 6.6%, overexpression 7.4%). Several links were found between different PKC genes; and also between the expression patterns of PKC genes and several classical pathological and clinical parameters. PKCι/λ alone was found to have prognostic significance (p = 0.043), whereas PKCα showed a trend towards an influence on relapse-free survival (p = 0.052). PKCι/λ retained its prognostic significance in Cox multivariate regression analysis (p = 0.031). These results reveal very complex expression patterns of PKC genes in breast tumors, and suggest that their expression should be considered together when evaluating anti-tumoral drugs. PKCι/λ seems to be the most promising therapeutic target in breast cancer.

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

    Directory of Open Access Journals (Sweden)

    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

  7. Bacterial diversity in a finished compost and vermicompost: differences revealed by cultivation-independent analyses of PCR-amplified 16S rRNA genes.

    Science.gov (United States)

    Fracchia, Letizia; Dohrmann, Anja B; Martinotti, Maria Giovanna; Tebbe, Christoph C

    2006-08-01

    Bacterial communities are important catalysts in the production of composts. Here, it was analysed whether the diversity of bacteria in finished composts is stable and specific for the production process. Single-strand conformation polymorphism (SSCP) based on polymerase chain reaction amplified partial 16S rRNA genes was used to profile and analyse bacterial communities found in total DNA extracted from finished composts. Different batches of compost samples stored over a period of 12 years and a 1-year-old vermicompost were compared to each other. According to digital image analysis, clear differences could be detected between the profiles from compost and vermicompost. Differences between three different periods of compost storage and between replicate vermicompost windrows were only minor. A total of 41 different 16S rRNA genes were identified from the SSCP profiles by DNA sequencing, with the vast majority related to yet-uncultivated bacteria. Sequences retrieved from compost mainly belonged to the phyla Actinobacteria and Firmicutes. In contrast, vermicompost was dominated by bacteria related to uncultured Chloroflexi, Acidobacteria, Bacteroidetes and Gemmatimonadetes. The differences were underscored with specific gene probes and Southern blot hybridizations. The results confirmed that different substrates and composting processes selected for specific bacterial communities in the finished products. The specificity and consistency of the bacterial communities inhabiting the compost materials suggest that cultivation-independent bacterial community analysis is a potentially useful indicator to characterize the quality of finished composts in regard to production processes and effects of storage conditions.

  8. Prognostic Relevance of Tumor Purity and TERT Promoter Mutations on MGMT Promoter Methylation in Glioblastoma.

    Science.gov (United States)

    Schulze Heuling, Eva; Knab, Felix; Radke, Josefine; Eskilsson, Eskil; Martinez-Ledesma, Emmanuel; Koch, Arend; Czabanka, Marcus; Dieterich, Christoph; Verhaak, Roel G; Harms, Christoph; Euskirchen, Philipp

    2017-02-01

    Promoter methylation status of O-6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme, is a critical biomarker in glioblastoma multiforme (GBM) as treatment decisions and clinical trial inclusion rely on its accurate assessment. However, interpretation of results is complicated by poor inter-assay reproducibility as well as weak a correlation between methylation status and expression levels of MGMT. The present study systematically investigates the influence of tumor purity on tissue subjected to MGMT analysis. A quantitative, allele-specific real-time PCR (qAS-PCR) assay was developed to determine genotype and mutant allele frequency of telomerase promoter (pTERT) mutations as a direct measure of tumor purity. We studied tumor purity, pTERT mutation by Sanger sequencing, MGMT methylation by pyrosequencing, IDH1 mutation status, and clinical parameters in a cohort of high-grade gliomas (n=97). The qAS-PCR reliably predicted pTERT genotype and tumor purity compared with independent methods. Tumor purity positively and significantly correlated with the extent of methylation in MGMT methylated GBMs. Extent of MGMT methylation differed significantly with respect to pTERT mutation hotspot (C228T vs. C250T). Interestingly, frontal lobe tumors showed greater tumor purity than those in other locations. Above all, tumor purity was identified as an independent prognostic factor in GBM. In conclusion, we determined mutual associations of tumor purity with MGMT methylation and pTERT mutations and found that the extent of MGMT methylation reflects tumor purity. In turn, tumor purity is prognostic in IDH1 wildtype GBM.

  9. Ketamine-Induced Apoptosis in Normal Human Urothelial Cells: A Direct, N-Methyl-d-Aspartate Receptor-Independent Pathway Characterized by Mitochondrial Stress.

    Science.gov (United States)

    Baker, Simon C; Shabir, Saqib; Georgopoulos, Nikolaos T; Southgate, Jennifer

    2016-05-01

    Recreational abuse of ketamine has been associated with the emergence of a new bladder pain syndrome, ketamine-induced cystitis, characterized by chronic inflammation and urothelial ulceration. We investigated the direct effects of ketamine on normal human urothelium maintained in organ culture or as finite cell lines in vitro. Exposure of urothelium to ketamine resulted in apoptosis, with cytochrome c release from mitochondria and significant subsequent caspase 9 and 3/7 activation. The anesthetic mode-of-action for ketamine is mediated primarily through N-methyl d-aspartate receptor (NMDAR) antagonism; however, normal (nonimmortalized) human urothelial cells were unresponsive to NMDAR agonists or antagonists, and no expression of NMDAR transcript was detected. Exposure to noncytotoxic concentrations of ketamine (≤1 mmol/L) induced rapid release of ATP, which activated purinergic P2Y receptors and stimulated the inositol trisphosphate receptor to provoke transient release of calcium from the endoplasmic reticulum into the cytosol. Ketamine concentrations >1 mmol/L were cytotoxic and provoked a larger-amplitude increase in cytosolic Ca(2+) concentration that was unresolved. The sustained elevation in cytosolic Ca(2+) concentration was associated with pathological mitochondrial oxygen consumption and ATP deficiency. Damage to the urinary barrier initiates bladder pain and, in ketamine-induced cystitis, loss of urothelium from large areas of the bladder wall is a reported feature. This study offers first evidence for a mechanism of direct toxicity of ketamine to urothelial cells by activating the intrinsic apoptotic pathway.

  10. 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.

  11. DNA methylation profiling in different phases of temporomandibular joint osteoarthritis in rats.

    Science.gov (United States)

    Xiao, Jia-Ling; Meng, Juan-Hong; Gan, Ye-Hua; Li, Ya-Li; Zhou, Chun-Yan; Ma, Xu-Chen

    2016-08-01

    Temporomandibular joint osteoarthritis (TMJOA) is a complex disease with strong genetic and epigenetic components in its pathogenesis. The aim of this study was to evaluate DNA methylation in mandibular head cartilage in different phases of experimentally-induced TMJOA in rats. DNA methylation was evaluated using microarrays in the mandibular head cartilage of early, intermediate and late stage experimentally-induced TMJOA, and of the normal age-matched control groups. Genes with differentially methylated CpG sites were analyzed to reveal the over-represented gene ontologies and pathways at different stages, and were compared with published expression profiles to assess their overlappings. The DNA methylation patterns of the target genes were validated by methylated DNA immunoprecipitation qPCR in additional independent cartilage samples and mRNA levels were analyzed by real-time PCR. We observed 9489 differentially methylated regions between the TMJOA and controls. A total of 440 consistently altered genes were revealed in all three stages; most (80%) were hypomethylated and many were associated with cell cycle regulation. We also detected different DNA methylation changes in early and late stage TMJOA (Rearly=0.68, Rlate=0.47), while the differences between age-matched healthy cartilage were subtle. Strong inverse changes between methylation status and mRNA levels were confirmed in Adamts5, Chad, Cldn11 and Tnf. Our data reveals dynamic DNA methylation patterns during the progression of TMJOA, with a different host of genes and pathways. The changes of cartilage DNA methylation patterns might contribute to understand the etiologic mechanisms of TMJOA epigenetically. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. New primers for methylation-specific polymerase chain reaction enhance specificity of detecting STAT1 methylation

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    Ming-Cheng Chang

    2012-03-01

    Conclusion: Specific primers for methylation-specific PCR are mandatory for the accurate detection of STAT1 gene methylation. Besides, specific primers can generate correct interpretation of STAT1 gene methylation, and its correlation with the clinicopathological characteristics and outcome of cancer patients.

  13. Methylation and Immunoexpression of p16(INK4a) Tumor Suppressor Gene in Primary Breast Cancer Tissue and Their Quantitative p16(INK4a) Hypermethylation in Plasma by Real-Time PCR.

    Science.gov (United States)

    Lee, Jae Jun; Ko, Eunkyung; Cho, Junhun; Park, Ha Young; Lee, Jeong Eon; Nam, Seok Jin; Kim, Duk-Hwan; Cho, Eun Yoon

    2012-12-01

    The p16(INK4a) gene methylation has been reported to be a major tumorigenic mechanism. We evaluated the methylation status of the p16(INK4a) genes in 231 invasive breast cancer and 90 intraductal carcinoma specimens using a methylation-specific polymerase chain reaction and p16 protein expression using immunohistochemistry. The quantity of cell-free methylated p16(INK4a) DNA in the plasma samples of 200 patients with invasive breast cancer was also examined using a fluorescence-based real-time polymerase chain reaction assay. The frequencies of p16(INK4a) methylation in invasive and intraductal tumors were 52.8% (122/231) and 57.8% (52/90), respectively. The p16 protein was overexpressed in 145 of the 231 invasive carcinomas (62.8%) and 63 of the 90 intraductal carcinomas (70%). High p16 expression in invasive carcinomas correlated significantly with a high histologic grade, a negative estrogen receptor and progesterone receptor status, p53 immunoreactivity and high Ki-67 expression with immunohistochemistry. In addition, the methylation index of p16(INK4a) was significantly higher in the cancer patients than the normal controls (pp16 immunoreactivity correlated with a loss of differentiation in breast carcinomas and high frequency of p16(INK4a) promoter methylation in both invasive and intraductal carcinomas, suggesting it may be involved in the pathogenesis of breast cancer.

  14. Detection of the TWIST1 promoter methylation state through fluorescent quantitative PCR in urine sediments of bladder cancer%荧光定量PCR检测膀胱癌患者尿沉淀细胞TWIST1基因启动子甲基化状态的临床价值

    Institute of Scientific and Technical Information of China (English)

    陆明; 钱麟; 潘彬; 陈建刚; 赵枰

    2012-01-01

    目的:评估尿沉淀细胞TWIST1基因启动子甲基化状态在膀胱癌诊断中的价值.方法:用实时荧光定量PCR的方法,对50例临床确诊的膀胱癌患者、13例非肿瘤性尿路疾病患者、7例健康志愿者检测了尿沉淀细胞TWIST1基因启动子的甲基化状态;同时行尿脱落细胞学检查.结果:50例膀胱癌患者中尿脱落细胞TWIST1基因启动子甲基化阳性率为64.0%(32/50),对照组均未发现甲基化改变,两者比较差异有统计学意义(P<0.01).TWIST1基因启动子甲基化率有随组织分期升高的趋势,但在不同分级、分期膀胱癌中的差异无显著性.实时荧光定量PCR法检测尿液中TWIST1基因启动子甲基化的敏感性和特异性分别为64.0%、100.0%.而尿脱落细胞学检查阳性率为48.0%( 24/50),其敏感性和特异性分别为48.0%和100.0%.结论:尿脱落细胞TWIST1基因启动子的甲基化检测诊断膀胱癌敏感性和特异性均较高,且无创、无痛苦,可作为早期诊断膀胱癌的敏感指标.%Objective:To assess the TWIST1 promoter methylation state in urine sediments and evaluate their value in diagnosis of bladder cancer. Methods: Fifty patients with bladder cancer, 13 patients with non tumorous urinary disease and 7 healthy volunteers were recruited in the study. The TWIST1 promoter methylation state in urine sediments was determined by real-time fluorescent quantitative PCR. The urine exfoliative cytologic examination was also performed. Results: The positive rate of TWIST1 promoter methylation was 64.0% (32/50) in exfoliated urothelial cells of 50 patients with bladder cancer, no one showed positive TWIST1 promoter methylation in the control group. The difference in the positive rate of TWIST1 promoter methylation between two groups was significant(P < 0.01). TWIST1 promoter methylation status did not correlate with stage and grade of bladder cancer,though there was a trend that more frequent methylation was detected

  15. 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.

  16. Promoter methylation and expression of CDH1 and susceptibility and prognosis of eyelid squamous cell carcinoma.

    Science.gov (United States)

    Wang, Yong-Qiang; Yuan, Ye; Jiang, Shan; Jiang, Hua

    2016-07-01

    Eyelid skin tumors are the most frequent type of cancer in ophthalmology. And, eyelid squamous cell carcinoma (SCC) accounts for a large part of it. CDH1 encodes E-cadherin, a glycoprotein that plays an important part in cell-cell interaction. Loss of CDH1 function was suspected to be associated with tumorigenesis. Methylation of CDH1 promotors can alter the expression of its protein and is also considered as a contributor to various cancers. In this study, CDH1 methylation and expression profile as well as prognosis of 38 cases of eyelid SCC and the corresponding adjacent tissues were analyzed to clarify the role of CDH1 methylation in SCC carcinogenesis and prognosis. Methylation was detected by PCR, and CDH1 expression was evaluated by immunohistochemistry. We observed that CDH1 methylation is significantly correlated with decreased CDH1 protein expression in eyelid SCC patients. Patients with methylation and low expression of CDH1 are significantly associated with advanced and aggressive phenotypes. Therefore, CDH1 methylation and CDH1 expression are both independent prognostic factors for prognosis of eyelid SCC patients.

  17. DNA Methylation Signatures of the Plant Chromomethyltransferases

    OpenAIRE

    Gouil, Quentin; Baulcombe, David C

    2016-01-01

    DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G). By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and...

  18. PCR thermocycler

    Science.gov (United States)

    Benett, William J.; Richards, James B.

    2003-01-01

    A sleeve-type silicon polymerase chain reaction (PCR) chamber or thermocycler having improved thermal performance. The silicon sleeve reaction chamber is improved in thermal performance by etched features therein that reduce thermal mass and increase the surface area of the sleeve for cooling. This improved thermal performance of the thermocycler enables an increase in speed and efficiency of the reaction chamber. The improvement is accomplished by providing grooves in the faces of the sleeve and a series of grooves on the interior surfaces that connect with grooves on the faces of the sleeve. The grooves can be anisotropically etched in the silicon sleeve simultaneously with formation of the chamber.

  19. Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes.

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    Ratna Karan

    Full Text Available BACKGROUND: Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant's responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. METHODOLOGY/PRINCIPAL FINDINGS: Methylation sensitive amplification polymorphism (MSAP technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. CONCLUSIONS/SIGNIFICANCE: The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not "directed". The natural genetic variation for salt tolerance observed in rice germplasm may be

  20. Methylation-mediated transcriptional repression of microRNAs during cervical carcinogenesis

    Science.gov (United States)

    Wilting, Saskia M.; Verlaat, Wina; Jaspers, Annelieke; Makazaji, Nour A.; Agami, Reuven; Meijer, Chris J.L.M.; Snijders, Peter J.F.

    2013-01-01

    Deregulated expression of microRNAs (miRNAs) is common and biologically relevant in cervical carcinogenesis and appears only partly related to chromosomal changes. We recently identified 32 miRNAs showing decreased expression in high-grade cervical intraepithelial neoplasia (CIN) and carcinomas not associated with a chromosomal loss, 6 of which were located within a CpG island. This study aimed to investigate to what extent these miRNAs are subject to DNA methylation-mediated transcriptional repression in cervical carcinogenesis.   Methylation-specific PCR (MSP) analysis on a cell line panel representing different stages of human papillomavirus (HPV) induced transformation revealed an increase in methylation of hsa-miR-149, -203 and -375 with progression to malignancy, whereas expression of these miRNAs was restored upon treatment with a demethylating agent. All three miRNAs showed significantly increased levels of methylation in cervical carcinomas, whereas methylation levels of hsa-miR-203 and -375 were also significantly increased in high-grade CIN. A pilot analysis showed that increased hsa-miR-203 methylation was also detectable in HPV-positive cervical scrapes of women with high-grade CIN compared with controls. Similar to recent findings on hsa-miR-375, ectopic expression of hsa-miR-203 in cervical cancer cells decreased both the proliferation rate and anchorage independent growth. We found evidence for methylation-mediated transcriptional repression of hsa-miR-149, -203 and -375 in cervical cancer. Methylation of the latter two was already apparent in precancerous lesions and represent functionally relevant events in HPV-mediated transformation. Increased hsa-miR-203 methylation was detectable in scrapes of women with high-grade CIN, indicating that methylated miRNAs may provide putative markers to assess the presence of (pre)cancerous lesions. PMID:23324622

  1. Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

    Science.gov (United States)

    A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

  2. Development and evaluation of a culture-independent method for source determination of fecal wastes in surface and storm waters using reverse transcriptase-PCR detection of FRNA coliphage genogroup gene sequences.

    Science.gov (United States)

    A complete method, incorporating recently improved reverse transcriptase-PCR primer/probe assays and including controls for determining interferences to phage recoveries from water sample concentrates and for detecting interferences to their analysis, was developed for the direct...

  3. 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

    Directory of Open Access Journals (Sweden)

    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

  4. 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

  5. Conventional and nanotechniques for DNA methylation profiling.

    Science.gov (United States)

    Shanmuganathan, Rajasree; Basheer, Nazeema B; Amirthalingam, Laxmi; Muthukumar, Harshiny; Kaliaperumal, Rajendran; Shanmugam, Kumaran

    2013-01-01

    DNA methylation is critical for gene silencing and is associated with the incidence of many diseases, including cancer. Underlying molecular mechanisms of human diseases and tissue-specific gene expression have been elucidated based on DNA methylation studies. This review highlights the advantages and drawbacks of various methylation screening techniques: blotting, genomic sequencing, bisulfite sequencing, methylation-specific PCR, methylated DNA immunoprecipitation, microarray analysis, matrix-assisted laser desorption ionization time-of-flight mass spectroscopy, nanowire transistor detection procedure, quantum dot-based nanoassay, single-molecule real-time detection, fluorimetric assay, electrochemical detection, and atomic force spectroscopy. The review provides insight for selecting a method or a combination of methods for DNA methylation analysis. Convergence of conventional and contemporary nanotechniques to enumerate methylation at specific CpG sites of oncogene would fill the gap in diagnosis of cancer.

  6. Digital PCR: A brief history

    OpenAIRE

    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...

  7. DNA Methylation

    OpenAIRE

    Alokail, Majed S.; Alenad, Amal M.

    2015-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication e...

  8. MGMT promoter methylation is associated with temozolomide response and prolonged progression-free survival in disseminated cutaneous melanoma.

    Science.gov (United States)

    Tuominen, Rainer; Jewell, Rosalyn; van den Oord, Joost J; Wolter, Pascal; Stierner, Ulrika; Lindholm, Christer; Hertzman Johansson, Carolina; Lindén, Diana; Johansson, Hemming; Frostvik Stolt, Marianne; Walker, Christy; Snowden, Helen; Newton-Bishop, Julia; Hansson, Johan; Egyházi Brage, Suzanne

    2015-06-15

    To investigate the predictive and prognostic value of O(6) -methylguanine DNA methyltransferase (MGMT) inactivation by analyses of promoter methylation in pretreatment tumor biopsies from patients with cutaneous melanoma treated with dacarbazine (DTIC) or temozolomide (TMZ) were performed. The patient cohorts consisted of Belgian and Swedish disseminated melanoma patients. Patients were subdivided into those receiving single-agent treatment with DTIC/TMZ (cohort S, n = 74) and those treated with combination chemotherapy including DTIC/TMZ (cohort C, n = 79). Median follow-up was 248 and 336 days for cohort S and cohort C, respectively. MGMT promoter methylation was assessed by three methods. The methylation-related transcriptional silencing of MGMT mRNA expression was assessed by real-time RT-PCR. Response to chemotherapy and progression-free survival (PFS) and overall survival were correlated to MGMT promoter methylation status. MGMT promoter methylation was detected in tumor biopsies from 21.5 % of the patients. MGMT mRNA was found to be significantly lower in tumors positive for MGMT promoter methylation compared to tumors without methylation in both treatment cohorts (p MGMT promoter methylation in cohort S (p = 0.0005), but did not reach significance in cohort C (p = 0.16). Significantly longer PFS was observed among patients with MGMT promoter-methylated tumors (p = 0.002). Multivariate Cox regression analysis identified presence of MGMT promoter methylation as an independent variable associated with longer PFS. Together, this implies that MGMT promoter methylation is associated with response to single-agent DTIC/TMZ and longer PFS in disseminated cutaneous melanoma.

  9. DNA Methylation in Thyroid Tumorigenesis

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    Stephen, Josena K., E-mail: jstephe2@hfhs.org [Department of Otolaryngology/Head and Neck Surgery, Henry Ford Hospital, Detroit, MI 48202 (United States); Chitale, Dhananjay [Department of Pathology, Henry Ford Hospital, Detroit, MI 48202 (United States); Narra, Vinod [Essex Surgical Associates, PC, Beverly, MA 01915 (United States); Chen, Kang Mei; Sawhney, Raja; Worsham, Maria J. [Department of Otolaryngology/Head and Neck Surgery, Henry Ford Hospital, Detroit, MI 48202 (United States)

    2011-03-29

    Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type.

  10. DNA Methylation-Guided Prediction of Clinical Failure in High-Risk Prostate Cancer.

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    Kirill Litovkin

    Full Text Available Prostate cancer (PCa is a very heterogeneous disease with respect to clinical outcome. This study explored differential DNA methylation in a priori selected genes to diagnose PCa and predict clinical failure (CF in high-risk patients.A quantitative multiplex, methylation-specific PCR assay was developed to assess promoter methylation of the APC, CCND2, GSTP1, PTGS2 and RARB genes in formalin-fixed, paraffin-embedded tissue samples from 42 patients with benign prostatic hyperplasia and radical prostatectomy specimens of patients with high-risk PCa, encompassing training and validation cohorts of 147 and 71 patients, respectively. Log-rank tests, univariate and multivariate Cox models were used to investigate the prognostic value of the DNA methylation.Hypermethylation of APC, CCND2, GSTP1, PTGS2 and RARB was highly cancer-specific. However, only GSTP1 methylation was significantly associated with CF in both independent high-risk PCa cohorts. Importantly, trichotomization into low, moderate and high GSTP1 methylation level subgroups was highly predictive for CF. Patients with either a low or high GSTP1 methylation level, as compared to the moderate methylation groups, were at a higher risk for CF in both the training (Hazard ratio [HR], 3.65; 95% CI, 1.65 to 8.07 and validation sets (HR, 4.27; 95% CI, 1.03 to 17.72 as well as in the combined cohort (HR, 2.74; 95% CI, 1.42 to 5.27 in multivariate analysis.Classification of primary high-risk tumors into three subtypes based on DNA methylation can be combined with clinico-pathological parameters for a more informative risk-stratification of these PCa patients.

  11. 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.

  12. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

    Sommer Kristensen, Lasse; 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....

  13. 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...

  14. Reverse-transcription PCR (RT-PCR).

    Science.gov (United States)

    Bachman, Julia

    2013-01-01

    RT-PCR is commonly used to test for genetic diseases and to characterize gene expression in various tissue types, cell types, and over developmental time courses. This serves as a form of expression profiling, but typically as a candidate approach. RT-PCR is also commonly used to clone cDNAs for further use with other molecular biology techniques (e.g., see Oligo(dT)-primed RT-PCR isolation of polyadenylated RNA degradation intermediates and Circularized RT-PCR (cRT-PCR): analysis of RNA 5' ends, 3' ends, and poly(A) tails).

  15. DNA Methylation Profiling Reveals Correlation of Differential Methylation Patterns with Gene Expression in Human Epilepsy.

    Science.gov (United States)

    Wang, Liang; Fu, Xinwei; Peng, Xi; Xiao, Zheng; Li, Zhonggui; Chen, Guojun; Wang, Xuefeng

    2016-05-01

    DNA methylation plays important roles in regulating gene expression and has been reported to be related with epilepsy. This study aimed to define differential DNA methylation patterns in drug-refractory epilepsy patients and to investigate the role of DNA methylation in human epilepsy. We performed DNA methylation profiling in brain tissues from epileptic and control patients via methylated-cytosine DNA immunoprecipitation microarray chip. Differentially methylated loci were validated by bisulfite sequencing PCR, and the messenger RNA (mRNA) levels of candidate genes were evaluated by reverse transcriptase PCR. We found 224 genes that showed differential DNA methylation between epileptic patients and controls. Among the seven candidate genes, three genes (TUBB2B, ATPGD1, and HTR6) showed relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 exhibited hypermethylation and decreased mRNA levels, whereas HTR6 displayed hypomethylation and increased mRNA levels in the epileptic samples. Our findings suggest that certain genes become differentially regulated by DNA methylation in human epilepsy.

  16. Whole genome methylation profiling by immunoprecipitation of methylated DNA.

    Science.gov (United States)

    Sharp, Andrew J

    2012-01-01

    I provide a protocol for DNA methylation profiling based on immunoprecipitation of methylated DNA using commercially available monoclonal antibodies that specifically recognize 5-methylcytosine. Quantification of the level of enrichment of the resulting DNA enables DNA methylation to be assayed for any genomic locus, including entire chromosomes or genomes if appropriate microarray or high-throughput sequencing platforms are used. In previous studies (1, 2), I have used hybridization to oligonucleotide arrays from Roche Nimblegen Inc, which allow any genomic region of interest to be interrogated, dependent on the array design. For example, using modern tiling arrays comprising millions of oligonucleotide probes, several complete human chromosomes can be assayed at densities of one probe per 100 bp or greater, sufficient to yield high-quality data. However, other methods such as quantitative real-time PCR or high-throughput sequencing can be used, giving either measurement of methylation at a single locus or across the entire genome, respectively. While the data produced by single locus assays is relatively simple to analyze and interpret, global assays such as microarrays or high-throughput sequencing require more complex statistical approaches in order to effectively identify regions of differential methylation, and a brief outline of some approaches is given.

  17. Study of the bacterial diversity of foods: PCR-DGGE versus LH-PCR.

    Science.gov (United States)

    Garofalo, Cristiana; Bancalari, Elena; Milanović, Vesna; Cardinali, Federica; Osimani, Andrea; Sardaro, Maria Luisa Savo; Bottari, Benedetta; Bernini, Valentina; Aquilanti, Lucia; Clementi, Francesca; Neviani, Erasmo; Gatti, Monica

    2017-02-02

    The present study compared two culture-independent methods, polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and length-heterogeneity polymerase chain reaction (LH-PCR), for their ability to reveal food bacterial microbiota. Total microbial DNA and RNA were extracted directly from fourteen fermented and unfermented foods, and domain A of the variable regions V1 and V2 of the 16S rRNA gene was analyzed through LH-PCR and PCR-DGGE. Finally, the outline of these analyses was compared with bacterial viable counts obtained after bacterial growth on suitable selective media. For the majority of the samples, RNA-based PCR-DGGE revealed species that the DNA-based PCR-DGGE was not able to highlight. When analyzing either DNA or RNA, LH-PCR identified several lactic acid bacteria (LAB) and coagulase negative cocci (CCN) species that were not identified by PCR-DGGE. This phenomenon was particularly evident in food samples with viable loadsPCR was able to detect a higher number of peaks in the analyzed food matrices relative to species identified by PCR-DGGE. In light of these findings, it may be suggested that LH-PCR shows greater sensitivity than PCR-DGGE. However, PCR-DGGE detected some other species (LAB included) that were not detected by LH-PCR. Therefore, certain LH-PCR peaks not attributed to known species within the LH-PCR database could be solved by comparing them with species identified by PCR-DGGE. Overall, this study also showed that LH-PCR is a promising method for use in the food microbiology field, indicating the necessity to expand the LH-PCR database, which is based, up to now, mainly on LAB isolates from dairy products. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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    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.

  19. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population.

    Science.gov (United States)

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun

    2016-01-01

    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p smoking for more than 40 pack-years (OR = 4.21, p smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC.

  20. Sex Determination Using PCR

    Science.gov (United States)

    Kima, Peter E.; Rasche, Madeline E.

    2004-01-01

    PCR has revolutionized many aspects of biochemistry and molecular biology research. In the following exercise, students learn PCR by isolating their own DNA, amplifying specific segments of the X and Y chromosomes, and estimating the sizes of the PCR products using agarose gel electrophoresis. Based on the pattern of PCR products, students can…

  1. Methyl gallate

    Directory of Open Access Journals (Sweden)

    Silvina Pagola

    2009-02-01

    Full Text Available The crystal structure of the title compound (systematic name: methyl 3,4,5-trihydroxybenzoate, C8H8O5, is composed of essentially planar molecules [maximum departures from the mean carbon and oxygen skeleton plane of 0.0348 (10 Å]. The H atoms of the three hydroxyl groups, which function as hydrogen-bond donors and acceptors simultaneously, are oriented in the same direction around the aromatic ring. In addition to two intramolecular hydrogen bonds, each molecule is hydrogen bonded to six others, creating a three-dimensional hydrogen-bonded network.

  2. 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

  3. DNA methylation of the LIN28 pseudogene family.

    Science.gov (United States)

    Davis, Aaron P; Benninghoff, Abby D; Thomas, Aaron J; Sessions, Benjamin R; White, Kenneth L

    2015-04-11

    DNA methylation directs the epigenetic silencing of selected regions of DNA, including the regulation of pseudogenes, and is widespread throughout the genome. Pseudogenes are decayed copies of duplicated genes that have spread throughout the genome by transposition. Pseudogenes are transcriptionally silenced by DNA methylation, but little is known about how pseudogenes are targeted for methylation or how methylation levels are maintained in different tissues. We employed bisulfite next generation sequencing to examine the methylation status of the LIN28 gene and four processed pseudogenes derived from LIN28. The objective was to determine whether LIN28 pseudogenes maintain the same pattern of methylation as the parental gene or acquire a methylation pattern independent of the gene of origin. In this study, we determined that the methylation status of LIN28 pseudogenes does not resemble the pattern evident for the LIN28 gene, but rather these pseudogenes appear to acquire methylation patterns independent of the parental gene. Furthermore, we observed that methylation levels of the examined pseudogenes correlate to the location of insertion within the genome. LIN28 pseudogenes inserted into gene bodies were highly methylated in all tissues examined. In contrast, pseudogenes inserted into genomic regions that are not proximal to genes were differentially methylated in various tissue types. Our analysis suggests that Lin28 pseudogenes do not acquire patterns of tissue-specific methylation as for the parental gene, but rather are methylated in patterns specific to the local genomic environment into which they were inserted.

  4. Methylation of IGF2 regulatory regions to diagnose adrenocortical carcinomas.

    Science.gov (United States)

    Creemers, S G; van Koetsveld, P M; van Kemenade, F J; Papathomas, T G; Franssen, G J H; Dogan, F; Eekhoff, E M W; van der Valk, P; de Herder, W W; Janssen, J A M J L; Feelders, R A; Hofland, L J

    2016-09-01

    Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis. Discrimination of ACCs from adrenocortical adenomas (ACAs) is challenging on both imaging and histopathological grounds. High IGF2 expression is associated with malignancy, but shows large variability. In this study, we investigate whether specific methylation patterns of IGF2 regulatory regions could serve as a valuable biomarker in distinguishing ACCs from ACAs. Pyrosequencing was used to analyse methylation percentages in DMR0, DMR2, imprinting control region (ICR) (consisting of CTCF3 and CTCF6) and the H19 promoter. Expression of IGF2 and H19 mRNA was assessed by real-time quantitative PCR. Analyses were performed in 24 ACCs, 14 ACAs and 11 normal adrenals. Using receiver operating characteristic (ROC) analysis, we evaluated which regions showed the best predictive value for diagnosis of ACC and determined the diagnostic accuracy of these regions. In ACCs, the DMR0, CTCF3, CTCF6 and the H19 promoter were positively correlated with IGF2 mRNA expression (P<0.05). Methylation in the most discriminating regions distinguished ACCs from ACAs with a sensitivity of 96%, specificity of 100% and an area under the curve (AUC) of 0.997±0.005. Our findings were validated in an independent cohort of 9 ACCs and 13 ACAs, resulting in a sensitivity of 89% and a specificity of 92%. Thus, methylation patterns of IGF2 regulatory regions can discriminate ACCs from ACAs with high diagnostic accuracy. This proposed test may become the first objective diagnostic tool to assess malignancy in adrenal tumours and facilitate the choice of therapeutic strategies in this group of patients.

  5. Inter-individual methylation variability in differentially methylated regions between maternal whole blood and first trimester CVS

    OpenAIRE

    Ioannides, Marios; Papageorgiou, Elisavet A.; Keravnou, Anna; Tsaliki, Evdokia; Spyrou, Christiana; Hadjidaniel, Michael; Sismani, Carolina; Koumbaris, George; Patsalis, Philippos C.

    2014-01-01

    Background DNA methylation is the most studied form of epigenetic regulation, a process by which chromatin composition and transcription factor binding is altered to influence tissue specific gene expression and differentiation. Such tissue specific methylation patterns are investigated as biomarkers for cancer and cell-free fetal DNA using various methodologies. Results We have utilized methylation DNA immunoprecipitation (MeDIP) and real-time quantitative PCR to investigate the inter-indivi...

  6. Electrochemical biosensing strategies for DNA methylation analysis.

    Science.gov (United States)

    Hossain, Tanvir; Mahmudunnabi, Golam; Masud, Mostafa Kamal; Islam, Md Nazmul; Ooi, Lezanne; Konstantinov, Konstantin; Hossain, Md Shahriar Al; Martinac, Boris; Alici, Gursel; Nguyen, Nam-Trung; Shiddiky, Muhammad J A

    2017-02-17

    DNA methylation is one of the key epigenetic modifications of DNA that results from the enzymatic addition of a methyl group at the fifth carbon of the cytosine base. It plays a crucial role in cellular development, genomic stability and gene expression. Aberrant DNA methylation is responsible for the pathogenesis of many diseases including cancers. Over the past several decades, many methodologies have been developed to detect DNA methylation. These methodologies range from classical molecular biology and optical approaches, such as bisulfite sequencing, microarrays, quantitative real-time PCR, colorimetry, Raman spectroscopy to the more recent electrochemical approaches. Among these, electrochemical approaches offer sensitive, simple, specific, rapid, and cost-effective analysis of DNA methylation. Additionally, electrochemical methods are highly amenable to miniaturization and possess the potential to be multiplexed. In recent years, several reviews have provided information on the detection strategies of DNA methylation. However, to date, there is no comprehensive evaluation of electrochemical DNA methylation detection strategies. Herein, we address the recent developments of electrochemical DNA methylation detection approaches. Furthermore, we highlight the major technical and biological challenges involved in these strategies and provide suggestions for the future direction of this important field.

  7. Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment.

    Science.gov (United States)

    Christensen, Geoff A; Wymore, Ann M; King, Andrew J; Podar, Mircea; Hurt, Richard A; Santillan, Eugenio U; Soren, Ally; Brandt, Craig C; Brown, Steven D; Palumbo, Anthony V; Wall, Judy D; Gilmour, Cynthia C; Elias, Dwayne A

    2016-10-01

    Two genes, hgcA and hgcB, are essential for microbial mercury (Hg) methylation. Detection and estimation of their abundance, in conjunction with Hg concentration, bioavailability, and biogeochemistry, are critical in determining potential hot spots of methylmercury (MeHg) generation in at-risk environments. We developed broad-range degenerate PCR primers spanning known hgcAB genes to determine the presence of both genes in diverse environments. These primers were tested against an extensive set of pure cultures with published genomes, including 13 Deltaproteobacteria, nine Firmicutes, and nine methanogenic Archaea genomes. A distinct PCR product at the expected size was confirmed for all hgcAB(+) strains tested via Sanger sequencing. Additionally, we developed clade-specific degenerate quantitative PCR (qPCR) primers that targeted hgcA for each of the three dominant Hg-methylating clades. The clade-specific qPCR primers amplified hgcA from 64%, 88%, and 86% of tested pure cultures of Deltaproteobacteria, Firmicutes, and Archaea, respectively, and were highly specific for each clade. Amplification efficiencies and detection limits were quantified for each organism. Primer sensitivity varied among species based on sequence conservation. Finally, to begin to evaluate the utility of our primer sets in nature, we tested hgcA and hgcAB recovery from pure cultures spiked into sand and soil. These novel quantitative molecular tools designed in this study will allow for more accurate identification and quantification of the individual Hg-methylating groups of microorganisms in the environment. The resulting data will be essential in developing accurate and robust predictive models of Hg methylation potential, ideally integrating the geochemistry of Hg methylation to the microbiology and genetics of hgcAB IMPORTANCE: The neurotoxin methylmercury (MeHg) poses a serious risk to human health. MeHg production in nature is associated with anaerobic microorganisms. The recent

  8. Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM.

    Directory of Open Access Journals (Sweden)

    Nameeta Shah

    Full Text Available O⁶-methylguanine DNA-methyltransferase (MGMT promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089-13.097], p<0.0001. To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301-7.27], p = 0.007. We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical

  9. 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.

  10. DNA methylation analysis in liquid-based cytology for cervical cancer screening.

    Science.gov (United States)

    Apostolidou, Sophia; Hadwin, Richard; Burnell, Matthew; Jones, Allison; Baff, Donna; Pyndiah, Nitisha; Mould, Tim; Jacobs, Ian J; Beddows, Simon; Kocjan, Gabrijela; Widschwendter, Martin

    2009-12-15

    Cervical cancer is the second most common type of cancer in women worldwide. Preinvasive disease can be detected by cervical cytology. All currently available cytology technologies rely on the visual analysis of exfoliated cells from the uterine cervix. Improvement of conventional cytological screening has been proposed by the introduction of molecular-based markers applied to liquid-based cytology (LBC), the suspension of cells collected from the cervix. DNA methylation changes occur very early in carcinogenesis and identification of appropriate DNA methylation markers in such samples should be able to distinguish high-grade squamous intraepithelial lesions (HSIL) from nonspecific cytology changes and the normal cervix. To address this potential, we have undertaken a proof-of-principle study of methylation status of LBC samples from HSIL cytology cases compared against matched normal controls. Using quantitative methylation-specific PCR on 28 genes, we found SOX1, HOXA11 and CADM1 to significantly discriminate between the groups analyzed (p<0.01). Area under the receiver operating characteristic (ROC) curve (AUC) demonstrated that methylation of SOX1, HOXA11 and CADM1 could discriminate between HSIL cases and controls with high sensitivity and specificity (AUC 0.910, 0.844 and 0.760, respectively). The results were further validated in an independent set. This proof-of-principle study is the first to validate the results in an independent case/control set and presents HOXA11, a gene that is important for cervical development, as a potentially useful DNA marker in LBC samples. Further assessment of these preliminary estimates will need to be performed in a larger cohort to confirm clinical utility.

  11. DNA methylation detection based on difference of base content

    Science.gov (United States)

    Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

    2016-04-01

    Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

  12. CODEHOP PCR and CODEHOP PCR primer design.

    Science.gov (United States)

    Staheli, Jeannette P; Boyce, Richard; Kovarik, Dina; Rose, Timothy M

    2011-01-01

    While PCR primer design for the amplification of known sequences is usually quite straightforward, the design, and successful application of primers aimed at the detection of as yet unknown genes is often not. The search for genes that are presumed to be distantly related to a known gene sequence, such as homologous genes in different species, paralogs in the same genome, or novel pathogens in diverse hosts, often turns into the proverbial search for the needle in the haystack. PCR-based methods commonly used to address this issue involve the use of either consensus primers or degenerate primers, both of which have significant shortcomings regarding sensitivity and specificity. We have developed a novel primer design approach that diminishes these shortcomings and instead takes advantage of the strengths of both consensus and degenerate primer designs, by combining the two concepts into a Consensus-Degenerate Hybrid Oligonucleotide Primer (CODEHOP) approach. CODEHOP PCR primers contain a relatively short degenerate 3' core and a 5' nondegenerate clamp. The 3' degenerate core consists of a pool of primers containing all possible codons for a 3-4 aminoacid motif that is highly conserved in multiply aligned sequences from known members of a protein family. Each primer in the pool also contains a single 5' nondegenerate nucleotide sequence derived from a codon consensus across the aligned aminoacid sequences flanking the conserved motif. During the initial PCR amplification cycles, the degenerate core is responsible for specific binding to sequences encoding the conserved aminoacid motif. The longer consensus clamp region serves to stabilize the primer and allows the participation of all primers in the pool in the efficient amplification of products during later PCR cycles. We have developed an interactive web site and algorithm (iCODEHOP) for designing CODEHOP PCR primers from multiply aligned protein sequences, which is freely available online. Here, we describe the

  13. 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...... about board independence in Western jurisdictions, a surprising disharmony prevails about the justification, extent and purpose of independence requirements. These considerations lead me to question the benefits of the current system. Instead, this paper proposes a new, ‘functional’ concept of board...

  14. DNA Methylation: Bisulphite Modification and Analysis

    OpenAIRE

    Patterson, Kate; Molloy, Laura; Qu, Wenjia; Clark, Susan

    2011-01-01

    Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expres...

  15. Choosing Independence

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Milo Djukanovic, Prime Minister of Montenegro, won a key referendum May 21 when voters in his tiny, mountainous nation endorsed a plan to split from Serbia and become an independent state. This marked a final step in the breakup of the former Yugoslavia formed by six republics.

  16. DNA Methylation Signatures of the Plant Chromomethyltransferases.

    Directory of Open Access Journals (Sweden)

    Quentin Gouil

    2016-12-01

    Full Text Available DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G. By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. In A. thaliana the biased representation of mCCG in heterochromatin is related to specificities of H3K9 methyltransferase SUVH family members. At CHH motifs there is an over-representation of different variant forms of mCHH that, similarly to mCCG hypomethylation, is partitioned into the pericentric regions of the two dicots but dispersed in the monocot chromosomes. The over-represented mCHH motifs in A. thaliana associate with specific types of transposon including both class I and II elements. At mCHH the contextual bias is due to the involvement of various chromomethyltransferases whereas the context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin. This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms associated with maintenance of methylation and the overlying chromatin structure.

  17. DNA Methylation Signatures of the Plant Chromomethyltransferases.

    Science.gov (United States)

    Gouil, Quentin; Baulcombe, David C

    2016-12-01

    DNA methylation in plants is traditionally partitioned into CG, CHG and CHH contexts (with H any nucleotide but G). By investigating DNA methylation patterns in trinucleotide contexts in four angiosperm species, we show that such a representation hides spatial and functional partitioning of different methylation pathways and is incomplete. CG methylation (mCG) is largely context-independent whereas, at CHG motifs, there is under-representation of mCCG in pericentric regions of A. thaliana and tomato and throughout the chromosomes of maize and rice. In A. thaliana the biased representation of mCCG in heterochromatin is related to specificities of H3K9 methyltransferase SUVH family members. At CHH motifs there is an over-representation of different variant forms of mCHH that, similarly to mCCG hypomethylation, is partitioned into the pericentric regions of the two dicots but dispersed in the monocot chromosomes. The over-represented mCHH motifs in A. thaliana associate with specific types of transposon including both class I and II elements. At mCHH the contextual bias is due to the involvement of various chromomethyltransferases whereas the context-independent CHH methylation in A. thaliana and tomato is mediated by the RNA-directed DNA methylation process that is most active in the gene-rich euchromatin. This analysis therefore reveals that the sequence context of the methylome of plant genomes is informative about the mechanisms associated with maintenance of methylation and the overlying chromatin structure.

  18. Genes and transposons are differentially methylated in plants, but not in mammals.

    Science.gov (United States)

    Rabinowicz, Pablo D; Palmer, Lance E; May, Bruce P; Hemann, Michael T; Lowe, Scott W; McCombie, W Richard; Martienssen, Robert A

    2003-12-01

    DNA methylation is found in many eukaryotes, but its function is still controversial. We have studied the methylation of plant and animal genomes using a PCR-based technique amenable for high throughput. Repetitive elements are methylated in both organisms, but whereas most mammalian exons are methylated, plant exons are not. Thus, targeting of methylation specifically to transposons appears to be restricted to plants. We propose that the mechanistic basis of this difference may involve RNA interference. Sequencing strategies that depend on differential methylation are predicted to have different outcomes in plant and mammalian genomes.

  19. 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...

  20. Culture independent PCR: an alternative enzyme discovery strategy

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    and glycosyl hydrolase family 45 though significant sequence divergence was observed. Glycosyl hydrolases from families 7 and 45 play a crucial role in biomass conversion to fuel ethanol. Research in this renewable energy source has two objectives: (i) To contribute to development of a renewable alternative...

  1. 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.

  2. Molecular insights into the association of obesity with breast cancer risk: relevance to xenobiotic metabolism and CpG island methylation of tumor suppressor genes.

    Science.gov (United States)

    Naushad, Shaik Mohammad; Hussain, Tajamul; Al-Attas, Omar S; Prayaga, Aruna; Digumarti, Raghunadha Rao; Gottumukkala, Suryanarayana Raju; Kutala, Vijay Kumar

    2014-07-01

    Obesity, genetic polymorphisms of xenobiotic metabolic pathway, hypermethylation of tumor suppressor genes, and hypomethylation of proapoptotic genes are known to be independent risk factors for breast cancer. The objective of this study is to evaluate the combined effect of these environmental, genetic, and epigenetic risk factors on the susceptibility to breast cancer. PCR-RFLP and multiplex PCR were used for the genetic analysis of six variants of xenobiotic metabolic pathway. Methylation-specific PCR was used for the epigenetic analysis of four genetic loci. Multifactor dimensionality reduction analysis revealed a significant interaction between the body mass index (BMI) and catechol-O-methyl transferase H108L variant alone or in combination with cytochrome P450 (CYP) 1A1m1 variant. Women with "Luminal A" breast cancer phenotype had higher BMI compared to other phenotypes and healthy controls. There was no association between the BMI and tumor grade. The post-menopausal obese women exhibited lower glutathione levels. BMI showed a positive association with the methylation of extracellular superoxide dismutase (r = 0.21, p obesity increases the breast cancer susceptibility by two possible mechanisms: (i) by interacting with xenobiotic genetic polymorphisms in inducing increased oxidative DNA damage and (ii) by altering the methylome of several tumor suppressor genes.

  3. 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.

  4. Associations of ChREBP and Global DNA Methylation with Genetic and Environmental Factors in Chinese Healthy Adults.

    Science.gov (United States)

    Gao, Jiajia; Qiu, Xueping; Wang, Xuebin; Peng, Chunyan; Zheng, Fang

    2016-01-01

    Age, gender, diet, gene and lifestyle have been reported to affect metabolic status and disease susceptibility through epigenetic pathway. But it remains indistinct that which factors account for certain epigenetic modifications. Our aim was to identify the influencing factors on inter-individual DNA methylation variations of carbohydrate response element binding protein (ChREBP) and global genome in peripheral blood leucocytes (PBLs). ChREBP DNA methylation was determined by bisulfite sequencing, and genomic 5mdC contents were quantified by capillary hydrophilic-interaction liquid chromatography/ in-source fragmentation/ tandem mass spectrometry system in about 300 healthy individuals. Eleven single nucleotide polymorphisms (SNPs) spanning ChREBP and DNA methyltransferase 1 (DNMT1) were genotyped by high resolution melting or PCR-restriction fragment length polymorphism. DNMT1 mRNA expression was analyzed by quantitative PCR. We found ChREBP DNA methylation levels were statistically associated with age (Beta (B) = 0.028, p = 0.006) and serum total cholesterol concentrations (TC) (B = 0.815, p = 0.010), independent of sex, concentrations of triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDL-C), fasting blood glucose and systolic blood pressure, diastolic blood pressure, PBLs counts and classifications. The DNMT1 haplotypes were related to ChREBP (odds ratio (OR) = 0.668, p = 0.029) and global (OR = 0.450, p = 0.015) DNA methylation as well as LDL-C, but not DNMT1 expression. However, only the relation to LDL-C was robust to correction for multiple testing (ORFDR = 1.593, pFDR = 0.013). These results indicated that the age and TC were independent influential factors of ChREBP methylation and DNMT1 variants could probably influence LDL-C to further modify ChREBP DNA methylation. Certainly, sequential comprehensive analysis of the interactions between genetic variants and blood lipid levels on ChREBP and global DNA methylation

  5. 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 obtaine...

  6. Association of TNFRSF10D DNA-methylation with the survival of melanoma patients.

    Science.gov (United States)

    Ratzinger, Gudrun; Mitteregger, Simone; Wolf, Barbara; Berger, Regina; Zelger, Bernhard; Weinlich, Georg; Fritsch, Peter; Goebel, Georg; Fiegl, Heidelinde

    2014-07-07

    In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE) melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated) as a prognostic marker for overall (p = 0.001) and for relapse-free survival (p = 0.008) in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012). In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR) of death, 4.6 (95% CI: 2.0-11.0; p < 0.001), RR of relapse, 7.2 (95% CI: 2.8-18.3; p < 0.001)). In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

  7. Association of TNFRSF10D DNA-Methylation with the Survival of Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Gudrun Ratzinger

    2014-07-01

    Full Text Available In this retrospective pilot study, the DNA-methylation status of genes that have been demonstrated to be involved in melanoma carcinogenesis was analyzed in order to identify novel biomarkers for the risk assessment of melanoma patients. We analyzed DNA extracted from punch-biopsies from 68 formalin-fixed paraffin-embedded (FFPE melanoma specimens. Using MethyLight PCR, we examined 20 genes in specimens from a training set comprising 36 melanoma patients. Selected candidate genes were validated in a test set using FFPE tissue samples from 32 melanoma patients. First, we identified the TNFRSF10D DNA-methylation status (TNFRSF10D methylated vs. unmethylated as a prognostic marker for overall (p = 0.001 and for relapse-free survival (p = 0.008 in the training set. This finding was confirmed in the independent test set (n = 32; overall survival p = 0.041; relapse-free survival p = 0.012. In a multivariate Cox-regression analysis including all patients, the TNFRSF10D DNA-methylation status remained as the most significant prognostic parameter for overall and relapse-free survival (relative-risk (RR of death, 4.6 (95% CI: 2.0–11.0; p < 0.001, RR of relapse, 7.2 (95% CI: 2.8–18.3; p < 0.001. In this study, we demonstrate that TNFRSF10D DNA-methylation analysis of a small tissue-punch from archival FFPE melanoma tissue is a promising approach to provide prognostic information in patients with melanoma.

  8. Study on the polymorphisms and promoter methylation and expression of the glutathione Stransferases P1 gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    张友才

    2006-01-01

    Objective To study the relationship between hepatocellular carcinoma (HCC) and the polymorphisms, promoter methylation, and expression of glutathione S-transferases P1 gene (GST)P1 gene. Methods Using methylation -special PCR (MSP), the methylated status of CpG islands of GSTP1 gene in tumor tissues of 53 HCC and its adjacent nontumor tissues were studied. The en-

  9. Metastatic suppressor genes inactivated by aberrant methylation in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To screen out the differentially methylated DNA sequences between gastric primary tumor and metastatic lymph nodes, test the methylation difference of gene PTPRG between primary gastric tumor and metastatic lymph nodes, and test the regulatory function of 5-aza-2-deoxycytidine which is an agent with suppression on methylation and the level of methylation in gastric cancer cell line.METHODS: Methylated DNA sequences in genome were enriched with methylated CpG islands amplification (MCA)to undergo representational difference analysis (RDA),with MCA production of metastatic lymph nodes as tester and that of primary tumor as driver. The obtained differentially methylated fragments were cloned and sequenced to acquire the base sequence, which was analyzed with bioinformatics. With methylation-specific PCR (MSP) and RT-PCR, methylation difference of gene PTPRG was detected between primary tumor and metastatic lymph nodes in 36 cases of gastric cancer.Methylation of gene PTPRG and its regulated expression were observed in gastric cancer cell line before and after being treated with methylation-suppressive agent.RESULTS: Nineteen differentially methylated sequences were obtained and located at 5' end, exons, introns and 3' end, in which KL59 was observed to be located at 9p21 as the first exon of gene p16 and KL22 to be located at promoter region of PRPRG. KL22, aS the probes, was hybridized with driver, tester and 3-round RDA products respectively with all positive signals except with the driver. Significant difference was observed in both methylation rate of gene PTPRG and PTPRG mRNA expression rate between primary tumor and metastatic lymph nodes. Demethylation of gene PTPRG, with recovered expression of PTPRG mRNA, was observed after gastric cancer cell line being treated with methylation-suppressive agent.CONCLUSION: Difference exists in DNA methylation between primary tumor and metastatic lymph nodes of gastric cancer, with MCA-RDA as one of the good analytical

  10. Enrichment of methylated DNA by methyl-CpG immunoprecipitation.

    Science.gov (United States)

    Sonnet, Miriam; Baer, Constance; Rehli, Michael; Weichenhan, Dieter; Plass, Christoph

    2013-01-01

    Normal DNA methylation is an epigenetic modification required for proper development. Aberrant DNA methylation, in contrast, is frequently observed in many different malignancies including leukemias and lymphomas. Global DNA methylation profiling addresses the methylated sequences (methylome) of patient genomes to identify disease-specific methylation patterns. Workload in methylome analyses can be considerably reduced by methylome enrichment using proteins or antibodies with high affinity to methylated DNA. Methyl-CpG Immunoprecipitation (MCIp) employs an immobilized recombinant human methyl-CpG binding domain protein 2, MBD2, which binds methylated CpGs in double-stranded DNA. Elution with increasing salt concentrations allows the fractionated enrichment of different degrees of methylation.

  11. Inverse PCR for Point Mutation Introduction.

    Science.gov (United States)

    Silva, Diogo; Santos, Gustavo; Barroca, Mário; Collins, Tony

    2017-01-01

    Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.

  12. 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.

  13. Detection of MGMT promoter methylation in glioblastoma using pyrosequencing.

    Science.gov (United States)

    Xie, Hao; Tubbs, Raymond; Yang, Bin

    2015-01-01

    Recent clinical trials on patients with glioblastoma revealed that O6-Methylguanine-DNA methyltransferase (MGMT) methylation status significantly predicts patient's response to alkylating agents. In this study, we sought to develop and validate a quantitative MGMT methylation assay using pyrosequencing on glioblastoma. We quantified promoter methylation of MGMT using pyrosequencing on paraffin-embedded fine needle aspiration biopsy tissues from 43 glioblastoma. Using a 10% cutoff, MGMT methylation was identified in 37% cases of glioblastoma and 0% of the non-neoplastic epileptic tissue. Methylation of any individual CpG island in MGMT promoter ranged between 33% and 95%, with a mean of 65%. By a serial dilution of genomic DNA of a homogenously methylated cancer cell line with an unmethylated cell line, the analytical sensitivity is at 5% for pyrosequencing to detect MGMT methylation. The minimal amount of genomic DNA required is 100 ng (approximately 3,000 cells) in small fine needle biopsy specimens. Compared with methylation-specific PCR, pyrosequencing is comparably sensitive, relatively specific, and also provides quantitative information for each CpG methylation.

  14. Apta-PCR.

    Science.gov (United States)

    Pinto, Alessandro; Polo, Pedro Nadal; Rubio, Miriam Jauest; Svobodova, Marketa; Lerga, Teresa Mairal; O'Sullivan, Ciara K

    2016-01-01

    Real-time Apta-PCR is a methodology that can be used for a wide variety of applications ranging from food quality control to clinical diagnostics. This method takes advantage of the combination of the sensitivity of nucleic acid amplification with the selectivity of aptamers. Ultra-low detection of target analyte can potentially be achieved, or, improved detection limits can be achieved with aptamers of low-medium affinity. Herein, we describe a generic methodology coined real-time Apta-PCR, using a model target (β-conglutin) and a competitive format, which can be adapted for the detection of any target which an aptamer has been selected for.

  15. Increased methylation of lung cancer-associated genes in sputum DNA of former smokers with chronic mucous hypersecretion

    OpenAIRE

    Bruse, Shannon; Petersen, Hans; Weissfeld, Joel; Picchi, Maria; Willink, Randall; Do, Kieu; Siegfried, Jill; Belinsky, Steven A.; Tesfaigzi, Yohannes

    2014-01-01

    Background Chronic mucous hypersecretion (CMH) contributes to COPD exacerbations and increased risk for lung cancer. Because methylation of gene promoters in sputum has been shown to be associated with lung cancer risk, we tested whether such methylation was more common in persons with CMH. Methods Eleven genes commonly silenced by promoter methylation in lung cancer and associated with cancer risk were selected. Methylation specific PCR (MSP) was used to profile the sputum of 900 individuals...

  16. P16 METHYLATION OF THE COLORECTAL CANCER AND ASSOCIATION WITH DUKES STAGES

    Institute of Scientific and Technical Information of China (English)

    王志伟; 易静; 仓辉; 邹鸿志; 郁宝铭; 汤雪明

    2001-01-01

    To explore whether methylation of the CpG island in the promoter of the p16 tumor suppressor gene was associated with clinicopathological characteristics of the colorectal cancer patients. Methods: Methylation- specific PCR (MSP) was used to detect p16 methylation of the colorectal cancer patients. Results: In 58 sporadic colorectal cancer, 43.1% of the tumors had detectable p16 methylation. Dukes' stage was associated with p16 methylation status. Dukes C, D patients (75%) were more likely to contain methylated p16 compared with Dukes A, B patients (13.3%). Conclusion: p16 methylation plays a role in the carcinogenesis of a subset of colorectal cancer. P16 methylation might be considered as a prognostic indicator.

  17. [DNA methylation and epigenetics].

    Science.gov (United States)

    Vaniushin, B F

    2006-09-01

    In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N6-methyladenine residues in the sequence TGATCA (TGATCA-->TGm6ATCA). Plants possess AdoMet-dependent endonucleases

  18. 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.

  19. Effects of arsenic trioxide on the methylation of TMS1 gene in K562 cells

    Institute of Scientific and Technical Information of China (English)

    李洪丽

    2014-01-01

    Objective To detect the methylation status of TMS1gene and its demethylation by arsenic trioxide(As2O2)in K562 cells.Methods K562 cells were treated with different concentrations of As2O2for 48 hours.Methylationspecific PCR(MSP)was used to determine the methylation status of TMS1.RT-PCR and Western blot were used to detect the levels of TMS1 mRNA and protein.

  20. LATS2 is De-methylated and Overexpressed in Nasopharyngeal Carcinoma and Predicts Poor Prognosis

    Directory of Open Access Journals (Sweden)

    Chen Jing

    2010-10-01

    Full Text Available Abstract Background LATS2, which encodes a novel serine/threonine kinase, is known to be important in centrosome duplication and in the maintenance of genomic stability. Recently, a potential role for LATS2 in cancer has been reported. In breast cancer and acute lymphoblastic leukemia (ALL, LATS2 mRNA is downregulated and has been suggested to be a tumor suppressor. However, the role of LATS2 in nasopharyngeal carcinoma has not been investigated. In this study, we aimed to investigate the expression pattern of LATS2 and its clinicopathological involvement in nasopharyngeal carcinoma to understand its effect on cell survival. Methods Using quantitative real time PCR and immunoblotting, the expression of LATS2 was detected in nasopharyngeal carcinoma cell lines and in the immortalized nasopharyngeal epithelial cell line NP69. Using immunohistochemistry, we analyzed LATS2 protein expression in 220 nasopharyngeal carcinoma cases. The association of LATS2 protein expression with the clinicopathological characteristics and the prognosis of nasopharyngeal carcinoma were subsequently assessed. Using methylation specific PCR, we detected the methylation status of the LATS2 promoter. RNA interference was performed by transfecting siRNA to specifically knock down LATS2 expression in 5-8F and CNE2. Results LATS2 protein was detected in 178 of 220 (80.91% cases of nasopharyngeal carcinoma. LATS2 overexpression was a significant, independent prognosis predictor (P = 0.037 in nasopharyngeal carcinoma patients. Methylation specific PCR revealed that 36.7% (11/30 of nasopharyngeal carcinoma tissues and all of the chronic nasopharyngeal inflammation samples were methylated. Functional studies showed that the suppression of LATS2 expression in nasopharyngeal carcinoma (5-8F and CNE2 cell lines by using specific small interfering (siRNA resulted in the inhibition of growth, induction of apoptosis and S-phase cell cycle increase. Overexpression of LATS2 in NP69

  1. Explanatory chapter: PCR primer design.

    Science.gov (United States)

    Álvarez-Fernández, Rubén

    2013-01-01

    This chapter is intended as a guide on polymerase chain reaction (PCR) primer design (for information on PCR, see General PCR and Explanatory Chapter: Troubleshooting PCR). In the next section, general guidelines will be provided, followed by a discussion on primer design for specific applications. A list of recommended software tools is shown at the end. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Comparative analysis of rubber seed methyl ester with other methyl ...

    African Journals Online (AJOL)

    Comparative analysis of rubber seed methyl ester with other methyl esters. ... In order to achieve a two-step transesterification process was developed to convert rubber seed oil to its methyl esters. The first step, acid catalyzed ... Article Metrics.

  3. 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.

  4. Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD.

    Science.gov (United States)

    Wielscher, Matthias; Vierlinger, Klemens; Kegler, Ulrike; Ziesche, Rolf; Gsur, Andrea; Weinhäusel, Andreas

    2015-08-01

    Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers. Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400 μl serum (n = 204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR. Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67-0.97) and specificity 90.2%, (95%CI: 0.65-0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57-1), and COPD from cancer with a specificity of 88% (95%CI: 0.64-0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4-0.78) and a specificity of 70% (95%CI: 0.54-0.81). The results were confirmed using an independent sample set (n = 46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72-0.95). This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

  5. Droplet digital polymerase chain reaction (PCR) outperforms real-time PCR in the detection of environmental DNA from an invasive fish species.

    Science.gov (United States)

    Doi, Hideyuki; Takahara, Teruhiko; Minamoto, Toshifumi; Matsuhashi, Saeko; Uchii, Kimiko; Yamanaka, Hiroki

    2015-05-01

    Environmental DNA (eDNA) has been used to investigate species distributions in aquatic ecosystems. Most of these studies use real-time polymerase chain reaction (PCR) to detect eDNA in water; however, PCR amplification is often inhibited by the presence of organic and inorganic matter. In droplet digital PCR (ddPCR), the sample is partitioned into thousands of nanoliter droplets, and PCR inhibition may be reduced by the detection of the end-point of PCR amplification in each droplet, independent of the amplification efficiency. In addition, real-time PCR reagents can affect PCR amplification and consequently alter detection rates. We compared the effectiveness of ddPCR and real-time PCR using two different PCR reagents for the detection of the eDNA from invasive bluegill sunfish, Lepomis macrochirus, in ponds. We found that ddPCR had higher detection rates of bluegill eDNA in pond water than real-time PCR with either of the PCR reagents, especially at low DNA concentrations. Limits of DNA detection, which were tested by spiking the bluegill DNA to DNA extracts from the ponds containing natural inhibitors, found that ddPCR had higher detection rate than real-time PCR. Our results suggest that ddPCR is more resistant to the presence of PCR inhibitors in field samples than real-time PCR. Thus, ddPCR outperforms real-time PCR methods for detecting eDNA to document species distributions in natural habitats, especially in habitats with high concentrations of PCR inhibitors.

  6. Genome-Wide Analysis of DNA Methylation in Human Amnion

    Directory of Open Access Journals (Sweden)

    Jinsil Kim

    2013-01-01

    Full Text Available The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3 gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

  7. Genome-Wide Analysis of DNA Methylation in Human Amnion

    Science.gov (United States)

    Kim, Jinsil; Pitlick, Mitchell M.; Christine, Paul J.; Schaefer, Amanda R.; Saleme, Cesar; Comas, Belén; Cosentino, Viviana; Gadow, Enrique; Murray, Jeffrey C.

    2013-01-01

    The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies. PMID:23533356

  8. 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....

  9. Genome-wide methylation analyses in glioblastoma multiforme.

    Directory of Open Access Journals (Sweden)

    Rose K Lai

    Full Text Available Few studies had investigated genome-wide methylation in glioblastoma multiforme (GBM. Our goals were to study differential methylation across the genome in gene promoters using an array-based method, as well as repetitive elements using surrogate global methylation markers. The discovery sample set for this study consisted of 54 GBM from Columbia University and Case Western Reserve University, and 24 brain controls from the New York Brain Bank. We assembled a validation dataset using methylation data of 162 TCGA GBM and 140 brain controls from dbGAP. HumanMethylation27 Analysis Bead-Chips (Illumina were used to interrogate 26,486 informative CpG sites in both the discovery and validation datasets. Global methylation levels were assessed by analysis of L1 retrotransposon (LINE1, 5 methyl-deoxycytidine (5m-dC and 5 hydroxylmethyl-deoxycytidine (5hm-dC in the discovery dataset. We validated a total of 1548 CpG sites (1307 genes that were differentially methylated in GBM compared to controls. There were more than twice as many hypomethylated genes as hypermethylated ones. Both the discovery and validation datasets found 5 tumor methylation classes. Pathway analyses showed that the top ten pathways in hypomethylated genes were all related to functions of innate and acquired immunities. Among hypermethylated pathways, transcriptional regulatory network in embryonic stem cells was the most significant. In the study of global methylation markers, 5m-dC level was the best discriminant among methylation classes, whereas in survival analyses, high level of LINE1 methylation was an independent, favorable prognostic factor in the discovery dataset. Based on a pathway approach, hypermethylation in genes that control stem cell differentiation were significant, poor prognostic factors of overall survival in both the discovery and validation datasets. Approaches that targeted these methylated genes may be a future therapeutic goal.

  10. 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.

  11. GSTP1 expression and promoter methylation in epithelial ovarian carcinoma

    Directory of Open Access Journals (Sweden)

    V Shilpa

    2014-01-01

    Full Text Available Context: GSTP1 is a subgroup of glutathione-S-transferase family, which provides cellular protection against free radical and carcinogenic compounds due to its detoxifying function. Altered GSTP1 activity due to down regulation of enzyme activity and DNA methylation has been reported in many tumors, although data for ovarian cancer are few. In this study, we aimed at determining the expression of GSTP1 in relation to the methylation of the GSTP1 promoter in epithelial ovarian cancer (EOC. Materials and Methods: GSTP1 mRNA expression and GSTP1 enzyme concentration were assessed by quantitative reverse transcriptase polymerase chain reaction (PCR and enzyme-linked immunosorbent assay, respectively, in 88 EOCs, 14 low malignant potential (LMP tumors, and 20 benign tumors. The promoter methylation of GSTP1 gene was evaluated by methylation-specific PCR. Results: Reduced GSTP1 mRNA expression was observed in 49% EOCs, 21.4% LMP, and 45% benign tumors. Significantly lower levels of plasma GSTP1 were observed in all tumor samples compared to normal. GSTP1 promoter methylation was detected in 10 (11.4% EOCs and 1 (7.3% LMP tumors. No methylation was observed in benign tumors and normal ovaries. Conclusions: Our results show that there is a significant down regulation of GSTP1 expression while hypermethylation of the GSTP1 gene promoter is not very frequent in EOC. Further studies are needed to study underlying mechanisms leading to decreased expression.

  12. 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.

  13. Methylation of Gene CHFR Promoter in Acute Leukemia Cells

    Institute of Scientific and Technical Information of China (English)

    GONG Hui; LIU Wengli; ZHOU Jianfeng; XU Huizhen

    2005-01-01

    Summary: In order to explore whether gene CHFR was inactivated by methylation in leukemia cells, the expression of CHFR was examined before and after treatment with demethylation agent in Molt-4, Jurkat and U937 leukemia cell lines by means of RT-PCR. The methylation of promoter in Molt-4, Jurkat and U937 cells as well as 41 acute leukemia patients was analyzed by MS-PCR. The results showed that methylation of CHFR promoter was inactivated and could be reversed by treatment with a demethylating agent in Molt-4, Jurkat and U937. CHFR promoter methylation was detected in 39 % of acute leukemia patients. There was no difference in incidence of CHFR promoter methylation between acute myelocytic leukemia and acute lymphocytic leukemia. In conclusion, CHFR is frequently inactivated in acute leukemia and is a good candidate for the leukemia supper gene. By affecting mitotic checkpoint function, CHFR inactivation likely plays a key role in tumorigenesis in acute leukemia. Moreover, the methylation of gene CHFR appears to be a good index with which to predict the sensitivity of acute leukemia to microtubule inhibitors.

  14. 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

    Oral lichen planus (OLP) is a common oral mucosal disease, which is generally considered a potentially malignant lesion. To identify efficiently prognostic biomarker, we investigated the microRNA-137 (miR-137) promoter methylation in OLP and compared with the samples from healthy volunteers...... 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...... between miR-137 and p16 methylation levels were statistically significant between healthy controls and patients. Methylation levels of the two promoters were also influenced by age, gender, and lesion duration. Interestingly, aberrant promoter methylation of the p16 and miR-137 genes was only found...

  15. Differential DNA methylation correlates with differential expression of angiogenic factors in human heart failure.

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    Mehregan Movassagh

    Full Text Available Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip, validated differential methylation loci by bisulfite-(BS PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01. Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogeneous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.

  16. Lead exposure suppressed ALAD transcription by increasing methylation level of the promoter CpG islands.

    Science.gov (United States)

    Li, Chunping; Xu, Ming; Wang, Sumeng; Yang, Xiaolin; Zhou, Shourong; Zhang, Jingping; Liu, Qizhan; Sun, Yujie

    2011-05-30

    DNA methylation provides a plausible link between the environment and alterations in gene expression that may lead to disease phenotypes. Lead exposure can change DNA methylation status. Here, we hypothesized that the methylation of the ALAD gene promoter may play an important role in lead toxicity. To determine whether the methylation level of the ALAD promoter is associated with the risk of lead poisoning, we conducted a case-control study of 103 workers from a battery plant and 103 healthy volunteers with matching age and gender distribution. We employed real-time PCR and methylation-specific PCR (MSP) in cell models to determine the relationship between ALAD methylation level and transcription level. We found lead exposure to increase the ALAD gene methylation level and down-regulate ALAD transcription. The difference in methylation frequencies between exposures and controls was statistically significant (p=0.002), and individuals with methylated ALAD gene showed an increased risk of lead poisoning (adjusted OR=3.57, 95% CI, 1.55-8.18). This study suggests that the lead-exposure-induced increases in ALAD methylation may be involved in the mechanism of lead toxicity. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  17. ENZYMOLOGY OF ARSENIC METHYLATION

    Science.gov (United States)

    Enzymology of Arsenic MethylationDavid J. Thomas, Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park...

  18. Lysine methylation: beyond histones

    Institute of Scientific and Technical Information of China (English)

    Xi Zhang; Hong Wen; Xiaobing Shi

    2012-01-01

    Posttranslational modifications (PTMs) of histone proteins,such as acetylation,methylation,phosphorylation,and ubiquitylation,play essential roles in regulating chromatin dynamics.Combinations of different modifications on the histone proteins,termed 'histone code' in many cases,extend the information potential of the genetic code by regulating DNA at the epigenetic level.Many PTMs occur on non-histone proteins as well as histones,regulating protein-protein interactions,stability,localization,and/or enzymatic activities of proteins involved in diverse cellular processes.Although protein phosphorylation,ubiquitylation,and acetylation have been extensively studied,only a few proteins other than histones have been reported that can be modified by lysine methylation.This review summarizes the current progress on lysine methylation of nonhistone proteins,and we propose that lysine methylation,like phosphorylation and acetylation,is a common PTM that regulates proteins in diverse cellular processes.

  19. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces.

    Science.gov (United States)

    Verhaegen, Bavo; De Reu, Koen; De Zutter, Lieven; Verstraete, Karen; Heyndrickx, Marc; Van Coillie, Els

    2016-05-18

    Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC) and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR), which has its specific limitations. Droplet digital PCR (ddPCR) has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014) for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan(®) Environmental Master Mix 2.0; UMM: TaqMan(®) Universal PCR Master Mix) were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  20. Comparison of Droplet Digital PCR and qPCR for the Quantification of Shiga Toxin-Producing Escherichia coli in Bovine Feces

    Directory of Open Access Journals (Sweden)

    Bavo Verhaegen

    2016-05-01

    Full Text Available Cattle are considered to be the main reservoir for Shiga toxin-producing Escherichia coli (STEC and are often the direct or indirect source of STEC outbreaks in humans. Accurate measurement of the concentration of shed STEC in cattle feces could be a key answer to questions concerning transmission of STEC, contamination sources and efficiency of treatments at farm level. Infected animals can be identified and the contamination level quantified by real-time quantitative PCR (qPCR, which has its specific limitations. Droplet digital PCR (ddPCR has been proposed as a method to overcome many of the drawbacks of qPCR. This end-point amplification PCR is capable of absolute quantification independent from any reference material and is less prone to PCR inhibition than qPCR. In this study, the qPCR-based protocol described by Verstraete et al. (2014 for Shiga toxin genes stx1 and stx2 and the intimin gene eae quantification was optimized for ddPCR analysis. The properties of ddPCR and qPCR using two different mastermixes (EMM: TaqMan® Environmental Master Mix 2.0; UMM: TaqMan® Universal PCR Master Mix were evaluated, using standard curves and both artificial and natural contaminated cattle fecal samples. In addition, the susceptibility of these assays to PCR-inhibitors was investigated. Evaluation of the standard curves and both artificial and natural contaminated cattle fecal samples suggested a very good agreement between qPCR using EMM and ddPCR. Furthermore, similar sensitivities and no PCR inhibition were recorded for both assays. On the other hand, qPCR using UMM was clearly prone to PCR inhibition. In conclusion, the ddPCR technique shows potential for the accurate absolute quantification of STEC on the farms, without relying on standardized reference material.

  1. Study of methyl transferase (G9aMT) and methylated histone (H3-K9) expressions in unexplained recurrent spontaneous abortion (URSA) and normal early pregnancy.

    Science.gov (United States)

    Fatima, Nishat; Ahmed, S H; Salhan, Sudha; Rehman, S M F; Kaur, Jatinder; Owais, M; Chauhan, Shyam S

    2011-11-01

    We investigated the expression of methyl transferase G9a and methylated histone H3-K9 in fresh human decidual/endometrial tissue of 12 normal early pregnancies and 15 unexplained recurrent spontaneous abortions (URSA). The samples were obtained through dilatation and curettage and collected as per strict inclusion-exclusion criteria. The tissue was subjected to immunohistochemical analysis (IHC), western blotting (WB) and RT-PCR analysis. The results demonstrated methyl transferase G9a to have a lower expression in abortions when compared with that in normal pregnancy (P K9 was significantly lower (P < 0.0001) in URSA tissues than in controls. This study suggests that methylation may cause URSA and indicates the need for further work to explore the role of methylation in URSA and its possible prevention through locally acting methylating/demethylating agents.

  2. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  4. 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.

  5. Promoter Methylation Precedes Chromosomal Alterations in Colorectal Cancer Development

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2006-01-01

    Full Text Available Background: Colorectal cancers are characterized by genetic and epigenetic alterations. This study aimed to explore the timing of promoter methylation and relationship with mutations and chromosomal alterations in colorectal carcinogenesis. Methods: In a series of 47 nonprogressed adenomas, 41 progressed adenomas (malignant polyps, 38 colorectal carcinomas and 18 paired normal tissues, we evaluated promoter methylation status of hMLH1, O6MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5, and CHFR using methylation-specific PCR. Mutation status of TP53, APC and KRAS were studied by p53 immunohistochemistry and sequencing of the APC and KRAS mutation cluster regions. Chromosomal alterations were evaluated by comparative genomic hybridization. Results: Our data demonstrate that nonprogressed adenomas, progressed adenomas and carcinomas show similar frequencies of promoter methylation for the majority of the genes. Normal tissues showed significantly lower frequencies of promoter methylation of APC, p16INK4A, GATA-4, and GATA-5 (P-values: 0.02, 0.02, 1.1×10−5 and 0.008 respectively. P53 immunopositivity and chromosomal abnormalities occur predominantly in carcinomas (P values: 1.1×10−5 and 4.1×10−10. Conclusions: Since promoter methylation was already present in nonprogressed adenomas without chromosomal alterations, we conclude that promoter methylation can be regarded as an early event preceding TP53 mutation and chromosomal abnormalities in colorectal cancer development.

  6. Outcome in unresectable glioblastoma: MGMT promoter methylation makes the difference.

    Science.gov (United States)

    Thon, Niklas; Thorsteinsdottir, Jun; Eigenbrod, Sabina; Schüller, Ulrich; Lutz, Jürgen; Kreth, Simone; Belka, Claus; Tonn, Jörg-Christian; Niyazi, Maximilian; Kreth, Friedrich Wilhelm

    2017-02-01

    In 2011, we reported a predominant prognostic/predictive role of MGMT promoter methylation status on progression-free survival (PFS) in unresectable glioblastoma patients undergoing upfront radiotherapy plus concomitant and maintenance temozolomide (RTX/TMZ → TMZ). We, here, present the final results of this prospective study focussing on the prognostic/predictive value of MGMT promoter methylation status for death risk stratification. Overall, 56 adult patients with unresectable, biopsy proven glioblastoma were prospectively assigned to upfront RTX/TMZ → TMZ treatment between March 2006 and August 2008. Last follow-up was performed in June 2016. MGMT promoter methylation was determined using methylation-specific PCR (MSP) and sodium bisulfite sequencing. Analyses were done by intention to treat. Prognostic factors were obtained from proportional hazard models. At the time of the final analysis 55 patients showed progressive disease and 53 patients had died. MGMT promoter was methylated (unmethylated) in 30 (26) patients. Methylation of the MGMT promoter was the strongest favorable predictor for overall survival (OS, median: 20.3 vs. 7.3 months, p MGMT promoter methylation status is essential for patients' counseling, prognostic evaluation, and for the design of future trials dealing with unresectable glioblastomas.

  7. Methyl 6-O-trityl-α-d-glucopyranoside methanol disolvate

    Directory of Open Access Journals (Sweden)

    Zeynep Gültekin

    2014-04-01

    Full Text Available The asymmetric unit of the title compound, C26H28O6·2CH3OH, contains two independent methyl 6-O-trityl-α-d-glucopyranoside molecules and four methanol solvent molecules. The rings of two methyl α-d-glucopyranoside adopt chair conformations. In the crystal, extensive intra- and intermolecular O—H...O and C—H...O hydrogen bonds link the molecules into a three-dimensional supramolecular architecture.

  8. Promoter hypermethylation of the p16 and Wif-1 genes as an independent prognostic marker in stage IA non-small cell lung cancers.

    Science.gov (United States)

    Yoshino, Mitsuru; Suzuki, Makoto; Tian, Lei; Moriya, Yasumitsu; Hoshino, Hidehisa; Okamoto, Tatsuro; Yoshida, Shigetoshi; Shibuya, Kiyoshi; Yoshino, Ichiro

    2009-11-01

    Hypermethylation of promoter CpG islands is a major inactivation mechanism of tumor suppressor genes, some of which are thought to be related to the prognosis of patients with non-small cell lung cancer (NSCLC). Therefore, hypermethylation of the specific genes may be expected to serve as a prognostic biomarker for NSCLC. In this study, the methylation status of 14 genes was analyzed in 44 stage IA NSCLC cases using methylation-specific PCR. Hypermethylation was detected in PTGER2 (70% of cases), DRM/Gremlin (66%), sFRP-2 (57%), IL-12Rbeta2 (48%), Reprimo (41%), APC (39%), CXCL12 (39%), HPP1 (30%), SPARC (30%), sFRP-5 (30%), p16 (25%), RUNX3 (20%), sFRP-1 (20%) and Wif-1 (16%). Patients with p16, sFRP-5, Wif-1 or CXCL12 methylation had a significantly shorter duration of relapse-free survival than their counterparts with an unmethylated gene (p16, P=0.011; sFRP-5, P=0.030, Wif-1, P=0.036; CXCL12, P=0.026). Also, those with methylated HPP1, p16 or Wif-1 had a significantly shorter duration of overall survival (HPP1, P=0.031; p16, P=0.026; Wif-1, P=0.008). Multivariate analysis revealed that p16 methylation in relapse-free survival and Wif-1 methylation in overall survival were the strongest independent prognostic factors (p16, P=0.036; Wif-1, P=0.035). In conclusion, the hypermethylation of the p16 and Wif-1 genes has potential as biomarkers that may be used to predict the prognosis of stage IA NSCLC.

  9. Long-term survival in glioblastoma: methyl guanine methyl transferase (MGMT promoter methylation as independent favourable prognostic factor

    Directory of Open Access Journals (Sweden)

    Smrdel Uros

    2016-12-01

    Full Text Available In spite of significant improvement after multi-modality treatment, prognosis of most patients with glioblastoma remains poor. Standard clinical prognostic factors (age, gender, extent of surgery and performance status do not clearly predict long-term survival. The aim of this case-control study was to evaluate immuno-histochemical and genetic characteristics of the tumour as additional prognostic factors in glioblastoma.

  10. Comparison of droplet digital PCR with quantitative real-time PCR for determination of zygosity in transgenic maize.

    Science.gov (United States)

    Xu, Xiaoli; Peng, Cheng; Wang, Xiaofu; Chen, Xiaoyun; Wang, Qiang; Xu, Junfeng

    2016-12-01

    This study evaluated the applicability of droplet digital PCR (ddPCR) as a tool for maize zygosity determination using quantitative real-time PCR (qPCR) as a reference technology. Quantitative real-time PCR is commonly used to determine transgene copy number or GMO zygosity characterization. However, its effectiveness is based on identical reaction efficiencies for the transgene and the endogenous reference gene. Additionally, a calibrator sample should be utilized for accuracy. Droplet digital PCR is a DNA molecule counting technique that directly counts the absolute number of target and reference DNA molecules in a sample, independent of assay efficiency or external calibrators. The zygosity of the transgene can be easily determined using the ratio of the quantity of the target gene to the reference single copy endogenous gene. In this study, both the qPCR and ddPCR methods were used to determine insect-resistant transgenic maize IE034 zygosity. Both methods performed well, but the ddPCR method was more convenient because of its absolute quantification property.

  11. Expression of SOCS-3 and Its Methylation in Renal Cancer

    Institute of Scientific and Technical Information of China (English)

    YU Jing; ZHAO Li-chun; YAO Song-mei; HE Cheng-yan; LI Hong-jun; KONG Xiang-bo

    2011-01-01

    The expression level of suppressor of cytokine signaling-3(SOCS-3) in human renal carcinoma and its methylation state were investigated. Reverse transcription-polymerase chain reaction(RT-PCR), immuocytochemistry,immunohistochemistry and Western blot were used to detect the expression level of SOCS-3, and the methylation of SOCS gene was investigated by methylation specific PCR in the tissues of i 5 cases of renal carcinoma. Compared to those of the normal renal cell line and specimens, the expression level of SOCS-3 in renal carcinoma was significantly lower or can't be detected(P<0.01). And the methylation of SOCS-3 gene in the tissue of renal carcinoma was significantly higher. The expression of SOCS-3 gene is significantly lower in renal carcinoma and the high methylation of the promoter island of SOCS-3 gene is associated with the lower expression of SOCS-3 gene. It may be one of main mechanisms for the development and progress of renal carcinoma.

  12. Culture-independent methods for identifying microbial communities in cheese

    OpenAIRE

    Jany, Jean-Luc; Barbier, Georges

    2008-01-01

    International audience; This review focuses on the culture-independent methods available for the description of both bacterial and fungal communities in cheese. Important steps of the culture-independent strategy, which relies on bulk DNA extraction from cheese and polymerase chain reaction (PCR) amplification of selected sequences, are discussed. We critically evaluate the identification techniques already used for monitoring microbial communities in cheese, including PCR-denaturing gradient...

  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. Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

    Science.gov (United States)

    Kraiczy, J; Nayak, K; Ross, A; Raine, T; Mak, T N; Gasparetto, M; Cario, E; Rakyan, V; Heuschkel, R; Zilbauer, M

    2016-05-01

    DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.

  15. Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

    Science.gov (United States)

    Kristensen, Lasse S; Michaelsen, Signe R; Dyrbye, Henrik; Aslan, Derya; Grunnet, Kirsten; Christensen, Ib J; Poulsen, Hans S; Grønbæk, Kirsten; Broholm, Helle

    2016-03-01

    Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma.

  16. Apoptosis and DNA Methylation

    Energy Technology Data Exchange (ETDEWEB)

    Meng, Huan X.; Hackett, James A. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Nestor, Colm [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Dunican, Donncha S.; Madej, Monika; Reddington, James P. [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Pennings, Sari [Queen' s Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ (United Kingdom); Harrison, David J. [Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Meehan, Richard R., E-mail: Richard.Meehan@hgu.mrc.ac.uk [MRC Human Genetics Unit, IGMM, Western General Hospital, Edinburgh EH4 2XU (United Kingdom); Breakthrough Research Unit, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU (United Kingdom)

    2011-04-01

    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  17. Apoptosis and DNA Methylation

    Directory of Open Access Journals (Sweden)

    Richard R. Meehan

    2011-04-01

    Full Text Available Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG.

  18. Infrequent methylation of the DUSP6 phosphatase in endometrial cancer.

    Science.gov (United States)

    Chiappinelli, Katherine B; Rimel, B J; Massad, L Stewart; Goodfellow, Paul J

    2010-10-01

    Dual-specificity phosphatase six (DUSP6, MKP3, or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on ERK-2 (MAPK1) to inactivate the ERK-2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. DNA methylation in the first intron of DUSP6 abrogates expression in a subset of pancreatic cancers. We sought to determine whether DUSP6 was similarly silenced by methylation in endometrial cancer, a tumor type in which there is frequent activation of the ERK pathway. One hundred and nine endometrial cancers were analyzed for DUSP6 methylation using combined bisulfite restriction analysis (COBRA). The cohort included 70 primary endometrioid endometrial cancers, 21 primary endometrial tumors of adverse histological types, and 18 endometrial cancer cell lines. Primary tumors, cell lines, and normal endometrial tissues were analyzed for DUSP6 mRNA levels using quantitative RT-PCR and pERK levels by Western blots and/or immunohistochemistry. Methylation of the first intron of the DUSP6 gene was seen in 1/91 primary endometrial cancers investigated. The methylated tumor was also methylated at the more 5' regulatory region of DUSP6. Q-RT-PCR revealed that DUSP6 transcript levels varied widely in primary endometrial tumors. DUSP6 mRNA levels did not correlate with pERK status in primary tumors, consistent with the existence of negative feedback loops activated by pERK that result in transcription of DUSP6. DUSP6 methylation is a rare event in endometrial cancer. Silencing of the DUSP6 phosphatase is unlikely to contribute to constitutive activation of the ERK kinase cascade in endometrial cancer. Copyright © 2010 Elsevier Inc. All rights reserved.

  19. Methylated DNA is over-represented in whole-genome bisulfite sequencing data

    Directory of Open Access Journals (Sweden)

    Lexiang eJi

    2014-10-01

    Full Text Available The development of whole-genome bisulfite sequencing (WGBS has led to a number of exciting discoveries about the role of DNA methylation leading to a plethora of novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced to the point that the bottleneck for experiments that use WGBS has shifted to data analysis and interpretation. Here we present empirical evidence for an over-representation of reads from methylated DNA in WGBS. This enrichment for methylated DNA is exacerbated by higher cycles of PCR and is influenced by the type of uracil-insensitive DNA polymerase used for amplifying the sequencing library. Future efforts to computationally correct for this enrichment bias will be essential to increasing the accuracy of determining methylation levels for individual cytosines. It is especially critical for studies that seek to accurately quantify DNA methylation levels in populations that may segregate for allelic DNA methylation states.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. Quality evaluation of methyl binding domain based kits for enrichment DNA-methylation sequencing.

    Directory of Open Access Journals (Sweden)

    Tim De Meyer

    Full Text Available DNA-methylation is an important epigenetic feature in health and disease. Methylated sequence capturing by Methyl Binding Domain (MBD based enrichment followed by second-generation sequencing provides the best combination of sensitivity and cost-efficiency for genome-wide DNA-methylation profiling. However, existing implementations are numerous, and quality control and optimization require expensive external validation. Therefore, this study has two aims: 1 to identify a best performing kit for MBD-based enrichment using independent validation data, and 2 to evaluate whether quality evaluation can also be performed solely based on the characteristics of the generated sequences. Five commercially available kits for MBD enrichment were combined with Illumina GAIIx sequencing for three cell lines (HCT15, DU145, PC3. Reduced representation bisulfite sequencing data (all three cell lines and publicly available Illumina Infinium BeadChip data (DU145 and PC3 were used for benchmarking. Consistent large-scale differences in yield, sensitivity and specificity between the different kits could be identified, with Diagenode's MethylCap kit as overall best performing kit under the tested conditions. This kit could also be identified with the Fragment CpG-plot, which summarizes the CpG content of the captured fragments, implying that the latter can be used as a tool to monitor data quality. In conclusion, there are major quality differences between kits for MBD-based capturing of methylated DNA, with the MethylCap kit performing best under the used settings. The Fragment CpG-plot is able to monitor data quality based on inherent sequence data characteristics, and is therefore a cost-efficient tool for experimental optimization, but also to monitor quality throughout routine applications.

  3. Methylated β-Cyclodextrins

    DEFF Research Database (Denmark)

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

    2011-01-01

    groups at O2 promote complexation by extending the hydrophobic cavity. Like in the case of 2-hydroxypropylated cyclodextrins, the methyl substituents cause an increased release of ordered water from the hydration shell of the bile salts, resulting in a strong increase in both the enthalpy and the entropy...

  4. Methylated β-Cyclodextrins

    DEFF Research Database (Denmark)

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

    2011-01-01

    groups at O2 promote complexation by extending the hydrophobic cavity. Like in the case of 2-hydroxypropylated cyclodextrins, the methyl substituents cause an increased release of ordered water from the hydration shell of the bile salts, resulting in a strong increase in both the enthalpy and the entropy...

  5. Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

    Directory of Open Access Journals (Sweden)

    Bo Li

    Full Text Available DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ and asthenozoospermia (AZ usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control, with only complete methylation and mild hypomethylation(0.05. However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05% and AZ (0.58±1.77% (P20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05. In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

  6. Deoxyribonucleic acid (DNA) methyltransferase contributes to p16 promoter CpG island methylation in lung adenocarcinoma with smoking.

    Science.gov (United States)

    Sun, Rongju; Liu, Jiahong; Wang, Bo; Ma, Lingyun; Quan, Xiaojiao; Chu, Zhixiang; Li, Tanshi

    2015-01-01

    In this study, the relationship between CpG island methylation and smoking and DNA methyltransferase in the occurrence and development of lung adenocarcinoma was explored by detecting p16 promoter methylation status. Protein and mRNA levels of p16 were detected by immunohistochemistry and in situ hybridization assays. p16 gene promoter and exon 1 CpG island locus Hap II sites methylation status was analyzed with the methylation-specific PCR. Only 4 of 40 p16-positive cases were detected to methylate on CpG islands with 10% methylating rate whereas 18 of p16-negative cases were methylated up to 36.73% of methylating rate. The methylating rates of both p16-positive and p16-negative groups were significantly different. 17 of 50 cases with smoking from total 89 lung adenocarcinoma cases were detected to methylate on CpG islands while only 5 of the remaining 39 non-smokers to methylate. The difference of the methylating rates in both smokers and non-smokers was significant to suggest the closely association of CpG island methylation of p16 with smoking. Furthermore, p16 promoter CpG islands were detected to methylate in 15 of 35 cases with higher DNA methyltransferase activity whereas only 7 detected to methylate in the remaining 54 cases with lower DNA methyltransferase activity. p16 promoter CpG island methylation likely made p16 expressing silence thus contributed to the tumorigenesis of lung adenocarcinoma. Smoking is likely to promote p16 CpG island methylation or by its effect of the activity and metabolism of DNA methyltransferase 1 (DNMT) on CpG island methylation status.

  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. Association between Long Interspersed Nuclear Element-1 Methylation and Relative Telomere Length in Wilms Tumor

    Institute of Scientific and Technical Information of China (English)

    Hui-Bo Chang; Ji-Zhen Zou; Cai He; Rui Zeng; Yuan-Yuan Li; Fei-Fei Ma; Zhuo Liu

    2015-01-01

    Background: DNA hypomethylation of long interspersed nuclear elements-1 (LINEs-1) occurs during carcinogenesis, whereas information addressing LINE-1 methylation in Wilms tumor (WT) is limited.The main purpose of our study was to quantify LINE-1 methylation levels and evaluate their relationship with relative telomere length (TL) in WT.Methods: We investigated LINE-1 methylation and relative TL using bisulfite-polymerase chain reaction (PCR) pyrosequencing and quantitative PCR, respectively, in 20 WT tissues, 10 normal kidney tissues and a WT cell line.Significant changes were analyzed by t-tests.Results: LINE-1 methylation levels were significantly lower (P < 0.05) and relative TLs were significantly shorter (P < 0.05) in WT compared with normal kidney.There was a significant positive relationship between LINE-1 methylation and relative TL in WT (r =0.671,P =0.001).LINE-1 Methylation levels were significantly associated with global DNA methylation (r =0.332, P < 0.01).In addition, relative TL was shortened and LINE-1 methylation was decreased in a WT cell line treated with the hypomethylating agent 5-aza-2'-deoxycytidine compared with untreated WT cell line.Conclusion: These results suggest that LINE-1 hypomethylation is common and may be linked to telomere shortening in WT.

  9. DNA Methylation Profiles of Protease Nexin 1 (SERPINE2) Gene in Human Cell Lines

    Institute of Scientific and Technical Information of China (English)

    Shan Gao; Peter A. Andreasen

    2011-01-01

    Objective: To investigated whether epigenetic mechanisms contribute to the variable expression of variable protease nexin1(PN-1) encoded by the SERPINE2 gene in different cell types. Methods: Working with 5 human cell lines, we determined the CpG methylation status within two CpG islands in the SERPINE2 gene by bisulphate sequencing and the PN-1 mRNA level by Q-RT PCR. Results: A CpG island spanning the transcription initiation site showed little methylation in 3 of the cell lines and substantial methylation in 2 of the cell lines. A CpG island covering the translation starting site showed full methylation in all investigated cell lines. Methylation within the CpG island was not randomly distributed, but showed accumulation at specific sites. However, we were not able to distinguish any patterns which related the methylation frequency to the gene expression level. Inhibition of CpG methylation with 5-aza-2′-deoxycytidine led to a several fold increase in PN-1 mRNA levels, but based on the results on CpG methylation in the CpG island spanning the transcript, the effect is most likely indirect. Conclusion: We have carefully mapped the CpG methylation pattern in two CpG islands in the 5′ part of the SERPINE2 gene without finding any obvious inverse correlation between methylation frequency and expression level.

  10. Association between GSTP1 CpG methylation and the early phase of lead exposure.

    Science.gov (United States)

    Li, Chunping; Yang, Xiaolin; Xu, Ming; Zhang, Jinlong; Sun, Na

    2014-02-01

    GSTP1 is induced by lead, and thus serves as a biomarker of lead exposure. Lead exposure changes DNA methylation status. We attempted to prove that the methylation of the GSTP1 promoter plays an important role in lead toxicity. We conducted a case-control study of 53 workers from a battery plant and 53 age and sex matched healthy volunteers to determine whether the methylation level of the GSTP1 promoter is associated with the risk of lead poisoning. We employed methylation-specific PCR (MSP) in cell models to determine the relationship between the GSTP1 methylation level and lead exposure. We found no association between GSTP1 methylation and lead exposure. The difference in methylation frequencies between the exposure group and the controls was not statistically significant (p = 0.401), and individuals with the methylated GSTP1 gene was not associated with the risk of lead poisoning (adjusted OR = 1.36, 95% CI, 0.22-8.24). This study suggests that GSTP1 methylation is not involved in the early phase of lead toxicity. Further studies should be performed to detect the association between GSTP1 methylation and the risk of lead poisoning in later phases.

  11. LINE-1 Methylation Status Correlates Significantly to Post-Therapeutic Recurrence in Stage III Colon Cancer Patients Receiving FOLFOX-4 Adjuvant Chemotherapy.

    Directory of Open Access Journals (Sweden)

    Yun-Ting Lou

    Full Text Available Methylation levels of long interspersed nucleotide elements (LINE-1 are representative of genome-wide methylation status and crucial in maintaining genomic stability and expression. Their prognostic impact on colon cancer patients receiving adjuvant chemotherapy has not been well established. We evaluated the association between LINE-1 methylation status and clinicopathologic features and postoperative oncological outcomes in stage III colon cancer patients.129 UICC stage III colon cancer patients who had received radical resection and FOLFOX adjuvant chemotherapy were enrolled. Global methylation was estimated by analyzing tumor LINE-1 methylation status using bisulfite-polymerase chain reaction (PCR and pyrosequencing assay. Demographics, clinicopathological data, and postoperative outcomes were recorded by trained abstractors. Outcome measurements included postoperative recurrence and disease-free survival. Univariate, multivariate, and survival analyses were conducted to identify prognostic factors of oncological outcomes.The LINE-1 methylation of all 129 patients was measured on a 0-100 scale (mean 63.3; median 63.7, standard deviation 7.1, LINE-1 hypomethylation was more common in patients aged 65 years and above (61.7%±7.6% vs. 64.6±6.4, p=0.019 and those with post-therapeutic recurrence (61.7±7.4 vs 64.3±6.7, p=0.041. Considering risk adjustment, LINE-1 hypomethylation was found to be an independent risk factor of post-therapeutic recurrence (Adjusted OR=14.1, p=0.012. Kaplan-Meier analysis indicated that patients in the low methylation group had shorter period of disease free survival (p=0.01. In a stratified analysis that included 48 patients with post-therapeutic recurrence, it was found that those who experienced shorter period of disease free survival (≦6 months appeared to have lower LINE-1 methylation levels than patients who reported of recurrence after 6 months (56.68±15.75 vs. 63.55±7.57, p=0.041.There was a

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

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    Weissfeld Joel L

    2007-05-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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

  13. Critical role of △DNMT3B4/2 in regulating RASSF1A promoter specific DNA methylation in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    WANG Shu-hang; LIU Nin-hong; WANG Jie; BAI Hua; MAO Li

    2008-01-01

    Background △DNMT3B (a new DNMT3B subfamily) expression is initiated through a novel promoter.We identified at least 7 transcription variants of △DNMT3B as a result of alternative pre-mRNA processing.The aim of this study was to detect the expression pattern of △DNMT3B variants in non-small cell lung cancer (NSCLC) and to explore the role of △DNMT3B variants in regulating the promoter-specific DNA methylation.Methods Specific polymerase chain reaction (PCR) primer sets were designed to distinguish individual △DNMT3B variants according to their splicing pattems.The expressions of seven △DNMT3B variants were measured in 13 cell lines,109 NSCLC patients,and the corresponding normal lung tissues using reverse transcription-PCR (RT-PCR).The status of the p16 and RASSF1A promoter methylations in the tumors was detected using a methylation specific PCR (MSP).The relationships of the expression patterns of the △DNMT3B variants were analyzed by observing the status of p16 and RASSF1A promoter methylations in the tumors.The siRNA and the anti-sense oligo-dioxynucleotide specifically targeting the junction of exon 5 and 7 of △DNMT3B were designed and transfected by lipofectmane 2000 into H1299 and H358 cell lines.RASSF1A promoter methylation from cells treated by siRNA-△DNMT3B4/2 was detected using MSP and Bisulfite sequencing,and Western blotting was used to detect the protein expression of DNMT3B and △DNMT3B.Cell growth and cell cycle distribution were measured by applying real-time cell growth analysis and flowcytometry,respectively.Results △DNMT3B variants,not DNMT3B,were the predominant transcripts in both NSCLC cell lines and primary tumors.The expression of △DNMT3B4 strongly correlated to the promoter methylation status of RASSFIA in a primary NSCLC.The knockdown of △DNMT3B4/2 by RNA-interference or anti-sense approaches resulted in a complete demethylation of RASSF1A promoter with the reactJvation of a RASSFIA gene expression in less than 12

  14. Identification of new differentially methylated genes that have potential functional consequences in prostate cancer.

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    Jin W Kim

    Full Text Available Many differentially methylated genes have been identified in prostate cancer (PCa, primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19 and adjacent normal tissues (n = 4 and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05, defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa. Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.

  15. Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism

    DEFF Research Database (Denmark)

    Hofman-Bang, Jacob; Gravesen, Eva; Olgaard, Klaus

    2012-01-01

    R in parathyroid cultures decreases rapidly. Methylation of promoter regions is often detected during epigenetic downregulation of gene expression. Therefore, using an experimental rat model, we examined changes in methylation levels of parathyroid CaR and VDR promoters in vivo and in vitro. Methods. Uremia...... was induced by 5/6 nephrectomy. Melting temperature profiling of CaR and VDR PCR products after bisulfite treatment of genomic DNA from rat parathyroids was performed. Real-time PCR measured expression of PTH, CaR, VDR, and klotho genes in vitro. Results. Parathyroids from uremic rats had similar low levels...... of methylation in vivo and in vitro. In culture, a significant downregulation of CaR, VDR, and klotho within two hours of incubation was observed, while housekeeping genes remained stable for 24 hours. Conclusion. In uremic s-HPT and in vitro, no overall changes in methylation levels in the promoter regions...

  16. Placental leptin gene methylation and macrosomia during normal pregnancy.

    Science.gov (United States)

    Xu, Xinyun; Yang, Xinjun; Liu, Ziwei; Wu, Kele; Liu, Zheng; Lin, Chong; Wang, Yuhuan; Yan, Hongtao

    2014-03-01

    The present study examined the placental leptin (LEP) DNA methylation and mRNA levels in macrosomic infants from normal pregnancies. In total, 49 neonates with macrosomia, i.e., high birth weights of ≥ 4,000 g, and 52 neonates with normal birth weights between 2,500 g and 4,000 g were recruited from The Second Affiliated Hospital of Wenzhou Medical University (Wenzhou, Zhejiang) in China. Placental LEP promoter methylation and LEP transcript levels were determined by Sequenom MassARRAY and quantitative PCR, respectively. LEP promoter methylation and mRNA levels were not significantly different between the individuals with macrosomia and the controls. However, stratification revealed that individual CpG dinucleotides were hypermethylated in macrosomia (Pmacrosomia following a normal pregnancy and under certain conditions. However, placental LEP expression was not affected.

  17. Profiling DNA Methylation and Hydroxymethylation at Retrotransposable Elements.

    Science.gov (United States)

    de la Rica, Lorenzo; Stanley, Jatinder S; Branco, Miguel R

    2016-01-01

    DNA methylation is a key epigenetic modification controlling the transcriptional activity of mammalian retrotransposable elements. Its oxidation to DNA hydroxymethylation has been linked to DNA demethylation and reactivation of retrotransposons. Here we describe in detail protocols for three methods to measure DNA methylation and hydroxymethylation at specific genomic targets: glucMS-qPCR, and two sequencing approaches (pyrosequencing and high-throughput sequencing) for analyzing bisulfite- and oxidative bisulfite-modified DNA. All three techniques provide absolute measurements of methylation and hydroxymethylation levels at single-base resolution. Differences between the methods are discussed, mainly with respect to throughput and target coverage. These constitute the core techniques that are used in our laboratory for accurately surveying the epigenetics of retrotransposable elements.

  18. 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.

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

    Science.gov (United States)

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

    2017-01-14

    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.

  20. Pros and cons of methylation-based enrichment methods for ancient DNA

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio

    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 contai...

  1. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  2. O-Methyl cyclolaudenol

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    Nisar Hussain

    2009-06-01

    Full Text Available The title compound (systematic name: 3-methoxy-24-methyl-9,19-cyclolanost-25-ene, C32H54O, is a triterpenoid which has been isolated from Skimmia laureola. The three six-membered rings adopt chair, slightly distorted half-chair and distorted boat conformations, and the five-membered ring adopts an envelope conformation. All the rings are trans fused.

  3. Epigenetic Patterns of PTSD: DNA Methylation In Serum of OIF/OEF Servicemembers

    Science.gov (United States)

    2011-01-01

    2116-2120. 54. Fenech M (2001): The role of folic acid and Vitamin B12 in genomic stability of human cells. Mutation Research. 475:57-67. 55...and PTSD, (4, 33-35) to our knowledge , this is the first study to investigate the association between DNA methylation in repetitive elements (LINE-1...simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res. 32:e38. 17. Robertson KD (2005

  4. Are Independent Fiscal Institutions Really Independent?

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    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.

  5. Abnormal promoter methylation of multiple genes in the malignant transformed PEP2D cells induced by alpha particles exposure

    Institute of Scientific and Technical Information of China (English)

    LiP; SuiJL

    2002-01-01

    The 5' promoter regions of some genes contain CpG-rich areas,known as CpG islands,Methylation of the cytosine in these dinuleotides has important regulatory effects on gene expression.The functional significance of promoter hypermethylation would play the same roles in carcinogenesis as gene mutations.The promoter methylations p14ARF,p16INK4a,MGMT,GSTP1,BUB3 and DAPK genes were analyzed with methylation specific PCR(MSP) in the transformed human bronchial epithelial cells(BEP2D) induced by α-particles.The results indicated that p14ARF gene was not methylated in BEP2D cells,but was methylated in the malignant transformed BERP35T-1 cells,and the level of its transcription was depressed remarkable in the latter.However p16INK4a gene,which shares two exons with p14ARF gene,was not methylated.MGMT gene was methylated in both BEP2D and BERP35T-1.DAPK gene was partially methylated in BEP2D cells and methylated completely in BERP35T1.GSTP1 was not methylated in BEP2D cells and was methylated partly in BERP35T-1.BUB3 gene was not methylated in BEP2D as well as BERP35T1 cells and was further proved by sequencing analysis.

  6. 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.

  7. Methylation in hepatocellular carcinoma

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    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

  8. Helicobacter pylori and EBV in gastric carcinomas:Methylation status and microsatellite instability

    Institute of Scientific and Technical Information of China (English)

    Adriana; Camargo; Ferrasi; Nídia; Alice; Pinheiro; Silvia; Helena; Barem; Rabenhorst; Otávia; Luisa; Caballero; Maria; Aparecida; Marchesan; Rodrigues; Fabrício; de; Carvalho; Celso; Vieira; de; Souza; Leite; Marcia; Valéria; Pitombeira; Ferreira; Marcos; Aurélio; Pessoa; Barros; Maria; Inês; de; Moura; Campos; Pardini

    2010-01-01

    AIM:To verify the methylation status of CDH1, DAPK, COX2, hMLH1 and CDKN2A genes and to evaluate their association with Helicobacter pylori (H. pylori)-cagA+ and Epstein Barr virus (EBV) infections in gastric adenocarcinomas.METHODS: Methylation-specific PCR (MSP) assay was performed in 89 primary gastric carcinomas (intestinal and diffuse types). Microsatellite instability (MSI) analysis was performed using the BAT26 primer set and PCR products were analyzed with the ABI PRISM 3100 Genetic Analyzer using G...

  9. Neurocryptococcosis: diagnosis by PCR method.

    Science.gov (United States)

    Paschoal, Regina Célia; Hirata, Mário Hiroyuki; Hirata, Rosário Crespo; Melhem, Márcia de Souza Carvalho; Dias, Amanda Latercia Tranches; Paula, Claudete Rodrigues

    2004-01-01

    Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.

  10. Methylation profiling using methylated DNA immunoprecipitation and tiling array hybridization.

    Science.gov (United States)

    Cheung, Hoi-Hung; Lee, Tin-Lap; Rennert, Owen M; Chan, Wai-Yee

    2012-01-01

    DNA methylation is an important epigenetic modification that regulates development and plays a role in the pathophysiology of many diseases. It is dynamically changed during germline development. Methylated DNA immunoprecipitation (MeDIP) is an efficient, cost-effective method for locus-specific and genome-wide analysis. Methylated DNA fragments are enriched by a 5-methylcytidine-recognizing antibody, therefore allowing the analysis of both CpG and non-CpG methylation. The enriched DNA fragments can be amplified and hybridized to tiling arrays covering CpG islands, promoters, or the entire genome. Comparison of different methylomes permits the discovery of differentially methylated regions that might be important in disease- or tissue-specific expression. Here, we describe an established MeDIP protocol and tiling array hybridization method for profiling methylation of testicular germ cells.

  11. Multiplexed Methylation Profiles of Tumor Suppressor Genes in Bladder Cancer

    Science.gov (United States)

    Cabello, Maria José; Grau, Laura; Franco, Noreli; Orenes, Esteban; Alvarez, Miguel; Blanca, Ana; Heredero, Oscar; Palacios, Alberto; Urrutia, Manuel; Fernández, Jesus María; López-Beltrán, Antonio; Sánchez-Carbayo, Marta

    2011-01-01

    Changes in DNA methylation of tumor suppressors can occur early in carcinogenesis and are potentially important early indicators of cancer. The objective of this study was to assess the methylation of 25 tumor suppressor genes in bladder cancer using a methylation-specific (MS) multiplex ligation-dependent probe amplification assay (MLPA). Initial analyses in bladder cancer cell lines (n = 14) and fresh-frozen primary bladder tumor specimens (n = 31) supported the panel of genes selected being altered in bladder cancer. The process of MS-MLPA was optimized for its application in body fluids using two independent training and validation sets of urinary specimens (n = 146), including patients with bladder cancer (n = 96) and controls (n = 50). BRCA1 (71.0%), WT1 (38.7%), and RARB (38.7%) were the most frequently methylated genes in bladder tumors, with WT1 methylation being significantly associated with tumor stage (P = 0.011). WT1 and PAX5A were identified as methylated tumor suppressors. In addition, BRCA1, WT1, and RARB were the most frequently methylated genes in urinary specimens. Receiver operating characteristic curve analyses revealed significant diagnostic accuracies in both urinary sets for BRCA1, RARB, and WT1. The novelty of this report relates to applying MS-MLPA, a multiplexed methylation technique, for tumor suppressors in bladder cancer and body fluids. Methylation profiles of tumor suppressor genes were clinically relevant for histopathological stratification of bladder tumors and offered a noninvasive diagnostic strategy for the clinical management of patients affected with uroepithelial neoplasias. PMID:21227392

  12. Event extraction for DNA methylation

    Directory of Open Access Journals (Sweden)

    Ohta Tomoko

    2011-10-01

    Full Text Available Abstract Background We consider the task of automatically extracting DNA methylation events from the biomedical domain literature. DNA methylation is a key mechanism of epigenetic control of gene expression and implicated in many cancers, but there has been little study of automatic information extraction for DNA methylation. Results We present an annotation scheme for DNA methylation following the representation of the BioNLP shared task on event extraction, select a set of 200 abstracts including a representative sample of all PubMed citations relevant to DNA methylation, and introduce manual annotation for this corpus marking nearly 3000 gene/protein mentions and 1500 DNA methylation and demethylation events. We retrain a state-of-the-art event extraction system on the corpus and find that automatic extraction of DNA methylation events, the methylated genes, and their methylation sites can be performed at 78% precision and 76% recall. Conclusions Our results demonstrate that reliable extraction methods for DNA methylation events can be created through corpus annotation and straightforward retraining of a general event extraction system. The introduced resources are freely available for use in research from the GENIA project homepage http://www-tsujii.is.s.u-tokyo.ac.jp/GENIA.

  13. 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.

  14. 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.

  15. Buoyancy-Driven Polymerase Chain Reaction (PCR) Devices

    Energy Technology Data Exchange (ETDEWEB)

    Ness, K D; Wheeler, E K; Benett, W; Stratton, P; Christian, A; Chen, A; Ortega, J; Weisgraber, T H; Goodson, K E

    2004-09-28

    Polymerase chain reaction (PCR) facilitates DNA detection by significantly increasing the concentration of specific DNA segments. A new class of PCR instruments uses a buoyancy-driven re-circulating flow to thermally cycle the DNA sample and benefits from reduced cycle times, low sample volumes, a miniaturized format, and low power consumption. This paper analyzes a specific buoyancy PCR device in a micro-channel ''race-track'' geometry to determine key parameters about PCR cycle times and other figures of merit as functions of device dimensions. The 1-D model balances the buoyancy driving force with frictional losses. A hydrostatic pressure imbalance concept is used between the left and right sides of the fluid loop to calculate the buoyancy driving force. Velocity and temperature distributions within the channels are determined from two-dimensional analysis of the channel section, with developing region effects included empirically through scaled values of the local Nusselt number. Good agreement between four independent verification steps validate the 1-D simulation approach: (1) analytical expressions for the thermal entrance length are compared against, (2) comparison with a full 3-D finite element simulation, (3) comparison with an experimental flow field characterization, and (4) calculation of the minimum PCR runtime required to get a positive PCR signal from the buoyancy-driven PCR device. The 1-D approach closely models an actual buoyancy-driven PCR device and can further be used as a rapid design tool to simulate buoyancy PCR flows and perform detailed design optimizations studies.

  16. Peptidyl arginine deiminase 4 participates in the pathogenesis of rheumatoid arthritis by influencing histone methylation

    Institute of Scientific and Technical Information of China (English)

    郭靖

    2013-01-01

    Objective To explore the probable function of peptidyl arginine deiminase 4 (PAD4) in rheumatoid arthritis (RA) .Methods Real-time quantitative polymerase chain reaction (PCR) was used to determine the expression of PAD4 mRNA in peripheral blood mononucleated cells (PBMCs) from 60 RA patients and 40 healthy individuals.Asymmetric di-methylation of histone H3R17,symmetric di-methylation and mono-methylation of H4R3were semi-quantified by Western blotting in 12 patients with osteoarthritis (OA) ,26 patients with RA and 10

  17. Identification and characterization of locus specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis

    DEFF Research Database (Denmark)

    Wojdacz, Tomasz K; Windeløv, Johanne Agerlin; Thestrup, Britta Boserup;

    2014-01-01

    biomarkers for early cancer detection and stratification of patients. METHODS: We used ultrahigh-resolution microarrays to compare genomewide methylation patterns of breast carcinomas (n = 20) and nonmalignant breast tissue (n = 5). Biomarker properties of a subset of discovered differentially methylated....... Analysis of the screening results showed that all DMRs tested displayed significant gains of methylation in the cancer tissue compared to the levels in control tissue. Interestingly, we observed two types of locus-specific methylation, with loci undergoing either predominantly full or heterogeneous...... methylation during carcinogenesis. Almost all tested DMRs (17 of 19) displayed low-level methylation in nonmalignant breast tissue, independently of locus-specific methylation patterns in cases. CONCLUSIONS: Specific loci can undergo either heterogeneous or full methylation during carcinogenesis, and loci...

  18. 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.

  19. Techniques of DNA methylation analysis with nutritional applications.

    Science.gov (United States)

    Mansego, Maria L; Milagro, Fermín I; Campión, Javier; Martínez, J Alfredo

    2013-01-01

    Epigenetic mechanisms are likely to play an important role in the regulation of metabolism and body weight through gene-nutrient interactions. This review focuses on methods for analyzing one of the most important epigenetic mechanisms, DNA methylation, from single nucleotide to global measurement depending on the study goal and scope. In addition, this study highlights the major principles and methods for DNA methylation analysis with emphasis on nutritional applications. Recent developments concerning epigenetic technologies are showing promising results of DNA methylation levels at a single-base resolution and provide the ability to differentiate between 5-methylcytosine and other nucleotide modifications such as 5-hydroxymethylcytosine. A large number of methods can be used for the analysis of DNA methylation such as pyrosequencing™, primer extension or real-time PCR methods, and genome-wide DNA methylation profile from microarray or sequencing-based methods. Researchers should conduct a preliminary analysis focused on the type of validation and information provided by each technique in order to select the best method fitting for their nutritional research interests.

  20. Detection of expression of methylation-related genes in mouse early embryonic cells using pre-amplification-based real time quantitative PCR%预扩增qPCR方法检测少量小鼠早期胚胎细胞中DNA甲基化相关基因的表达

    Institute of Scientific and Technical Information of China (English)

    程琳; 孙平楠; 谢庆东; 周小玲

    2016-01-01

    目的:比较3种实时定量PCR(qPCR)方法对少量小鼠早期胚胎细胞中甲基化相关基因表达水平的检测效果,以期获得适合日常检测的方法.方法:分别应用常规qPCR方法、基于等温预扩增的qPCR方法、基于PCR预扩增的qPCR方法3种方案对少量小鼠早期胚胎细胞样本中甲基化相关基因TET1、TET2、TET3、DNMT3A的mRNA表达进行检测.结果:3种方法的灵敏度由高到低依次为基于等温预扩增的qPCR方法、基于PCR预扩增qPCR方法、常规qPCR方法.前两种方法均能在合适的循环数下对少量小鼠胚胎细胞中的多个甲基化相关基因表达进行定量.而基于PCR预扩增的qPCR方法比基于等温预扩增的qPCR方法更经济,适合日常检测.因此,我们选择应用基于PCR预扩增的qPCR方法,检测了小鼠卵细胞以及卵细胞受精后22 h甲基化相关基因表达的变化,结果发现该过程中TET1表达量很低,不易检测到;TET2表达呈降低趋势;TET3和DNMT3A表达显著升高(P<0.01),与文献报道基本一致.结论:基于等温预扩增的qPCR方法检测少量细胞样品中甲基化相关基因的表达的灵敏度在3种qPCR方法中最高.而基于PCR预扩增的qPCR方法操作简单、灵敏度较高、成本相对低,适合少量细胞样本中多个相关基因表达的实时定量日常检测.

  1. Tissue factor expression and methylation regulation in differentiation of embryonic stem cells into trophoblast

    Institute of Scientific and Technical Information of China (English)

    Lin-Xin Liu; Hui Zeng; En-Yi Liu; Fang-Ping Chen

    2014-01-01

    Objective:To explore tissue factor(TF) expression and methylation regulation in differentiation of human embryonic stem cells(hESCs) into trophoblast.Methods:Differentiation of hESCs into trophoblast was induced by bone morphogenetic protein4(BMP4).Expression of gene, protein of TF andDNA methylation at different time points during induction process was detected byRT-PCT,Western blot, flow cytometry andMSP-PCR method.Results:The expression of mRNA, protein level ofTF could be detected during directional differentiation of hESCs to trophoblast cells, semi methylation-semi non methylation expression appeared atTFDNA promoter region, and it showed decreased methylation level and increased non methylation level with formation of trophoblast cell and increased expression ofTF.Conclusions:It shows that during differentiation of hESCs into trophoblast, the differential expression ofTF is related withDNA methylation level, and it is changed with the methylation or non methylated degree.It provids new platform to furtherly explore the regulation mechanisms of specific expression of tissue factor in the process of the embryonic stem cell development.

  2. Epigenetic marker (LINE-1 promoter) methylation level was associated with occupational lead exposure.

    Science.gov (United States)

    Li, Chunping; Yang, Xiaolin; Xu, Ming; Zhang, Jinlong; Sun, Na

    2013-05-01

    Occupational and environmental exposures to lead (Pb) are a worldwide concern. DNA methylation plays an important role in the development of Pb toxicity. Here, we try to find out the evidence to prove that the methylation of the LINE-1 promoter may be involved in Pb toxicity. To determine whether the methylation level of the LINE-1 is associated with the risk of Pb poisoning, we first constructed a Pb acetate-treated cell model to detect the association between LINE-1 methylation and Pb exposure. A case-control study involving 53 workers from a battery plant and 57 healthy volunteers with matching age and gender distribution was carried out. We employed methylation-specific real-time PCR to determine the relationship between LINE-1 methylation level and Pb exposure. In the cell model, Pb exposure significantly decreased the level of LINE-1 methylation (p = 0.009). Significant difference in methylation frequencies was found between the exposed and control samples (p promoter methylation might contribute to the risk of Pb poisoning and identified a possible epigenetic biomarker for Pb toxicity, especially in individuals occupationally exposed to Pb.

  3. 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.

  4. A high-throughput DNA methylation analysis of a single cell.

    Science.gov (United States)

    Kantlehner, Martin; Kirchner, Roland; Hartmann, Petra; Ellwart, Joachim W; Alunni-Fabbroni, Marianna; Schumacher, Axel

    2011-04-01

    In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.

  5. Expression and methylation pattern of p16 in neuroblastoma tumorigenesis.

    Science.gov (United States)

    Aktas, Safiye; Celebiler, Aydan Cavusoglu; Zadeoğlulari, Zeynep; Diniz, Gulden; Kargi, Aydanur; Olgun, Nur

    2010-03-01

    Understanding migration, population and differentiation of primordial neural crest cells will help in evolving biology of neuroblastoma. P16 is a tumour suppressor gene contributing in cell cycle arrest as cyclin dependent kinase inhibitor. Methylation is an important mechanism for silencing tumor suppressor genes. The aim of this study was to evaluate the role of p16 and its methylation pattern in neuroblastoma tumorigenesis. This study included 23 cases (11 male; 12 female) and 31 samples from archival paraffin embedded tissues. P16 was studied in 5 samples of normal adrenal medullar tissue, 5 samples of adrenal tissue including blastic rests, 5 samples of neuroblastoma in situ tissue and in 8 samples of neuroblastoma tissues primary and after chemotherapy in each group. The adrenal gland tissues were obtained from paediatric autopsy cases. Expression of p16 was searched by immunohistochemistry. Methylation specific PCR was used to detect the methylation rate of p16. The age range of autopsy cases was between 20 weeks of foetal age and 36 months of infant age. The mean age of neuroblastoma cases was 45 months. P16 expression was positive in normal adrenal tissues, in one of 5 samples of adrenal blastic rest tissue and in all of samples of after chemotherapy; while no expression was observed in neuroblastoma and neuroblastoma in situ tissues. P16 methylation was observed in samples of neuroblastoma in situ and primary neuroblastoma tissues. Our results suggest that p16 and its methylation seems to play role in neuroblastoma tumorigenesis and in the migration, population and differentiation of primordial neural crest cells. Inhibitors of DNA methylation may provide a useful tool for restoring p16 activity in neuroblastoma treatment.

  6. Role of DNA Methylation in Type 2 Diabetes Etiology: Using Genotype as a Causal Anchor.

    Science.gov (United States)

    Elliott, Hannah R; Shihab, Hashem A; Lockett, Gabrielle A; Holloway, John W; McRae, Allan F; Smith, George Davey; Ring, Susan M; Gaunt, Tom R; Relton, Caroline L

    2017-06-01

    Several studies have investigated the relationship between genetic variation and DNA methylation with respect to type 2 diabetes, but it is unknown if DNA methylation is a mediator in the disease pathway or if it is altered in response to disease state. This study uses genotypic information as a causal anchor to help decipher the likely role of DNA methylation measured in peripheral blood in the etiology of type 2 diabetes. Illumina HumanMethylation450 BeadChip data were generated on 1,018 young individuals from the Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. In stage 1, 118 unique associations between published type 2 diabetes single nucleotide polymorphisms (SNPs) and genome-wide methylation (methylation quantitative trait loci [mQTLs]) were identified. In stage 2, a further 226 mQTLs were identified between 202 additional independent non-type 2 diabetes SNPs and CpGs identified in stage 1. Where possible, associations were replicated in independent cohorts of similar age. We discovered that around half of known type 2 diabetes SNPs are associated with variation in DNA methylation and postulated that methylation could either be on a causal pathway to future disease or could be a noncausal biomarker. For one locus (KCNQ1), we were able to provide further evidence that methylation is likely to be on the causal pathway to disease in later life. © 2017 by the American Diabetes Association.

  7. Real-time PCR in Food Science: Introduction.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Hernandez, Marta

    2013-01-01

    Food safety and quality control programmes are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostics have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

  8. Independent candidates in Mexico

    OpenAIRE

    Campos, Gonzalo Santiago

    2014-01-01

    In this paper we discuss the issue of independent candidates in Mexico, because through the so-called political reform of 2012 was incorporated in the Political Constitution of the Mexican United States the right of citizens to be registered as independent candidates. Also, in September 2013 was carried out a reform of Article 116 of the Political Constitution of the Mexican United States in order to allow independent candidates in each state of the Republic. However, prior to the constitutio...

  9. Central bank Financial Independence

    OpenAIRE

    J.Ramon Martinez-Resano

    2004-01-01

    Central bank independence is a multifaceted institutional design. The financial component has been seldom analysed. This paper intends to set a comprehensive conceptual background for central bank financial independence. Quite often central banks are modelled as robot like maximizers of some goal. This perspective neglects the fact that central bank functions are inevitably deployed on its balance sheet and have effects on its income statement. A financially independent central bank exhibits ...

  10. Methylation of PTCH1a gene in a subset of gastric cancers

    Institute of Scientific and Technical Information of China (English)

    Peng Du; Hai-Rong Ye; Jun Gao; Wei Chen; Zhong-Chuan Wang; Hong-Hua Jiang; Ji Xu; Ji-We Zhang; Jian-Cheng Zhang; Long Cui

    2009-01-01

    AIM: To establish if PTCH1a transcriptional regulation region (TRR) is methylated in gastric cancer and its influence in gastric tumorigenesis. METHODS: The CpG islands in PTCH1a TRR were analyzed by Methyl Primer Express v1.0 software. The region from -643 to -355 bp (the transcription initiation site of PTCH1a was designated as 0) that contained 19 CpG sites was chosen for bisulfitesequencing PCR (BSP) and methylation-specific PCR (MSP) detection. The gastric cancer cell line AGS was treated with 5-aza-2'-deoxycytidine (5-Aza-dC; 1 μmol/L) for 3 d. Alterations in PTCH1a TRR methylation in treated AGS cells was measured through BSP clone sequences, and their PTCH1 expression was measured by quantitative RT-PCR. The cell cycle and apoptosis were observed with flow cytometry through propidium iodide (PI) staining or annexin V/PI double staining. The prevalence of PTCH1a TRR methylation was investigated in 170 gastric cancer tissue samples and the adjacent normal tissues by MSP. The correlation of PTCH1a TRR methylation with PTCH1 expression or with patients' clinical features was analyzed. RESULTS: Methylation of PTCH1a TRR was observed in AGS cells and a subset of gastric cancer tissues (32%, 55/170), while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1a TRR was correlated negatively with PTCH1 expression (Spearman's r = -0.380, P = 0.000). However, methylation of PTCH1a TRR was not related to the gastric cancer patients' clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCH1a TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression (5.3 ± 2.5 times; n = 3) and apoptosis rate (3.0 ± 0.26 times; P < 0.05; n = 3). CONCLUSION: Methylation of PTCH1a TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in

  11. Identification of methylated genes associated with aggressive bladder cancer.

    Directory of Open Access Journals (Sweden)

    Carmen J Marsit

    Full Text Available Approximately 500,000 individuals diagnosed with bladder cancer in the U.S. require routine cystoscopic follow-up to monitor for disease recurrences or progression, resulting in over $2 billion in annual expenditures. Identification of new diagnostic and monitoring strategies are clearly needed, and markers related to DNA methylation alterations hold great promise due to their stability, objective measurement, and known associations with the disease and with its clinical features. To identify novel epigenetic markers of aggressive bladder cancer, we utilized a high-throughput DNA methylation bead-array in two distinct population-based series of incident bladder cancer (n = 73 and n = 264, respectively. We then validated the association between methylation of these candidate loci with tumor grade in a third population (n = 245 through bisulfite pyrosequencing of candidate loci. Array based analyses identified 5 loci for further confirmation with bisulfite pyrosequencing. We identified and confirmed that increased promoter methylation of HOXB2 is significantly and independently associated with invasive bladder cancer and methylation of HOXB2, KRT13 and FRZB together significantly predict high-grade non-invasive disease. Methylation of these genes may be useful as clinical markers of the disease and may point to genes and pathways worthy of additional examination as novel targets for therapeutic treatment.

  12. Gravity Independent Compressor Project

    Data.gov (United States)

    National Aeronautics and Space Administration — We propose to develop and demonstrate a small, gravity independent, vapor compression refrigeration system using a linear motor compressor which effectively...

  13. 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.

  14. DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

    Directory of Open Access Journals (Sweden)

    Andreas C Jenke

    Full Text Available BACKGROUND: Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs from 6 healthy individuals. MATERIALS AND METHODS: Epithelial cells were isolated from small bowel (i.e. terminal ileum biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP. RESULTS: While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples. CONCLUSIONS: Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.

  15. Temporal stability of epigenetic markers: sequence characteristics and predictors of short-term DNA methylation variations.

    Directory of Open Access Journals (Sweden)

    Hyang-Min Byun

    Full Text Available BACKGROUND: DNA methylation is an epigenetic mechanism that has been increasingly investigated in observational human studies, particularly on blood leukocyte DNA. Characterizing the degree and determinants of DNA methylation stability can provide critical information for the design and conduction of human epigenetic studies. METHODS: We measured DNA methylation in 12 gene-promoter regions (APC, p16, p53, RASSF1A, CDH13, eNOS, ET-1, IFNγ, IL-6, TNFα, iNOS, and hTERT and 2 of non-long terminal repeat elements, i.e., L1 and Alu in blood samples obtained from 63 healthy individuals at baseline (Day 1 and after three days (Day 4. DNA methylation was measured by bisulfite-PCR-Pyrosequencing. We calculated intraclass correlation coefficients (ICCs to measure the within-individual stability of DNA methylation between Day 1 and 4, subtracted of pyrosequencing error and adjusted for multiple covariates. RESULTS: Methylation markers showed different temporal behaviors ranging from high (IL-6, ICC = 0.89 to low stability (APC, ICC = 0.08 between Day 1 and 4. Multiple sequence and marker characteristics were associated with the degree of variation. Density of CpG dinucleotides nearby the sequence analyzed (measured as CpG(o/e or G+C content within ±200 bp was positively associated with DNA methylation stability. The 3' proximity to repeat elements and range of DNA methylation on Day 1 were also positively associated with methylation stability. An inverted U-shaped correlation was observed between mean DNA methylation on Day 1 and stability. CONCLUSIONS: The degree of short-term DNA methylation stability is marker-dependent and associated with sequence characteristics and methylation levels.

  16. Exposures in early life: associations with DNA promoter methylation in breast tumors.

    Science.gov (United States)

    Tao, M-H; Marian, C; Shields, P G; Potischman, N; Nie, J; Krishnan, S S; Berry, D L; Kallakury, B V; Ambrosone, C; Edge, S B; Trevisan, M; Winston, J; Freudenheim, J L

    2013-04-01

    There is evidence that epigenetic changes occur early in breast carcinogenesis. We hypothesized that early-life exposures associated with breast cancer would be associated with epigenetic alterations in breast tumors. In particular, we examined DNA methylation patterns in breast tumors in association with several early-life exposures in a population-based case-control study. Promoter methylation of E-cadherin, p16 and RAR-β2 genes was assessed in archived tumor blocks from 803 cases with real-time methylation-specific PCR. Unconditional logistic regression was used for case-case comparisons of those with and without promoter methylation. We found no differences in the prevalence of DNA methylation of the individual genes by age at menarche, age at first live birth and weight at age 20. In case-case comparisons of premenopausal breast cancer, lower birth weight was associated with increased likelihood of E-cadherin promoter methylation (OR = 2.79, 95% CI, 1.15-6.82, for ⩽2.5 v. 2.6-2.9 kg); higher adult height with RAR-β2 methylation (OR = 3.34, 95% CI, 1.19-9.39, for ⩾1.65 v. <1.60 m); and not having been breastfed with p16 methylation (OR = 2.75, 95% CI, 1.14-6.62). Among postmenopausal breast cancers, birth order was associated with increased likelihood of p16 promoter methylation. Being other than first in the birth order was inversely associated with likelihood of ⩾1 of the three genes being methylated for premenopausal breast cancers, but positively associated with methylation in postmenopausal women. These results suggest that there may be alterations in methylation associated with early-life exposures that persist into adulthood and affect breast cancer risk.

  17. RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in arabidopsis.

    Directory of Open Access Journals (Sweden)

    Simon W-L Chan

    2006-06-01

    Full Text Available Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes.

  18. Expression and DNA methylation analysis of SNRPN in Prader-Willi patients

    Energy Technology Data Exchange (ETDEWEB)

    Glenn, C.C.; Jong, M.T.C.; Driscoll, D.J. [Univ. of Florida, Gainesville, FL (United States)] [and others

    1994-09-01

    The human SNRPN gene is one of a gene family that encode proteins involved in pre-mRNA splicing and maps to the Prader-Willi syndrome critical region in 15q11-q13. We have previously demonstrated functional imprinting of SNRPN, with absent expression in PWS skin fibroblasts and lymphoblasts. We now show a similar lack of expression in blood of PWS patients, which appear to correlate with DNA methylation of NotI sites in the 5{prime} region of the gene. RNA and DNA was extracted from peripheral blood of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) deletion patients to be used for RT-PCR with SNRPN gene-specific primers and DNA methylation analysis. Either no or highly reduced levels of SNRPN RT-PCR product were detected in nine PWS samples but was present in normals, AS patients, and one clinically typical PWS patient. Parent-of-origin DNA methylation imprints are also present within the SNRPN gene. PWS patients having only a maternal contribution of SNRPN have several NotI restriction sites near the transcription start site which are methylated, while these same sites are unmethylated on the paternal chromosome (i.e., AS samples). Several CpG sites approximately 22 kb downstream of the transcription start site are methylated preferentially on the paternal allele. These observations for human SNRPN are similar to those of the mouse imprinted gene Igf2r, which exhibits DNA methylation of a CpG island 27 kb from the transcription start site on the expressed allele, and DNA methylation in the promoter region of the repressed allele. We suggest that RT-PCR and/or DNA methylation analysis from blood of PWS patients may be the most accurate means of diagnosing classical PWS. These results further indicate a role for SNRPN in the pathogenesis of PWS, and may serve as a model to study other human imprinted genes.

  19. Heterogeneous patterns of DNA methylation-based field effects in histologically normal prostate tissue from cancer patients.

    Science.gov (United States)

    Møller, Mia; Strand, Siri Hundtofte; Mundbjerg, Kamilla; Liang, Gangning; Gill, Inderbir; Haldrup, Christa; Borre, Michael; Høyer, Søren; Ørntoft, Torben Falck; Sørensen, Karina Dalsgaard

    2017-01-13

    Prostate cancer (PC) diagnosis is based on histological evaluation of prostate needle biopsies, which have high false negative rates. Here, we investigated if cancer-associated epigenetic field effects in histologically normal prostate tissue may be used to increase sensitivity for PC. We focused on nine genes (AOX1, CCDC181 (C1orf114), GABRE, GAS6, HAPLN3, KLF8, MOB3B, SLC18A2, and GSTP1) known to be hypermethylated in PC. Using quantitative methylation-specific PCR, we analysed 66 malignant and 134 non-malignant tissue samples from 107 patients, who underwent ultrasound-guided prostate biopsy (67 patients had at least one cancer-positive biopsy, 40 had exclusively cancer-negative biopsies). Hypermethylation was detectable for all genes in malignant needle biopsy samples (AUC: 0.80 to 0.98), confirming previous findings in prostatectomy specimens. Furthermore, we identified a four-gene methylation signature (AOX1xGSTP1xHAPLN3xSLC18A2) that distinguished histologically non-malignant biopsies from patients with vs. without PC in other biopsies (AUC = 0.65; sensitivity = 30.8%; specificity = 100%). This signature was validated in an independent patient set (59 PC, 36 adjacent non-malignant, and 9 normal prostate tissue samples) analysed on Illumina 450 K methylation arrays (AUC = 0.70; sensitivity = 40.6%; specificity = 100%). Our results suggest that a novel four-gene signature may be used to increase sensitivity for PC diagnosis through detection of epigenetic field effects in histologically non-malignant prostate tissue samples.

  20. 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…

  1. Independence of Internal Auditors.

    Science.gov (United States)

    Montondon, Lucille; Meixner, Wilda F.

    1993-01-01

    A survey of 288 college and university auditors investigated patterns in their appointment, reporting, and supervisory practices as indicators of independence and objectivity. Results indicate a weakness in the positioning of internal auditing within institutions, possibly compromising auditor independence. Because the auditing function is…

  2. Fostering Musical Independence

    Science.gov (United States)

    Shieh, Eric; Allsup, Randall Everett

    2016-01-01

    Musical independence has always been an essential aim of musical instruction. But this objective can refer to everything from high levels of musical expertise to more student choice in the classroom. While most conceptualizations of musical independence emphasize the demonstration of knowledge and skills within particular music traditions, this…

  3. Microarray-based method for detecting methylation changes of p16Ink4a gene 5'-CpG islands in gastric carcinomas

    Institute of Scientific and Technical Information of China (English)

    Peng Hou; Jia-Yao Shen; Mei-Ju Ji; Nong-Yue He; Zu-Hong Lu

    2004-01-01

    AIM: Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation of the CpG sites of the tumor suppressor gene is closely associated with carcinogenesis. However, large-scale analysis of candidate genes has so far been hampered by the lack of highthroughput approach for analyzing DNA methylation. The aim of this study was to describe a microarray-based method for detecting changes of DNA methylation in cancer.METHODS: This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Therefore, the amplified product might contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status.Nine sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of p16 gene CpG islands in gastric carcinomas. The results were further validated by methylation-specific PCR (MSP).RESULTS: The experimental results showed that the microarray assay could successfully detect methylation changes of p16 gene in 18 gastric tumor samples. Moreover,it could also potentially increase the frequency of detecting p16 methylation from tumor samples than MSP.CONCLUSION: Microarray assay could be applied as a useful tool for mapping methylation changes in multiple CpG loci and for generating epigenetic profiles in cancer.

  4. On Background Independence

    CERN Document Server

    Anderson, Edward

    2013-01-01

    This paper concerns what Background Independence itself is (as opposed to some particular physical theory that is background independent). The notions presented mostly arose from a layer-by-layer analysis of the facets of the Problem of Time in Quantum Gravity. Part of this coincides with two relational postulates which are thus identified as classical precursors of two of the facets of the Problem of Time. These are furthemore tied to the forms of each of the GR Hamiltonian and momentum constraints. Other aspects of Background Independence include the algebraic closure of these constraints, expressing physics in terms of beables, foliation independence as implemented by refoliation invariance, the reconstruction of spacetime from space. The final picture is that Background Independence - a philosophically desirable and physically implementable feature for a theory to have - has the facets of the Problem of Time among its consequences. Thus these arise naturally and are problems to be resolved, as opposed to ...

  5. 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.

  6. Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1 is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

    Directory of Open Access Journals (Sweden)

    Meyer-Schwesinger Catherine

    2011-10-01

    Full Text Available Abstract Background We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1 in prostate cancer (PCa compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways. Results Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20 of tumor tissue compared to 15% (3/20 of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2. Conclusion From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.

  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. DNA Methylation in Schizophrenia.

    Science.gov (United States)

    Pries, Lotta-Katrin; Gülöksüz, Sinan; Kenis, Gunter

    2017-01-01

    Schizophrenia is a highly heritable psychiatric condition that displays a complex phenotype. A multitude of genetic susceptibility loci have now been identified, but these fail to explain the high heritability estimates of schizophrenia. In addition, epidemiologically relevant environmental risk factors for schizophrenia may lead to permanent changes in brain function. In conjunction with genetic liability, these environmental risk factors-likely through epigenetic mechanisms-may give rise to schizophrenia, a clinical syndrome characterized by florid psychotic symptoms and moderate to severe cognitive impairment. These pathophysiological features point to the involvement of epigenetic processes. Recently, a wave of studies examining aberrant DNA modifications in schizophrenia was published. This chapter aims to comprehensively review the current findings, from both candidate gene studies and genome-wide approaches, on DNA methylation changes in schizophrenia.

  9. Comparison between two methods of qPCR for detection of methylation status of CDX2 gene in serrated adenoma%检测锯齿状腺瘤中CDX2基因甲基化率的两种qPCR方法比较

    Institute of Scientific and Technical Information of China (English)

    许春伟; 葛畅; 王鲁平; 张玉萍

    2014-01-01

    目的 应用两种方法检测锯齿状腺瘤中CDX2基因甲基化状态,比较这两种检测方法的差异.方法 采用Taqman探针qPCR (MethyLight)和SYBR Green qPCR方法检测129例锯齿状腺瘤(包括61例SSA/P、68例TSA)和42例正常结直肠黏膜组织中CDX2基因CpG岛甲基化状态.结果 MethyLight方法:SSA/P组织中CDX2基因甲基化率(75.41%,46/61)高于对照组(53.38%,22/42),两者差异显著(P<0.05).TSA组织中CDX2基因甲基化率(80.88%,55/68)高于对照组(53.38%,22/42),两者差异显著(P<0.05).SYBR Green qPCR方法:SSA/P、TSA组和对照组的组织中CDX2基因均呈高甲基化状态,SSA/P组织中CDX2基因甲基化率(81.97%,50/61)高于对照组(73.81%,31/42),两者比较差异不显著(P>0.05);TSA组织中CDX2基因甲基化率(86.76%,59/68)高于对照组(73.81%,31/42),两者比较差异不显著(P>0.05);结论 MethyLight消除了引物二聚体和非特异性扩增对试验结果的影响,提高了结果的特异性和准确性,被证实是进行异常DNA甲基化状态检测的可靠方法.

  10. DNA methylation and carcinogenesis.

    Science.gov (United States)

    Lichtenstein, A V; Kisseljova, N P

    2001-03-01

    In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

  11. PCR

    African Journals Online (AJOL)

    ELO

    2012-01-05

    mail: ... Plates transferred to the laboratory were incubated at 25°C for 48 h for growing the .... Use of a probiotic to control lactococcosis and streptococcosis in ... Streptococcus difficile: two new streptococcal species causing a.

  12. Persistent infection of hepatitis B virus is involved in high rate of p16 methylation in hepatocellular carcinoma.

    Science.gov (United States)

    Jicai, Zhang; Zongtao, Yü; Zongtao, Yu; Jun, Lü; Jun, Lu; Haiping, Li; Jianmin, Wu; Lihua, Hu

    2006-07-01

    High rate of chronic hepatitis B virus (HBV) infection and p16 promoter methylation were found in the majority of hepatocellular carcinoma (HCC). To investigate the potential linkage between high rate of p16 methylation and HBV infection, p16 methylation was detected with methylation-specific polymerase chain reaction (PCR), and HBV markers were examined with real-time PCR and immunologic method. p16 methylation was detected in 5.5% of patients with hepatitis B, 9.1% of noncancerous liver, 36.6% of cirrhotic liver tissue, and 70.5% of cancerous tissue of HCC, primarily in cirrhotic (46.7%) and cancerous tissue (90.6%) with HBV infection. In noncancerous tissue, p16 methylation could only be detected in samples with HBV infection, although no significant difference, the frequency of p16 methylation in noncancerous tissue with HBV infection was higher than those without it. The results showed that, in cancerous, cirrhotic, or noncancerous tissues, the frequency of p16 methylation in samples with HBV infection was higher than those without it, suggesting possible association between HBV infection and p16 methylation. The result of HBV-DNA analysis showed that 96.1% (49/51) samples with p16 methylation also showed detectable HBV-DNA; it signifies that replication and/or integration of HBV may contribute to high rate of p16 methylation in hepatocarcinogenesis. Generally, these results indicate that persistent HBV infection may be associated with high rate of p16 methylation, and involved in development of HCC through this way.

  13. 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.

  14. 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.

  15. 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

    2016-01-01

    AIM To clarify whether Agtr1a methylation is involved in the development of nonalcoholic steatohepatitis (NASH)-related liver fibrosis in adult rats. METHODS 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. RESULTS 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 renin-angiotensin system-related gene expression during liver fibrosis. PMID:27729955

  16. Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach.

    Science.gov (United States)

    Kupcinskaite-Noreikiene, Rita; Ugenskiene, Rasa; Noreika, Alius; Rudzianskas, Viktoras; Gedminaite, Jurgita; Skieceviciene, Jurgita; Juozaityte, Elona

    2016-01-26

    There is considerable information on the methylation of the promoter regions of different genes involved in gastric carcinogenesis. However, there is a lack of information on how this epigenetic process differs in tumors originating at different sites in the stomach. The aim of this study is to assess the methylation profiles of the MLH1, MGMT, and DAPK-1 genes in cancerous tissues from different stomach sites. Samples were acquired from 81 patients suffering stomach adenocarcinoma who underwent surgery for gastric cancer in the Lithuanian University of Health Sciences Hospital Kaunas Clinics in 2009-2012. Gene methylation was investigated with methylation-specific PCR. The study was approved by the Lithuanian Biomedical Research Ethics Committee. The frequencies of methylation in cancerous tissues from the upper, middle, and lower thirds of the stomach were 11.1, 23.1, and 45.4%, respectively, for MLH1; 22.2, 30.8, and 57.6%, respectively, for MGMT; and 44.4, 48.7, and 51.5%, respectively, for DAPK-1. MLH1 and MGMT methylation was observed more often in the lower third of the stomach than in the upper third (p stomach (coefficient, -0.48; p = 0.01). DAPK-1 and MLH1 methylation correlated inversely in tumors in the middle-third of the stomach (coefficient, -0.41; p = 0.01). Gene promoter methylation depends on the gastric tumor location.

  17. Evaluation of Methylation Biomarkers for Detection of Circulating Tumor DNA and Application to Colorectal Cancer

    Directory of Open Access Journals (Sweden)

    Susan M. Mitchell

    2016-12-01

    Full Text Available Solid tumors shed DNA into circulation, and there is growing evidence that the detection of circulating tumor DNA (ctDNA has broad clinical utility, including monitoring of disease, prognosis, response to chemotherapy and tracking tumor heterogeneity. The appearance of ctDNA in the circulating cell-free DNA (ccfDNA isolated from plasma or serum is commonly detected by identifying tumor-specific features such as insertions, deletions, mutations and/or aberrant methylation. Methylation is a normal cell regulatory event, and since the majority of ccfDNA is derived from white blood cells (WBC, it is important that tumour-specific DNA methylation markers show rare to no methylation events in WBC DNA. We have used a novel approach for assessment of low levels of DNA methylation in WBC DNA. DNA methylation in 29 previously identified regions (residing in 17 genes was analyzed in WBC DNA and eight differentially-methylated regions (DMRs were taken through to testing in clinical samples using methylation specific PCR assays. DMRs residing in four genes, BCAT1, GRASP, IKZF1 and IRF4, exhibited low positivity, 3.5% to 7%, in the plasma of colonoscopy-confirmed healthy subjects, with the sensitivity for detection of ctDNA in colonoscopy-confirmed patients with colorectal cancer being 65%, 54.5%, 67.6% and 59% respectively.

  18. Nucleosome Positions and Differential Methylation Status of Various Regions within MLH1 CpG Island

    Institute of Scientific and Technical Information of China (English)

    BAI Hua; ZHOU Jing; DENG Da-jun

    2008-01-01

    Objective:To determine the relationship between nucleosome positions and formation of differential methylation of the reported region A,B,C,and D within the MLH1 CpG island. Methods:Methylation of the MLH1 promoter was analyzed by combined of bisulfite restriction assay.Chromatin of RKO and MGC803 cells were extracted and digested by Mnase.Mononucleosomal DNA fragment was isolated and used as templates for detection of nucleosomal distribution by a battery of quantitative PCRs covering the full MLH1 promoter region. Results:The MLH1 was methylated in RKO and unmethylated in MGC803.At the region B,where methylation of CpG sites did not correlated with transcription of this gene well,qPCR product of the M-3(-599nt~-475nt)fragment was amplified in both RKO and MGC803 cells.However,at the region C and D within the core promoter,where methylation of CpG sites correlated with loss of MLH1 transcription well,the M-7(-257nt~-153nt)and M-8(-189nt~-71nt)fragments were amplified remarkably only in RKO cells. Conclusion:Nucleosome may be the basic unit for both CpG methylation and methylation-related regulation of gene transcription.Methylation status of CpG sites within the same nucleosome may be homogeneous;between different nucleosomes,homogeneous or heterogeneous.

  19. Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

    Directory of Open Access Journals (Sweden)

    Owczarek-Lipska Marta

    2011-05-01

    Full Text Available Abstract Background Porcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2. Results Bisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele. Conclusions Our results indicate that the IGF2 imprinting status is transcript

  20. Promoter Methylation and Relative mRNA Expression of the p16 Gene in Cervical Cancer in North Indians.

    Science.gov (United States)

    Gupta, Amita; Ahmad, Mohammad Kaleem; Mahndi, Abbas Ali; Singh, Renu; Pradeep, Yashodhara

    2016-01-01

    Cervical carcinoma is one of the main causes of mortality in women worldwide as well as in India. It occurs as a result of various molecular events that develop from the combined influences of an individual's genetic predisposition and external agents such as smoking and menstrual hygiene, for example. However, infection with human papillomavirus (HPV) is the established major risk factor. The aim of the current study was to investigate p16 CpG island methylation and establish any correlation with mRNA expression in a north Indian population. We analyzed 196 woman volunteers out of which 98 were cases and 98 healthy controls. For the analysis of methylation pattern, DNA extracted from blood samples was modified with a bisulfate kit and used as template for methylation specific PCR (MSP). Quantitative real-time PCR (QRT-PCR) was performed to check mRNA expression. Correlation between methylation status of p16 gene and poor menstrual hygiene was significant (p=0.006), high parity cases showed methylation of p16 gene (p=0.031) with increased risk up to 1.86 times for cervical cancer and smoking was a strong risk factor associated with cervical cancer. We analyzed methylation pattern and found 60.3% methylation in cases with low mRNA expression level (0.014) as compared to controls (1.24). It was also observed that promoter methylation of p16 gene was significantly greater in FIGO stage III. We conclude that p16 methylation plays an important role in cervical cancer in the north Indian population and its methylation decreases mRNA expression. It can be used as an important and consistent blood biomarker in cervical cancer patients.

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

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    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.

  3. CpG island methylator phenotype of multigene in serum of sporadic breast carcinoma.

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    Jing, Feng; Yuping, Wang; Yong, Chen; Jie, Luo; Jun, Lu; Xuanbing, Tang; Lihua, Hu

    2010-08-01

    CpG island methylator phenotype (CIMP) involves methylation targeted toward the promoters of multiple genes. We determined a methylation profile of tumor-related genes in serum of sporadic breast cancer (SBC). The multigene methylation was examined by methylation-specific polymerase chain reaction assay in serum of 50 SBCs and 50 paired nontumors, and CIMP+ was defined as having three genes that are concordantly methylated. The methylation frequency of ten genes in serum of 50 SBCs varied from 10% in FHIT to 74% in RASSF1A. The methylation status of RASSF1A, BRCA1, p16, CDH1, ER, RARbeta2, APC, and DAPK was significantly correlated with SBC and nontumor serum (P < 0.05). Methylation of at least one gene was found in 92% SBC; CIMP was more frequent in SBC than nontumor serum (P < 0.001). There was a significant association between CIMP and methylation of RASSF1A, BRCA1, p16, CDH1, ER, RARbeta2, APC, and DAPK (P < 0.05); the methylation link profile of CDH1, RASSF1A, BRCA1, and RARbeta2 as breast cancer marker may contribute high sensitivity (90%) and specificity (88%). ER and RARbeta2 methylation was associated with elevated serum CA153 levels in 39 SBC samples with CIMP+ (P < 0.05). Multivariate analysis showed that living area of patients was found to provide independent prognostic information associated with a relative risk of tumor recurrence of 5.3. Multigene-specific methylation profile in serum was association with the recurrence risk of rural SBC, and positive correlation of CIMP can serve as a promising molecular marker of SBC.

  4. DNA methylation in psychosis: insights into etiology and treatment.

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    Castellani, Christina A; Melka, Melkaye G; Diehl, Eric J; Laufer, Benjamin I; O'Reilly, Richard L; Singh, Shiva M

    2015-01-01

    Evidence for involvement of DNA methylation in psychosis forms the focus of this perspective. Of interest are results from two independent sets of experiments including rats treated with antipsychotic drugs and monozygotic twins discordant for schizophrenia. The results show that DNA methylation is increased in rats treated with antipsychotic drugs, reflecting the global effect of the drugs. Some of these changes are also seen in affected schizophrenic twins that were treated with antipsychotics. The genes and pathways identified in the unrelated experiments are relevant to neurodevelopment and psychiatric disorders. The common cause is hypothesized to be aberrations resulting from medication use. However, this needs to be established by future studies that address the origin of methylation changes in psychosis.

  5. Assessment of 'one-step' versus 'sequential' embryo culture conditions through embryonic genome methylation and hydroxymethylation changes.

    Science.gov (United States)

    Salvaing, J; Peynot, N; Bedhane, M N; Veniel, S; Pellier, E; Boulesteix, C; Beaujean, N; Daniel, N; Duranthon, V

    2016-11-01

    In comparison to in vivo development, how do different conditions of in vitro culture ('one step' versus 'sequential medium') impact DNA methylation and hydroxymethylation in preimplantation embryos? Using rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used. Correct regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations. The study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference. Immunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing

  6. Contribution of LATS1 and LATS2 promoter methylation in OSCC development.

    Science.gov (United States)

    Ladiz, Mohammad Ayoub Rigi; Najafi, Maryam; Kordi-Tamandani, Dor Mohammad

    2017-03-01

    The aberrant DNA methylation of the tumor suppressor genes involved in DNA Damage Response (DDR) signaling and cell cycle regulation may lead to the tumorigenesis. Our purpose here is to analyze the promoter methylation and mRNA expression levels of LATS1 and LATS2 (LATS1/2) genes in OSCC. Promoter methylation status of LATS1/2 genes was evaluated in 70 OSCC paraffin-embedded tissues and 70 normal oral samples, using Methylation Specific PCR (MSP). LATS1/2 mRNA expression profiles were also investigated in 14 OSCC patients and 14 normal samples, using real-time PCR. In both candidate genes, promoter methylation assessment revealed significant relationship between cases and controls (OR = 2.24, 95 % CI = 1.40-3.54, P = 0.001; LATS1 and OR = 15.5, 95%CI = 3.64-64.76, P promoter methylation in OSCC. It is suggested to explore the down-stream transcription factors of both genes for finding the molecular mechanism of this deregulation in OSCC.

  7. 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 (p<0.0003). There was a variable frequency and individual methylation quotient detected, depending on the TSG and the tumor type. When comparing normal, benign, and malignant SGTs, there was a statistically significant trend 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

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

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    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. Identification of novel high-frequency DNA methylation changes in breast cancer.

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    Jared M Ordway

    Full Text Available Recent data have revealed that epigenetic alterations, including DNA methylation and chromatin structure changes, are among the earliest molecular abnormalities to occur during tumorigenesis. The inherent thermodynamic stability of cytosine methylation and the apparent high specificity of the alterations for disease may accelerate the development of powerful molecular diagnostics for cancer. We report a genome-wide analysis of DNA methylation alterations in breast cancer. The approach efficiently identified a large collection of novel differentially DNA methylated loci (approximately 200, a subset of which was independently validated across a panel of over 230 clinical samples. The differential cytosine methylation events were independent of patient age, tumor stage, estrogen receptor status or family history of breast cancer. The power of the global approach for discovery is underscored by the identification of a single differentially methylated locus, associated with the GHSR gene, capable of distinguishing infiltrating ductal breast carcinoma from normal and benign breast tissues with a sensitivity and specificity of 90% and 96%, respectively. Notably, the frequency of these molecular abnormalities in breast tumors substantially exceeds the frequency of any other single genetic or epigenetic change reported to date. The discovery of over 50 novel DNA methylation-based biomarkers of breast cancer may provide new routes for development of DNA methylation-based diagnostics and prognostics, as well as reveal epigenetically regulated mechanism involved in breast tumorigenesis.

  10. luxS Mutant Regulation: Quorum Sensing Impairment or Methylation Disorder?

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    Zhengwei Huang

    2012-05-01

    Full Text Available AI-2–mediated quorum sensing has been identified in various bacteria, including both Gram-negative and Gram-positive species, and numerous phenotypes have been reported to be regulated by this mechanism, using the luxS-mutant strain. But the AI-2 production process confused this regulatory function; some considered this regulation as the result of a metabolic change, which refers to an important metabolic cycle named activated methyl cycle (AMC, caused by luxS-mutant simultaneously with the defect of AI-2. Herein we hypothesized that the quorum sensing system—not the metabolic aspect—is responsible for such a regulatory function. In this study, we constructed plasmids infused with sahH and induced protein expression in the luxS-mutant strain to make the quorum-sensing system and metabolic system independent. The biofilm-related genes were investigated by real-time polymerase chain reaction (PCR, and the results demonstrated that the quorum-sensing completed strain restored the gene expression of the defective strain, but the metabolically completed one did not. This evidence supported our hypothesis that the autoinducer-2-mediated, quorum-sensing system, not the AMC, was responsible for luxS mutant regulation.

  11. Determination of Arabidopsis thaliana telomere length by PCR.

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    Vaquero-Sedas, María I; Vega-Palas, Miguel A

    2014-07-02

    In humans, telomere length studies have acquired great relevance because the length of telomeres has been related to natural processes like disease, aging and cancer. However, very little is known about the influence of telomere length on the biology of wild type plants. The length of plant telomeres has been usually studied by Terminal Restriction Fragment (TRF) analyses. This technique requires high amounts of tissue, including multiple cell types, which might be the reason why very little is known about the influence of telomere length on plant natural processes. In contrast, many of the human telomere length studies have focused on homogenous cell populations. Most of these studies have been performed by PCR, using telomeric degenerated primers, which allow the determination of telomere length from small amounts of human cells. Here, we have adapted the human PCR procedure to analyze the length of Arabidopsis thaliana telomeres. This PCR approach will facilitate the analysis of telomere length from low amounts of tissue. We have used it to determine that CG and non CG DNA methylation positively regulates Arabidopsis telomere length.

  12. 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....... Interestingly, HIF3A expression in SAT, but not in muscle, associated negatively with BMI and whole-body insulin resistance. We found a significant effect of familiality on HIF3A methylation levels in the blood and HIF3A expression levels in skeletal muscle. CONCLUSIONS: Our findings are in line...... 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...

  13. Correlation between methylation of the p16 promoter and cervical cancer incidence.

    Science.gov (United States)

    Wang, F-L; Yang, Y; Liu, Z-Y; Qin, Y; Jin, T

    2017-05-01

    To study the methylation of the promoter of the p16 gene in cervical cancer patients and explore the correlation between methylation and the incidence of cervical cancer. We recruited 78 patients with cervical cancer and 48 healthy individuals. The methylation-specific PCR was used to detect the methylation status in the promoter of the p16 gene. The mRNA expression of p16 was studied by quantitative fluorescence PCR. The protein expression of p16 was monitored by Enzyme-linked immunosorbent assay (ELISA) and Western blot. Immunohistochemistry was applied to detect the expression and distribution of p16 in cervical tissues. The methylation sequencing results showed that samples from cervical cancer patients had a methylation rate of 78.52% in the p16 gene promoter region compared with a much lower rate of 9.8% in the control group (9.8%). Quantitative fluorescence PCR indicated that the p16 mRNA expression was significantly reduced in cervical cancer patients compared with controls. ELISA and Western blot results showed that expression of the p16 protein in cancer tissue was 0.81 ± 0.12 µg/l, whereas in the healthy controls it was 3.21 ± 0.24 µg/l. Immunohistochemical results showed that the p16 protein was mainly present in the cytoplasm. The rate of p16 positive cells in the healthy cervical tissue 83.29% was higher than in cervical cancer 10.18%. The methylation of the p16 gene promoter could significantly reduce p16 expression, losing its tumor suppressor activity and promoting the development of cervical cancer.

  14. H19-DMR allele-specific methylation analysis reveals epigenetic heterogeneity of CTCF binding site 6 but not of site 5 in head-and-neck carcinomas

    DEFF Research Database (Denmark)

    De Castro Valente Esteves, Leda Isabel; De Karla Cervigne, Nilva; Do Carmo Javaroni, Afonso

    2006-01-01

    Aberrant methylation of seven potential binding sites of the CTCF factor in the differentially methylated region upstream of the H19 gene (H19-DMR) has been suggested as critical for the regulation of IGF2 and H19 imprinted genes. In this study, we analyzed the allele-specific methylation pattern...... of CTCF binding sites 5 and 6 using methylation-sensitive restriction enzyme PCR followed by RFLP analysis in matched tumoral and lymphocyte DNA from head-and-neck squamous cell carcinoma (HNSCC) patients, as well as in lymphocyte DNA from control individuals who were cancer-free. The monoallelic...... methylation pattern was maintained in CTCF binding site 5 in 22 heterozygous out of 91 samples analyzed. Nevertheless, a biallelic methylation pattern was detected in CTCF binding site 6 in a subgroup of HNSCC patients as a somatic acquired feature of tumor cells. An atypical biallelic methylation was also...

  15. A genome-wide methylation study on obesity

    Science.gov (United States)

    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 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. PMID:23644594

  16. LINE-1 methylation is inherited in familial testicular cancer kindreds

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    Gadalla Shahinaz M

    2010-05-01

    Full Text Available Abstract Background Testicular germ cell tumors (TGCT are the most frequent cancers among young men. There is a clear familial component to TGCT etiology, but no high-penetrance susceptibility gene has been identified. Epigenetic aberrations of the genome represent an alternative mechanism for cancer susceptibility; and, studies suggest that epigenetic changes that influence cancer risk can be inherited through the germline. Global DNA hypomethylation has been associated with the risk of cancers of the bladder and head/neck. Methods We performed a pilot study of global methylation at long interspersed nuclear elements-1 (LINE-1 in peripheral blood DNA isolated from 466 family members of 101 multiple-case testicular cancer families. Results Investigating the correlation of LINE-1 methylation levels among parent-child pairs independent of affection status (n = 355 revealed a strong positive association only between mother-daughter (r = 0.48, P = r = 0.31, P = 0.02, suggesting gender-specific inheritance of methylation. Incorporating cancer status, we observed a strong correlation in LINE-1 methylation levels only among affected father-affected son pairs (r = 0.49, P = 0.03. There was a marginally significant inverse association between lower LINE-1 methylation levels and increased TGCT risk, compared with healthy male relatives (P = 0.049. Conclusions Our data suggest that heritability of LINE-1 methylation may be gender-specific. Further, the strong correlation between LINE-1 methylation levels among affected father-affected son pairs suggests that transgenerational inheritance of an epigenetic event may be associated with disease risk. Larger studies are needed to clarify these preliminary observations.

  17. RT-PCR detection of HIV in Republic of Macedonia.

    Science.gov (United States)

    Bosevska, Golubinka; Panovski, Nikola; Dokić, Eleni; Grunevska, Violeta

    2008-11-01

    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 masculineC, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20 masculineC for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  18. DNA Methylation, Epigenetics, and Evolution in Vertebrates: Facts and Challenges

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    Annalisa Varriale

    2014-01-01

    Full Text Available DNA methylation is a key epigenetic modification in the vertebrate genomes known to be involved in biological processes such as regulation of gene expression, DNA structure and control of transposable elements. Despite increasing knowledge about DNA methylation, we still lack a complete understanding of its specific functions and correlation with environment and gene expression in diverse organisms. To understand how global DNA methylation levels changed under environmental influence during vertebrate evolution, we analyzed its distribution pattern along the whole genome in mammals, reptiles and fishes showing that it is correlated with temperature, independently on phylogenetic inheritance. Other studies in mammals and plants have evidenced that environmental stimuli can promote epigenetic changes that, in turn, might generate localized changes in DNA sequence resulting in phenotypic effects. All these observations suggest that environment can affect the epigenome of vertebrates by generating hugely different methylation patterns that could, possibly, reflect in phenotypic differences. We are at the first steps towards the understanding of mechanisms that underlie the role of environment in molding the entire genome over evolutionary times. The next challenge will be to map similarities and differences of DNA methylation in vertebrates and to associate them with environmental adaptation and evolution.

  19. Differential DNA Methylation Analysis without a Reference Genome

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    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.

  20. Differential DNA Methylation Analysis without a Reference Genome.

    Science.gov (United States)

    Klughammer, Johanna; Datlinger, Paul; Printz, Dieter; Sheffield, Nathan C; Farlik, Matthias; Hadler, Johanna; Fritsch, Gerhard; Bock, Christoph

    2015-12-22

    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.

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

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    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

  2. Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA).

    Science.gov (United States)

    Zhan, Leyun; Zhang, Bingyi; Tan, Yaojun; Yang, Chengliang; Huang, Chenhong; Wu, Qiongya; Zhang, Yulin; Chen, Xiaobo; Zhou, Mi; Shu, Aihua

    2017-02-01

    Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer.

  3. Quantitative assessment of the relationship between RASSF1A gene promoter methylation and bladder cancer (PRISMA)

    Science.gov (United States)

    Zhan, Leyun; Zhang, Bingyi; Tan, Yaojun; Yang, Chengliang; Huang, Chenhong; Wu, Qiongya; Zhang, Yulin; Chen, Xiaobo; Zhou, Mi; Shu, Aihua

    2017-01-01

    Abstract Background: Methylation of the Ras-association domain family 1 isoform A (RASSF1A) gene promoter region is thought to participate in the initiation and development of many different cancers. However, in bladder cancer the role of RASSF1A methylation was unclear. To evaluate the relationship between RASSF1A methylation and bladder cancer, a quantitative assessment of an independent meta-analysis was performed. In addition, a DNA methylation microarray database from the cancer genome atlas (TCGA) project was used to validate the results of the meta-analysis. Methods: We searched published articles from computerized databases, and DNA methylation data were extracted from TCGA project. All data were analyzed by R software. Results: The results of the meta-analysis indicated that the frequency of RASSF1A gene methylation in bladder cancer patients is significantly higher than in healthy controls. The hazard ratio (HR) was 2.24 (95% CI = [1.45; 3.48], P = 0.0003) for overall survival (OS), and the RASSF1A gene promoter methylation status was strongly associated with the TNM stage and differentiation grade of the tumor. The similar results were also found by the data from TCGA project. Conclusion: There was a significant relationship between the methylation of the RASSF1A gene promoter and bladder cancer. Therefore, RASSF1A gene promoter methylation will be a potential biomarker for the clinical diagnosis of bladder cancer. PMID:28207521

  4. Clinical characteristics and prognosis of acute myeloid leukemia associated with DNA-methylation regulatory gene mutations.

    Science.gov (United States)

    Ryotokuji, Takeshi; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kurosawa, Saiko; Kobayashi, Yutaka; Kawata, Eri; Tajika, Kenji; Gomi, Seiji; Kanda, Junya; Kobayashi, Anna; Omori, Ikuko; Marumo, Atsushi; Fujiwara, Yusuke; Yui, Shunsuke; Terada, Kazuki; Fukunaga, Keiko; Hirakawa, Tsuneaki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Tamai, Hayato; Nakayama, Kazutaka; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

    2016-09-01

    In recent years, it has been reported that the frequency of DNA-methylation regulatory gene mutations - mutations of the genes that regulate gene expression through DNA methylation - is high in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. We studied 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutations were observed in 135 of the 308 cases (43.8%). Acute myeloid leukemia associated with a DNA-methylation regulatory gene mutation was more frequent in older patients (Pgene mutation was an unfavorable prognostic factor for overall survival in the whole cohort (P=0.0018), in patients aged ≤70 years, in patients with intermediate cytogenetic risk, and in FLT3-ITD-negative patients (P=0.0409). Among the patients with DNA-methylation regulatory gene mutations, 26.7% were found to have two or more such mutations and prognosis worsened with increasing number of mutations. In multivariate analysis DNA-methylation regulatory gene mutation was an independent unfavorable prognostic factor for overall survival (P=0.0424). However, patients with a DNA-methylation regulatory gene mutation who underwent allogeneic stem cell transplantation in first remission had a significantly better prognosis than those who did not undergo such transplantation (P=0.0254). Our study establishes that DNA-methylation regulatory gene mutation is an important unfavorable prognostic factor in acute myeloid leukemia.

  5. 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.

  6. Distro’: Independent Creativity for Independent Industr

    Directory of Open Access Journals (Sweden)

    Wiwik Sri Wulandari

    2014-11-01

    Full Text Available To shortened this introduction, ‘Distro’ is one of cultural phenomenon in theyoung generation nowadays. The word of ‘Distro’ is the shortened of DistributionOutlet. The phenomenon of ‘Distro’ has been some kind of new trends inproducing and distributing creative design products of goods amongst theyoungsters independently, in an independence industry that open for challengingand competitiveness for everyone. This field research has been done in the city ofYogyakarta, reknown as the second city in creative design products after the cityof Bandung. Yogyakarta is welknown as the students’ city as well as the capital cityof culture of Indonesia. As a students’ city it is normal that Yogyakarta is growingin numbers of young people who pursued to study here and enriched the cultureof the city to become more multicultural and the varieties of pluralism as well.This sociocultural phenomenon not only brought some dynamic changing tosociety, economy and cultural life of the city, but also social problems that needsto be overcome. My first research question then is about how the existence of‘Distro’ in Yogyakarta can be a positive answer for social problems that may arisesfrom the hegemony of globalization markets domestically? My second questionis how the creative product designs are being made and distributed creatively inindependent industry? Lastly, my third question is dealling with the genres ofthe design products and how it can be a new trend in art expression? ‘Distro’ is aproduct of culture and it is also creating cultural change in some aspects of the lifeof the youngsters who are ‘Distro’ enthusiasts. ‘Distro’ phenomenon basically is anoffensive to the hegemony of internationally branded product design which turnsto become more over-dominated to the domestic markets and industry and thus,‘Distro’ has the spirit of survival whilts at the same time producing opportunity ofenterpreneurship

  7. Metal Complexation Studies of 1-(4-Carboxy-3-hydroxy-N-methyl phenyl- amino methyl 2-methyl perimidine

    Directory of Open Access Journals (Sweden)

    Umang N. Patel

    2009-01-01

    Full Text Available Aminomethylation of 2-methyl perimidine was carried out by treating 2-methyl perimidine with formaldehyde and 4-aminosalicylic acid. The resultant compound was designed as 1-(4-caroxy-3-hydroxy-N-methyl phenylamino methyl2-methyl perimidine. The transition metal complexes of Cu2+, Co2+, Ni2+, Mn2+ and Zn2+ have been prepared and characterized by elemental analysis, spectral studies, magnetic moment determination, molar conductivity measurement and antimicrobial activity.

  8. iMethyl-PseAAC: Identification of Protein Methylation Sites via a Pseudo Amino Acid Composition Approach

    Directory of Open Access Journals (Sweden)

    Wang-Ren Qiu

    2014-01-01

    Full Text Available Before becoming the native proteins during the biosynthesis, their polypeptide chains created by ribosome’s translating mRNA will undergo a series of “product-forming” steps, such as cutting, folding, and posttranslational modification (PTM. Knowledge of PTMs in proteins is crucial for dynamic proteome analysis of various human diseases and epigenetic inheritance. One of the most important PTMs is the Arg- or Lys-methylation that occurs on arginine or lysine, respectively. Given a protein, which site of its Arg (or Lys can be methylated, and which site cannot? This is the first important problem for understanding the methylation mechanism and drug development in depth. With the avalanche of protein sequences generated in the postgenomic age, its urgency has become self-evident. To address this problem, we proposed a new predictor, called iMethyl-PseAAC. In the prediction system, a peptide sample was formulated by a 346-dimensional vector, formed by incorporating its physicochemical, sequence evolution, biochemical, and structural disorder information into the general form of pseudo amino acid composition. It was observed by the rigorous jackknife test and independent dataset test that iMethyl-PseAAC was superior to any of the existing predictors in this area.

  9. Symmetry implies independence

    CERN Document Server

    Renner, R

    2007-01-01

    Given a quantum system consisting of many parts, we show that symmetry of the system's state, i.e., invariance under swappings of the subsystems, implies that almost all of its parts are virtually identical and independent of each other. This result generalises de Finetti's classical representation theorem for infinitely exchangeable sequences of random variables as well as its quantum-mechanical analogue. It has applications in various areas of physics as well as information theory and cryptography. For example, in experimental physics, one typically collects data by running a certain experiment many times, assuming that the individual runs are mutually independent. Our result can be used to justify this assumption.

  10. Cigarette smoking and DNA methylation

    Science.gov (United States)

    Lee, Ken W. K.; Pausova, Zdenka

    2013-01-01

    DNA methylation is the most studied epigenetic modification, capable of controlling gene expression in the contexts of normal traits or diseases. It is highly dynamic during early embryogenesis and remains relatively stable throughout life, and such patterns are intricately related to human development. DNA methylation is a quantitative trait determined by a complex interplay of genetic and environmental factors. Genetic variants at a specific locus can influence both regional and distant DNA methylation. The environment can have varying effects on DNA methylation depending on when the exposure occurs, such as during prenatal life or during adulthood. In particular, cigarette smoking in the context of both current smoking and prenatal exposure is a strong modifier of DNA methylation. Epigenome-wide association studies have uncovered candidate genes associated with cigarette smoking that have biologically relevant functions in the etiology of smoking-related diseases. As such, DNA methylation is a potential mechanistic link between current smoking and cancer, as well as prenatal cigarette-smoke exposure and the development of adult chronic diseases. PMID:23882278

  11. PCR bias in amplification of androgen receptor alleles, a trinucleotide repeat marker used in clonality studies.

    OpenAIRE

    Mutter, G L; Boynton, K A

    1995-01-01

    Trinucleotide CAG repeats in the X-linked human androgen receptor gene (HUMARA) have proved a useful means of determining X chromosome haplotypes, and when combined with methylation analysis of nearby cytosine residues permits identification of non-random X inactivation in tumors of women. Co-amplification of two alleles in a heterozygote generates PCR products which differ in the number of CAG units, and thus their melting and secondary structure characteristics. We have shown that under opt...

  12. Variation in genomic methylation in natural populations of chinese white poplar.

    Directory of Open Access Journals (Sweden)

    Kaifeng Ma

    Full Text Available BACKGROUND: It is thought that methylcytosine can be inherited through meiosis and mitosis, and that epigenetic variation may be under genetic control or correlation may be caused by neutral drift. However, DNA methylation also varies with tissue, developmental stage, and environmental factors. Eliminating these factors, we analyzed the levels and patterns, diversity and structure of genomic methylcytosine in the xylem of nine natural populations of Chinese white poplar. PRINCIPAL FINDINGS: On average, the relative total methylation and non-methylation levels were approximately 26.567% and 42.708% (P<0.001, respectively. Also, the relative CNG methylation level was higher than the relative CG methylation level. The relative methylation/non-methylation levels were significantly different among the nine natural populations. Epigenetic diversity ranged from 0.811 (Gansu to 1.211 (Shaanxi, and the coefficients of epigenetic differentiation (GST  = 0.159 were assessed by Shannon's diversity index. Co-inertia analysis indicated that methylation-sensitive polymorphism (MSP and genomic methylation pattern (CG-CNG profiles gave similar distributions. Using a between-group eigen analysis, we found that the Hebei and Shanxi populations were independent of each other, but the Henan population intersected with the other populations, to some degree. CONCLUSIONS: Genome methylation in Populus tomentosa presented tissue-specific characteristics and the relative 5'-CCGG methylation level was higher in xylem than in leaves. Meanwhile, the genome methylation in the xylem shows great epigenetic variation and could be fixed and inherited though mitosis. Compared to genetic structure, data suggest that epigenetic and genetic variation do not completely match.

  13. Construction of Differential-Methylation Subtractive Library

    Directory of Open Access Journals (Sweden)

    Wei Hu

    2014-01-01

    Full Text Available Stress-induced ROS changes DNA methylation patterns. A protocol combining methylation-sensitive restriction endonuclease (MS-RE digestion with suppression subtractive hybridization (SSH to construct the differential-methylation subtractive library was developed for finding genes regulated by methylation mechanism under cold stress. The total efficiency of target fragment detection was 74.64%. DNA methylation analysis demonstrated the methylation status of target fragments changed after low temperature or DNA methyltransferase inhibitor treatment. Transcription level analysis indicated that demethylation of DNA promotes gene expression level. The results proved that our protocol was reliable and efficient to obtain gene fragments in differential-methylation status.

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

    Institute of Scientific and Technical Information of China (English)

    Mian-Yang Li; Yuan-Yuan Xu; Hui-Yuan Kang; Xin-Rong Wang; Li Gao; Jian Cen; Wei Wang

    2015-01-01

    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 ofID4 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 ofID4 methylation positivity rates and levels could be a useful biomarker for MDS diagnosis.

  15. The Critical Independence Number and an Independence Decomposition

    CERN Document Server

    Larson, Craig Eric

    2009-01-01

    An independent set $I_c$ is a \\textit{critical independent set} if $|I_c| - |N(I_c)| \\geq |J| - |N(J)|$, for any independent set $J$. The \\textit{critical independence number} of a graph is the cardinality of a maximum critical independent set. This number is a lower bound for the independence number and can be computed in polynomial-time. Any graph can be decomposed into two subgraphs where the independence number of one subgraph equals its critical independence number, where the critical independence number of the other subgraph is zero, and where the sum of the independence numbers of the subgraphs is the independence number of the graph. A proof of a conjecture of Graffiti.pc yields a new characterization of K\\"{o}nig-Egervary graphs: these are exactly the graphs whose independence and critical independence numbers are equal.

  16. Analysis of MEFV exon methylation and expression patterns in familial Mediterranean fever

    Directory of Open Access Journals (Sweden)

    Ozdogan Huri

    2011-08-01

    Full Text Available Abstract Background MEFV mutations and decreased expression level of the gene are related to FMF pathology. DNA methylation at CpG islands is a well-known mechanism for transcriptional silencing. MEFV has a CpG island, spanning a part of the first intron and the whole of the second exon of the gene covering 998 bp region. Here, we tested the hypothesis that the MEFV transcript level in FMF patients correlates with its methylation level, and methylation, by allowing transcription silencing, has a role in FMF ethiopathogenesis. Methods The study group was composed of pediatric FMF patients (N = 51 and age-gender matched healthy controls (N = 21. The relative expression level of MEFV was assessed via quantitative real-time PCR (qRT-PCR and bisulfite sequencing (BS was performed to analyse the methylation level quantitatively. Results MEFV expression in FMF patients were decreased compared to healthy controls (P = 0.031. Methylation level of exon 2 of MEFV was found to be slightly higher in FMF patients compared to healthy controls (76% versus 74% (P = 0.049. The expression level of the MEFV was negatively correlated with the methylation level of the CpG island in both FMF and healthy controls groups (cor = -0.29, P = 0.041 but more so in the FMF only group (cor = -0.36, P = 0.035. Conclusions In this study, the relation between reduced MEFV expression level and FMF was confirmed. Observed slight increase in methylation in FMF patients, and correlation of methylation with expression might be indicative of its role in FMF, however a larger dataset is needed to confirm our preliminary findings.

  17. Pros and cons of methylation-based enrichment methods for ancient DNA

    DEFF Research Database (Denmark)

    Seguin-Orlando, Andaine; Gamba, Cristina; Der Sarkissian, Clio;

    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...

  18. Characterisation of IS900 loci in Mycobacterium avium subspeciesparatuberculosis and development of a rapid multiplex PCR typing system

    Institute of Scientific and Technical Information of China (English)

    Tim Bull; Ivo Pavlik; Fouad El-Zaatari; John Hermon-Taylor

    2000-01-01

    AIM To characterize genomic DNA flanking IS900 insertions and develop a rapid Multiplex PCR IS900Locus (MPIL) typing method for MAP reporting the presence or absence of the element at each locus,METHODS Genomic DNA flanking 14 of the 18 IS900 loci was sequenced and compared with databasehomologues. An MPIL typing method was developed using a common IS900 primer and individual locus-specific primers designed to produce amplification products differing by about 50bp which could be easilyresolved on a single gel. MPIL was applied to a panel of 81 MAP isolates and compared with RFLP profiles.RESULTS Genes flanking IS900 loci included homologues of transcription regulators, a sigma factor, anitrate reductase, a polyketide synthase and an O6-methylguanine-methyl transferase. MPIL rapidly andconsistently identified 10 individual types of MAP from the panel of 81 isolates, and distinguished betweenbovine and ovine strains. Nine MPIL types corresponded directly to single RFLP types previously identified.CONCLUSION Isg00 insertions in MAP may affect the expression of genes critically associated with thepathogenic phenotype. MPIL typing can identify bovine and ovine strains independent of the need for cultureand may contribute to studies of the molecular epidemiology of these difficult organisms.

  19. High-definition DNA methylation profiles from breast and ovarian carcinoma cell lines with differing doxorubicin resistance.

    Directory of Open Access Journals (Sweden)

    Michael Boettcher

    Full Text Available Acquired drug resistance represents a frequent obstacle which hampers efficient chemotherapy of cancers. The contribution of aberrant DNA methylation to the development of drug resistant tumor cells has gained increasing attention over the past decades. Hence, the objective of the presented study was to characterize DNA methylation changes which arise from treatment of tumor cells with the chemotherapeutic drug doxorubicin. DNA methylation levels from CpG islands (CGIs linked to twenty-eight genes, whose expression levels had previously been shown to contribute to resistance against DNA double strand break inducing drugs or tumor progression in different cancer types were analyzed. High-definition DNA methylation profiles which consisted of methylation levels from 800 CpG sites mapping to CGIs around the transcription start sites of the selected genes were determined. In order to investigate the influence of CGI methylation on the expression of associated genes, their mRNA levels were investigated via qRT-PCR. It was shown that the employed method is suitable for providing highly accurate methylation profiles, comparable to those obtained via clone sequencing, the gold standard for high-definition DNA methylation studies. In breast carcinoma cells with acquired resistance against the double strand break inducing drug doxorubicin, changes in methylation of specific cytosines from CGIs linked to thirteen genes were detected. Moreover, similarities between methylation profiles obtained from breast and ovarian carcinoma cell lines with acquired doxorubicin resistance were found. The expression levels of a subset of analyzed genes were shown to be linked to the methylation levels of the analyzed CGIs. Our results provide detailed DNA methylation information from two separate model systems for acquired doxorubicin resistance and suggest the occurrence of similar methylation changes in both systems upon exposure to the drug.

  20. Independent School Administration.

    Science.gov (United States)

    Springer, E. Laurence

    This book deals with the management of privately supported schools and offers guidelines on how these schools might be operated more effectively and economically. The discussions and conclusions are based on observations and data from case studies of independent school operations. The subjects discussed include the role and organization of…

  1. Independence in appearance

    DEFF Research Database (Denmark)

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

    2011-01-01

    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...

  2. The association between global DNA methylation and telomere length in a longitudinal study of boilermakers.

    Science.gov (United States)

    Wong, Jason Y Y; De Vivo, Immaculata; Lin, Xihong; Grashow, Rachel; Cavallari, Jennifer; Christiani, David C

    2014-04-01

    The objectives of this study were to determine if global DNA methylation, as reflected in LINE-1 and Alu elements, is associated with telomere length and whether it modifies the rate of telomeric change. A repeated-measures longitudinal study was performed with a panel of 87 boilermaker subjects. The follow-up period was 29 months. LINE-1 and Alu methylation was determined using pyrosequencing. Leukocyte relative telomere length was assessed via real-time qPCR. Linear-mixed models were used to estimate the association between DNA methylation and telomere length. A structural equation model (SEM) was used to explore the hypothesized relationship between DNA methylation, proxies of particulate matter exposure, and telomere length at baseline. There appeared to be a positive association between both LINE-1 and Alu methylation levels, and telomere length. For every incremental increase in LINE-1 methylation, there was a statistically significant 1.0 × 10(-1) (95% CI: 4.6 × 10(-2), 1.5 × 10(-1), P telomere length, controlling for age at baseline, current and past smoking status, work history, BMI (log kg/m(2) ) and leukocyte differentials. Furthermore, for every incremental increase in Alu methylation, there was a statistically significant 6.2 × 10(-2) (95% CI: 1.0 × 10(-2), 1.1 × 10(-1), P = 0.02) unit increase in relative telomere length. The interaction between LINE-1 methylation and follow-up time was statistically significant with an estimate -9.8 × 10(-3) (95% CI: -1.8 × 10(-2), -1.9 × 10(-3), P = 0.02); suggesting that the rate of telomeric change was modified by the degree of LINE-1 methylation. No statistically significant association was found between the cumulative PM exposure construct, with global DNA methylation and telomere length at baseline.

  3. DNA methylation modifies the association between obesity and survival after breast cancer diagnosis.

    Science.gov (United States)

    McCullough, Lauren E; Chen, Jia; Cho, Yoon Hee; Khankari, Nikhil K; Bradshaw, Patrick T; White, Alexandra J; Garbowski, Gail; Teitelbaum, Susan L; Terry, Mary Beth; Neugut, Alfred I; Hibshoosh, Hanina; Santella, Regina M; Gammon, Marilie D

    2016-02-01

    Mechanisms underlying the poor breast cancer prognosis among obese women are unresolved. DNA methylation levels are linked to obesity and to breast cancer survival. We hypothesized that obesity may work in conjunction with the epigenome to alter prognosis. Using a population-based sample of women diagnosed with first primary breast cancer, we examined modification of the obesity-mortality association by DNA methylation. In-person interviews were conducted approximately 3 months after diagnosis. Weight and height were assessed [to estimate body mass index (BMI)], and blood samples collected. Promoter methylation of 13 breast cancer-related genes was assessed in archived tumor by methylation-specific PCR and Methyl Light. Global methylation in white blood cell DNA was assessed by analysis of long interspersed elements-1 (LINE-1) and with the luminometric methylation assay (LUMA). Vital status among 1308 patients (with any methylation biomarker and complete BMI assessment) was determined after approximately 15 years of follow-up (N = 194/441 deaths due to breast cancer-specific/all-cause mortality). We used Cox proportional hazards regression to estimate hazard ratios (HRs) and 95 % confidence intervals (CIs) using two-sided p values of 0.05. Breast cancer-specific mortality was higher among obese (BMI ≥ 30) patients with promoter methylation in APC (HR = 2.47; 95 % CI = 1.43-4.27) and TWIST1 (HR = 4.25; 95 % CI = 1.43-12.70) in breast cancer tissue. Estimates were similar, but less pronounced, for all-cause mortality. Increased all-cause (HR = 1.81; 95 % CI = 1.19-2.74) and breast cancer-specific (HR = 2.61; 95 % CI = 1.45-4.69) mortality was observed among obese patients with the lowest LUMA levels. The poor breast cancer prognosis associated with obesity may depend on methylation profiles, which warrants further investigation.

  4. Genomic distribution and inter-sample variation of non-CpG methylation across human cell types.

    Directory of Open Access Journals (Sweden)

    Michael J Ziller

    2011-12-01

    Full Text Available DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.

  5. 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

  6. Methylation Status of Vitamin D Receptor Gene Promoter in Benign and Malignant Adrenal Tumors

    Directory of Open Access Journals (Sweden)

    Catia Pilon

    2015-01-01

    Full Text Available We previously showed a decreased expression of vitamin D receptor (VDR mRNA/protein in a small group of adrenocortical carcinoma (ACC tissues, suggesting the loss of a protective role of VDR against malignant cell growth in this cancer type. Downregulation of VDR gene expression may result from epigenetics events, that is, methylation of cytosine nucleotide of CpG islands in VDR gene promoter. We analyzed methylation of CpG sites in the VDR gene promoter in normal adrenals and adrenocortical tumor samples. Methylation of CpG-rich 5′ regions was assessed by bisulfite sequencing PCR using bisulfite-treated DNA from archival microdissected paraffin-embedded adrenocortical tissues. Three normal adrenals and 23 various adrenocortical tumor samples (15 adenomas and 8 carcinomas were studied. Methylation in the promoter region of VDR gene was found in 3/8 ACCs, while no VDR gene methylation was observed in normal adrenals and adrenocortical adenomas. VDR mRNA and protein levels were lower in ACCs than in benign tumors, and VDR immunostaining was weak or negative in ACCs, including all 3 methylated tissue samples. The association between VDR gene promoter methylation and reduced VDR gene expression is not a rare event in ACC, suggesting that VDR epigenetic inactivation may have a role in adrenocortical carcinogenesis.

  7. Aberrant CBFA2T3B gene promoter methylation in breast tumors

    Directory of Open Access Journals (Sweden)

    Bais Anthony J

    2004-08-01

    Full Text Available Abstract Background The CBFA2T3 locus located on the human chromosome region 16q24.3 is frequently deleted in breast tumors. CBFA2T3 gene expression levels are aberrant in breast tumor cell lines and the CBFA2T3B isoform is a potential tumor suppressor gene. In the absence of identified mutations to further support a role for this gene in tumorigenesis, we explored whether the CBFA2T3B promoter region is aberrantly methylated and whether this correlates with expression. Results Aberrant hypo and hypermethylation of the CBFA2T3B promoter was detected in breast tumor cell lines and primary breast tumor samples relative to methylation index interquartile ranges in normal breast counterpart and normal whole blood samples. A statistically significant inverse correlation between aberrant CBFA2T3B promoter methylation and gene expression was established. Conclusion CBFA2T3B is a potential breast tumor suppressor gene affected by aberrant promoter methylation and gene expression. The methylation levels were quantitated using a second-round real-time methylation-specific PCR assay. The detection of both hypo and hypermethylation is a technicality regarding the methylation methodology.

  8. Aberrant gene promoter methylation in sputum from individuals exposed to smoky coal emissions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Y.; Lan, Q.; Shen, M.; Jin, J.; Mumford, J.; Ren, D.X.; Keohavong, P. [University of Pittsburgh, Pittsburgh, PA (United States). Dept. of Environment and Occupational Health

    2008-07-15

    Recent studies suggested the potential for aberrant gene promoter methylation in sputum as a predictive marker for lung cancer. Here, the promoter methylation of p16, MGMT, RASSF1A and DAPK genes was investigated in sputum of individuals exposed to smoky coal emissions in Xuan Wei, China, where the lung cancer rate is more than 6 times the Chinese national average. Sputum DNA of 107 noncancer individuals and 58 lung cancer patients was screened for promoter methylation using methylation-specific PCR. Promoter methylation of the p16 gene was detected in about half (51.4% (551107)) of sputum DNA from noncancer individuals, a frequency higher than that observed for the RASSF1A (29.9%), MGMT (17.8%) and DAPK (15.9%) genes. Furthermore, the p16 gene was affected by promoter methylation at a frequency even higher among the lung cancer group, compared with the noncancer group (70.7% (41/58) versus 51.7% (55/107), p=0.017). Individuals exposed to smoky coal emissions in this region harbored frequent promoter methylation of these genes in their sputum and some of such alterations may be involved in lung tumor development.

  9. Independent Generation and Reactivity of Thymidine Radical Cations.

    Science.gov (United States)

    Sun, Huabing; Taverna Porro, Marisa L; Greenberg, Marc M

    2017-10-10

    Thymidine radical cation (1) is produced by ionizing radiation and has been invoked as an intermediate in electron transfer in DNA. Previous studies on its structure and reactivity have utilized thymidine as a precursor, which limits quantitative product analysis because thymidine is readily reformed from 1. In this investigation, radical cation 1 is independently generated via β-heterolysis of a pyrimidine radical generated photochemically from an aryl sulfide. Thymidine is the major product (33%) from 1 at pH 7.2. Diastereomeric mixtures of thymidine glycol and the corresponding 5-hydroxperoxides resulting from water trapping of 1 are formed. Significantly lower yields of products such as 5-formyl-2'-deoxyuridine that are ascribable to deprotonation from the C5-methyl group of 1 are observed. Independent generation of the N3-methyl analogue of 1 (NMe-1) produces considerably higher yields of products derived from water trapping, and these products are formed in much higher yields than those attributable to the C5-methyl group deprotonation in NMe-1. N3-Methyl-thymidine is, however, the major product and is produced in as high as 70% yield when the radical cation is produced in the presence of excess thiol. The effects of exogenous reagents on product distributions are consistent with the formation of diffusively free radical cations (1, NMe-1). This method should be compatible with producing radical cations at defined positions within DNA.

  10. A simple modification to improve the accuracy of methylation-sensitive restriction enzyme quantitative polymerase chain reaction.

    Science.gov (United States)

    Krygier, Magdalena; Podolak-Popinigis, Justyna; Limon, Janusz; Sachadyn, Paweł; Stanisławska-Sachadyn, Anna

    2016-05-01

    DNA digestion with endonucleases sensitive to CpG methylation such as HpaII followed by polymerase chain reaction (PCR) quantitation is commonly used in molecular studies as a simple and inexpensive solution for assessment of region-specific DNA methylation. We observed that the results of such analyses were highly overestimated if mock-digested samples were applied as the reference. We determined DNA methylation levels in several promoter regions in two setups implementing different references: mock-digested and treated with a restriction enzyme that has no recognition sites within examined amplicons. Fragmentation of reference templates allowed removing the overestimation effect, thereby improving measurement accuracy.

  11. Differentially Methylated DNA Regions in Monozygotic Twin Pairs Discordant for Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Svendsen, Anders J; Gervin, Kristina; Lyle, Robert;

    2016-01-01

    OBJECTIVES: In an explorative epigenome-wide association study (EWAS) to search for gene independent, differentially methylated DNA positions and regions (DMRs) associated with rheumatoid arthritis (RA) by studying monozygotic (MZ) twin pairs discordant for RA. METHODS: Genomic DNA was isolated......: We identified several differentially methylated regions associated with RA, which may represent environmental effects or consequences of the disease and plausible biological pathways pertinent to the pathogenesis of RA....

  12. Effects of methylation status of caspase-8 promoter on antitumor activity of TRAIL to human gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ru-gang; FANG Dian-chun; YANG Liu-qin; LUO Yuan-gang

    2004-01-01

    Objective: To study the effects of the methylation status of caspase-8 promoter on the antitumor activity of TRAIL to the human gastric cancer cells. Methods: The methylation of caspase-8 was measured with methylation specific PCR (MSP) and the antitomor capability of TRAIL to human gastric cancer cells was determined with MTT. Results: No methylation of caspase-8 in the human gastric cancer cells was found. The sensitivity of 5 lines of gastric cancer cells to the antitumor activity of TRAIL was different. The administration of the demethylation agent 5-Aza-2'-deoxycytidine ( 5-AzaCdR) increased the sensitivity of gastric cancer cells to TRAIL but did not change the methylation status of caspase-8 promoter in gastric cancer cells. Conclusion: 5-Aza-CdR increases the sensitivity of most of gastric cancer cells to TRAIL but caspase-8 is not involved in the antitumor activity of TRAIL.

  13. Methylization analysis of the FMR1 gene in carrier females

    Energy Technology Data Exchange (ETDEWEB)

    Meyers, S.; Cappon, S.; Khalifa, M.M. [Kingston General Hospital (Canada)] [and others

    1994-09-01

    The fragile X syndrome mutation is associated with an expansion of a CGG repeat sequence and methylation of the CpG island in the promoter of the FMR1 gene. Methylation of the CpG island silences the FMR1 gene, thereby generating the disease phenotypes. Previous studies suggest that the normal FMR1 gene has the properties of an X-linked housekeeping gene that is subject to X inactivation, i.e., its CpG island is unmethylated on the active X chromosome and methylated on the inactive X. Because methylation of the mutant FMR1 gene occurs in both males and females with the full mutation, inactivating the FMR1 gene in these females might be a localized event independent from X inactivation. To test this hypothesis we compared the methylation pattern of two housekeeping genes, PGK1 and androgen receptor (AR) with that of the FMR1 in 46 female carriers of the fragile X syndrome. Twenty eight females were in the premutation range (63-193 repeats) and 16 were carriers of the full mutation (263-996 repeats). The data revealed complete correlation between the methylation pattern of PGK1 and AR. There was also a close correlation between X inactivation pattern detected by PGK1 and/or AR and that detected by FMR1 in female carriers of the premutation. In all female carriers of the full mutation there was complete methylation of the BssHII site in the expanded FMR1 allele. The X chromosome inactivation pattern in these females as detected by PGK1 and/or AR was as follows: in 10 cases the X inactivation was skewed in favor of the mutant FMR1, i.e. the mutant allele was on the inactive X chromosome, in 3 the inactivation was random and in 3 the inactivation was skewed in favor of the normal allele. These data suggest that the methylation of the FMR1 gene in females with the full mutation is a localized event and methylation of the FMR1 gene in these females cannot be used as a predictor of X inactivation.

  14. 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.

  15. Field Independent Cosmic Evolution

    Directory of Open Access Journals (Sweden)

    Nayem Sk

    2013-01-01

    Full Text Available It has been shown earlier that Noether symmetry does not admit a form of corresponding to an action in which is coupled to scalar-tensor theory of gravity or even for pure theory of gravity taking anisotropic model into account. Here, we prove that theory of gravity does not admit Noether symmetry even if it is coupled to tachyonic field and considering a gauge in addition. To handle such a theory, a general conserved current has been constructed under a condition which decouples higher-order curvature part from the field part. This condition, in principle, solves for the scale-factor independently. Thus, cosmological evolution remains independent of the form of the chosen field, whether it is a scalar or a tachyon.

  16. TO BE INDEPENDENT

    Institute of Scientific and Technical Information of China (English)

    石波

    2000-01-01

    @@ Ⅰ. Introduction At present, in the college, English extensive reading class, most students are not used to being independent. They always ask the teacher to explain the passages sentence by sentence and they need a lot of time to use the dictionary. Yet, we should take the responsibility for the students by making clear the difference between intensive and extensive reading. The traditional teaching approaches pays more attention to the teacher-centered way;the teacher always plays a monodrama, and the teacher dominates the class. The students are lack initiative. Some students do not know where they could start from, and the others are short of fast-reading skills, always fixing their eyes on one word or one sentence. Under the new situation and new thinking, students should learn to be more independent.

  17. Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

    Directory of Open Access Journals (Sweden)

    Rajeevan Mangalathu S

    2009-06-01

    Full Text Available Abstract Background Differential expression of perforin (PRF1, a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction. Findings We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA or DNA extracted from peripheral blood mononuclear cells (PBMC DNA from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18% to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92% in the distal enhancer region (CpG sites 1–10, a variable methylation (range 49%–83% in the methylation sensitive region (CpG sites 11–17, and a progressively declining methylation level (range 12%–80% in the proximal promoter region (CpG sites 18–32 of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA. Conclusion This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune

  18. Real-time PCR in Food Science: PCR Diagnostics.

    Science.gov (United States)

    Rodriguez-Lazaro, David; Cook, Nigel; Hernandez, Marta

    2013-01-01

    A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labelling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programmes of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

  19. 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...... in a Matlab toolbox, is demonstrated for non-negative decompositions and compared with non-negative matrix factorization....

  20. Random Independence Systems

    Science.gov (United States)

    1981-11-01

    Whitney provided a set of axioms for a structure commonly called i a matroid. Matroid theory (see ( Tutte , 19711, [Lawler, 19761) has applications to a wide...applicable in this case. On the other hand, there is no known efficient ( polynomial time) algorithm for constructing cliques of size 2 log n with...intersection. The problem of constructing a maximal independent set in the intersection of k matroids has a polynomial time (in tEl) algorithm [Lawler, 197

  1. Methylation and mutation analysis of p16 gene in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Yi Ding; Xiao-Ping Le; Qin-Xian Zhang; Peng Du

    2003-01-01

    AIM: To study methylation, frequencies of homozygous deletion and mutation of p16 gene in gastric carcinoma.METHODS: The methylation pattern in exon 1 and exon 2of p16 gene was studied with polymerase chain reaction (PCR), using methylation sensitive restriction endonuclease HpaⅡ and methylation insensitive restriction endonudease MspⅠ. PCR technique was used to detect homozygous deletions of exon 1 and exon 2 of p16 gene and single strand conformation polymorphism (SSCP) technique was used to detect the mutation of the gene.RESULTS: Hypermethylation changes in exon 1 and exon 2 of p16 gene were observed in 25 % and 45 % of 20gastric cancer tissues, respectively, while no methylation abnormality was found in normal tissues. The homozygous deletion frequency of exon 1 and exon 2 of p16 gene in 20gastric cancer tissues was 20 % and 10 %, respectively. No mutation was found in exon 1 of p16 gene, while abnormal single strands were found in 2 (10 %) cases in exon 2 as detected by SSCP.CONCLUSION: The results suggest that hypermethylation and abnormality of p16 gene may play a key role in the progress of gastric cancer. Hypermethylation of exon 2 of p16 gene may have effects on the carcinogenesis of gastric mucosa and may be a later event.

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

    Directory of Open Access Journals (Sweden)

    Zhe Chao

    2016-07-01

    Full Text Available 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.

  3. Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism.

    Science.gov (United States)

    Hofman-Bang, Jacob; Gravesen, Eva; Olgaard, Klaus; Lewin, Ewa

    2012-01-01

    Secondary hyperparathyroidism (s-HPT) in uremia is characterized by decreased expression in the parathyroids of calcium sensing (CaR) and vitamin D receptors (VDR). Parathyroid hormone (PTH) is normalized despite low levels of CaR and VDR after experimental reversal of uremia. The expression of CaR in parathyroid cultures decreases rapidly. Methylation of promoter regions is often detected during epigenetic downregulation of gene expression. Therefore, using an experimental rat model, we examined changes in methylation levels of parathyroid CaR and VDR promoters in vivo and in vitro. Methods. Uremia was induced by 5/6 nephrectomy. Melting temperature profiling of CaR and VDR PCR products after bisulfite treatment of genomic DNA from rat parathyroids was performed. Real-time PCR measured expression of PTH, CaR, VDR, and klotho genes in vitro. Results. Parathyroids from uremic rats had similar low levels of methylation in vivo and in vitro. In culture, a significant downregulation of CaR, VDR, and klotho within two hours of incubation was observed, while housekeeping genes remained stable for 24 hours. Conclusion. In uremic s-HPT and in vitro, no overall changes in methylation levels in the promoter regions of parathyroid CaR and VDR genes were found. Thus, epigenetic methylation of these promoters does not explain decreased parathyroid expression of CaR and VDR genes in uremic s-HPT.

  4. Epigenetic Methylation of Parathyroid CaR and VDR Promoters in Experimental Secondary Hyperparathyroidism

    Directory of Open Access Journals (Sweden)

    Jacob Hofman-Bang

    2012-01-01

    Full Text Available Secondary hyperparathyroidism (s-HPT in uremia is characterized by decreased expression in the parathyroids of calcium sensing (CaR and vitamin D receptors (VDR. Parathyroid hormone (PTH is normalized despite low levels of CaR and VDR after experimental reversal of uremia. The expression of CaR in parathyroid cultures decreases rapidly. Methylation of promoter regions is often detected during epigenetic downregulation of gene expression. Therefore, using an experimental rat model, we examined changes in methylation levels of parathyroid CaR and VDR promoters in vivo and in vitro. Methods. Uremia was induced by 5/6 nephrectomy. Melting temperature profiling of CaR and VDR PCR products after bisulfite treatment of genomic DNA from rat parathyroids was performed. Real-time PCR measured expression of PTH, CaR, VDR, and klotho genes in vitro. Results. Parathyroids from uremic rats had similar low levels of methylation in vivo and in vitro. In culture, a significant downregulation of CaR, VDR, and klotho within two hours of incubation was observed, while housekeeping genes remained stable for 24 hours. Conclusion. In uremic s-HPT and in vitro, no overall changes in methylation levels in the promoter regions of parathyroid CaR and VDR genes were found. Thus, epigenetic methylation of these promoters does not explain decreased parathyroid expression of CaR and VDR genes in uremic s-HPT.

  5. Methylation Inactivation Mechanism of Parkin Gene mRNA Expression in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    Ni Haifeng; Jiang Bo; Zhou Zhen; Li Yong; Huang Guangwu

    2014-01-01

    Objective:To investigate the methylation inactivation and the clinical signiifcance of Parkin gene mRNA expression in nasopharyngeal carcinoma (NPC). Methods: The methylation-speciifc PCR (MSP) and semi-quantitative reverse transcription PCR (RT-PCR) were used to detect methylation and the mRNA expression level of Parkin gene in 54 cases of NPC tissues and 16 cases of normal nasopharyngeal epithelial (NNE) tissues.The mRNA expression of Parkin gene in two NPC cell lines (CNE1 and CNE2) were detected before and after treatment with the methyltransferase inhibitor 5-aza-2-deoxycytidine so as to analyze the effects of Parkin gene methylation and demethylation on Parkin gene mRNA expression and the relationship between Parkin gene mRNA expression and clinical factors. Results: The methylation frequency of Parkin gene in human NPC tissues was 62.96% (34/54), but didn’t happen in any of 16 cases of NNE tissues. The mRNA expression level was (0.3430±0.4947) in 54 cases of NPC tissues and (1.0052±0.4911) in NNE tissues, showing that the mRNA expression level of NPC tissues was significantly down-regulated (P0.05), but was closely related to lymph node metastasis (P<0.05). Conclusion:Parkin gene mRNA expression, serving as a cancer suppressor gene in the occurrence and development of NPC, is inactivated and regulated by methylation, which also has a negative correlation with lymph node metastasis and could be considered as the judgment of predictive index of clinical prognosis of NPC.

  6. A Study of Mercury Methylation Genetics: Qualitative and Quantitative Analysis of hgcAB in Pure Culture

    Science.gov (United States)

    Christensen, G. A.; Wymore, A. M.; King, A. J.; Podar, M.; Hurt, R. A., Jr.; Santillan, E. F. U.; Gilmour, C. C.; Brandt, C. C.; Brown, S. D.; Palumbo, A. V.; Elias, D. A.

    2015-12-01

    Two proteins (HgcA and HgcB) have been determined to be essential for mercury (Hg)-methylation and either one alone is not sufficient for this process. Detection and quantification of these genes to determine at risk environments is critical. Universal degenerate polymerase chain reaction (PCR) primers spanning hgcAB were developed to ascertain organismal diversity and validate that both genes were present as an established prerequisite for Hg-methylation. To confirm this approach, an extensive set of pure cultures with published genomes (including methylators and non-methylators: 13 Deltaproteobacteria, 9 Firmicutes, and 10 methanogenic Archaea) were assayed with the newly designed universal hgcAB primer set. A single band within an agarose gel was observed for the majority of the cultures with known hgcAB and confirmed via Sanger sequencing. For environmental applications, once the potential for Hg-methylation is established from PCR amplification with the universal hgcAB primer set, quantification of clade-specific hgcAB gene abundance is desirable. We developed quantitative polymerase chain reaction (qPCR) degenerate primers targeting hgcA from each of the three dominate clades (Deltaproteobacteria, Firmicutes and methanogenic Archaea) known to be associated with anaerobic Hg-methylation. The qPCR primers amplify virtually all hgcA positive cultures overall and are specific for their designed clade. Finally, to ensure the procedure is robust and sensitive in complex environmental matrices, cells from all clades were mixed in different combinations and ratios to assess qPCR primer specificity. The development and validation of these high fidelity quantitative molecular tools now allows for rapid and accurate risk management assessment in any environment.

  7. Variation in genomic methylation in natural populations of chinese white poplar.

    Science.gov (United States)

    Ma, Kaifeng; Song, Yuepeng; Yang, Xiaohui; Zhang, Zhiyi; Zhang, Deqiang

    2013-01-01

    It is thought that methylcytosine can be inherited through meiosis and mitosis, and that epigenetic variation may be under genetic control or correlation may be caused by neutral drift. However, DNA methylation also varies with tissue, developmental stage, and environmental factors. Eliminating these factors, we analyzed the levels and patterns, diversity and structure of genomic methylcytosine in the xylem of nine natural populations of Chinese white poplar. On average, the relative total methylation and non-methylation levels were approximately 26.567% and 42.708% (Pdifferentiation (GST  = 0.159) were assessed by Shannon's diversity index. Co-inertia analysis indicated that methylation-sensitive polymorphism (MSP) and genomic methylation pattern (CG-CNG) profiles gave similar distributions. Using a between-group eigen analysis, we found that the Hebei and Shanxi populations were independent of each other, but the Henan population intersected with the other populations, to some degree. Genome methylation in Populus tomentosa presented tissue-specific characteristics and the relative 5'-CCGG methylation level was higher in xylem than in leaves. Meanwhile, the genome methylation in the xylem shows great epigenetic variation and could be fixed and inherited though mitosis. Compared to genetic structure, data suggest that epigenetic and genetic variation do not completely match.

  8. DNA methylation profiles and their relationship with cytogenetic status in adult acute myeloid leukemia.

    Directory of Open Access Journals (Sweden)

    Sara Alvarez

    Full Text Available BACKGROUND: Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. METHODOLOGY/PRINCIPAL FINDINGS: We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. CONCLUSIONS/SIGNIFICANCE: Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.

  9. Gene Methylation Biomarkers in Sputum and Plasma as Predictors for Lung Cancer Recurrence.

    Science.gov (United States)

    Belinsky, Steven A; Leng, Shuguang; Wu, Guodong; Thomas, Cynthia L; Picchi, Maria A; Lee, Sandra J; Aisner, Seena; Ramalingam, Suresh; Khuri, Fadlo R; Karp, Daniel D

    2017-09-13

    Detection of methylated genes in exfoliated cells from the lungs of smokers provides an assessment of the extent of field cancerization, is a validated biomarker for predicting lung cancer, and provides some discrimination when interrogated in blood. The potential utility of this 8-gene methylation panel for predicting tumor recurrence has not been assessed. The Eastern Cooperative Oncology Group initiated a prevention trial (ECOG-ACRIN5597) that enrolled resected Stage I non-small cell lung cancer patients who were randomized 2:1 to receive selenized yeast versus placebo for four years. We conducted a correlative biomarker study to assess prevalence for methylation of the 8-gene panel in longitudinally collected sputum and blood after tumor resection to determine if selenium alters their methylation profile and whether this panel predicts local and/or distant recurrence. Patients (n=1561) were enrolled into the prevention trial, 565 participated in the biomarker study with 122 recurrences among that group. Assessing the association between recurrence and risk of gene methylation longitudinally for up to 48 months showed a 1.4-fold increase in odds ratio for methylation in sputum in the placebo group independent of location (local or distant). Kaplan Meier curves evaluating the association between number of methylated genes and time to recurrence showed no increased risk in sputum, while a significant hazard ratio of 1.5 was seen in plasma. Methylation detection in sputum and blood is associated with risk for recurrence. Copyright ©2017, American Association for Cancer Research.

  10. DNA methylation profiling of well-differentiated thyroid cancer uncovers markers of recurrence free survival.

    Science.gov (United States)

    Mancikova, Veronika; Buj, Raquel; Castelblanco, Esmeralda; Inglada-Pérez, Lucía; Diez, Anna; de Cubas, Aguirre A; Curras-Freixes, Maria; Maravall, Francisco Xavier; Mauricio, Didac; Matias-Guiu, Xavier; Puig-Domingo, Manel; Capel, Ismael; Bella, María Rosa; Lerma, Enrique; Castella, Eva; Reverter, Jordi Lluis; Peinado, Miguel Ángel; Jorda, Mireia; Robledo, Mercedes

    2014-08-01

    Thyroid cancer is a heterogeneous disease with several subtypes characterized by cytological, histological and genetic alterations, but the involvement of epigenetics is not well understood. Here, we investigated the role of aberrant DNA methylation in the development of well-differentiated thyroid tumors. We performed genome-wide DNA methylation profiling in the largest well-differentiated thyroid tumor series reported to date, comprising 83 primary tumors as well as 8 samples of adjacent normal tissue. The epigenetic profiles were closely related to not only tumor histology but also the underlying driver mutation; we found that follicular tumors had higher levels of methylation, which seemed to accumulate in a progressive manner along the tumorigenic process from adenomas to carcinomas. Furthermore, tumors harboring a BRAF or RAS mutation had a larger number of hypo- or hypermethylation events, respectively. The aberrant methylation of several candidate genes potentially related to thyroid carcinogenesis was validated in an independent series of 52 samples. Furthermore, through the integration of methylation and transcriptional expression data, we identified genes whose expression is associated with the methylation status of their promoters. Finally, by integrating clinical follow-up information with methylation levels we propose etoposide-induced 2.4 and Wilms tumor 1 as novel prognostic markers related to recurrence-free survival. This comprehensive study provides insights into the role of DNA methylation in well-differentiated thyroid cancer development and identifies novel markers associated with recurrence-free survival. © 2013 UICC.

  11. 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.

  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. 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…

  14. High-density sampling differential display of prokaryotic mRNAs with RAP-PCR.

    Science.gov (United States)

    Walters, Dana M; Rouvière, Pierre E

    2006-01-01

    A high-throughput approach to prokaryotic differential display has been developed. A large number of reverse transcription polymerase chain reactions (RT-PCR) are performed on total RNA isolated from induced and control bacterial cultures. Each RT-PCR reaction uses a single oligonucleotide primer and constitutes an independent sampling of the mRNA population. The large number of reactions performed allows the repeated sampling of the targeted polycistronic mRNA, which is clearly identified among possible false positives.

  15. Highly sensitive detection of DNA methylation levels by using a quantum dot-based FRET method

    Science.gov (United States)

    Ma, Yunfei; Zhang, Honglian; Liu, Fangming; Wu, Zhenhua; Lu, Shaohua; Jin, Qinghui; Zhao, Jianlong; Zhong, Xinhua; Mao, Hongju

    2015-10-01

    DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR amplification for the incorporation of Alexa Fluor-647 (A647) fluorophores. DNA methylation levels can be detected qualitatively through gel analysis and quantitatively by the signal amplification from QDs to A647 during FRET. Furthermore, the methylation levels of three tumor suppressor genes, PCDHGB6, HOXA9 and RASSF1A, in 20 lung adenocarcinoma and 20 corresponding adjacent nontumorous tissue (NT) samples were measured to verify the feasibility of the QD-based FRET method and a high sensitivity for cancer detection (up to 90%) was achieved. Our QD-based FRET method is a convenient, continuous and high-throughput method, and is expected to be an alternative for detecting DNA methylation as a biomarker for certain human cancers.DNA methylation is the most frequently studied epigenetic modification that is strongly involved in genomic stability and cellular plasticity. Aberrant changes in DNA methylation status are ubiquitous in human cancer and the detection of these changes can be informative for cancer diagnosis. Herein, we reported a facile quantum dot-based (QD-based) fluorescence resonance energy transfer (FRET) technique for the detection of DNA methylation. The method relies on methylation-sensitive restriction enzymes for the differential digestion of genomic DNA based on its methylation status. Digested DNA is then subjected to PCR

  16. 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

  17. Genome-wide DNA methylation analysis in permanent atrial fibrillation.

    Science.gov (United States)

    Zhao, Guochang; Zhou, Jian; Gao, Jie; Liu, Yan; Gu, Song; Zhang, Xitao; Su, Pixiong

    2017-10-01

    Atrial fibrillation (AF) is a highly heterogeneous genetic disease; however, the pathogenesis of AF cannot be explained by genetic variants alone. DNA methylation is a heritable method of gene expression regulation, and may be a potential regulatory mechanism in AF. Therefore, in the present study, the genome‑wide DNA methylation pattern in cells derived from the left atrium of patients with permanent AF (n=7) was compared with that of healthy heart donors (n=4) with a normal sinus rhythm (SR). Enriched biological functions of the differentially methylated genes were assessed. Integrated analysis of genome‑wide methylation and mRNA expression profiles was performed, and reverse transcription quantitative‑polymerase chain reaction (RT‑qPCR) was used to determine the expression levels of four selected genes. A total of 417 differentially methylated CpG sites were identified in the fibrillating atrium (P0.17); the majority of which were located in gene‑body and intergenic regions outside of CpG islands. Aberrantly methylated genes participated in the activation of inflammation, sodium and potassium ion transport, fibrosis and the reduction of lipid metabolism. Hypermethylation in the AF susceptible loci, paired‑like homeodomain transcription factor 2 (chromosome 4q25) and coiled‑coil domain containing 141 (chromosome 2q31), as well as hypomethylation in the calcium voltage‑gated channel subunit α1C (chromosome 12p13) locus, were identified in all patients with AF. Of the 420 upregulated and 567 downregulated genes previously identified in patients with AF relative to those with normal SR (fold‑change >2.0; P≤0.05), 12 genes were hypomethylated and eight genes were hypermethylated in each group, respectively (|β|>0.2: Peffect of DNA methylation on gene expression. These results suggest that DNA methylation‑mediated regulation of gene expression may serve an important role in AF pathogenesis, and several susceptible AF CpG loci were

  18. Quantitative assay of photoinduced DNA strand breaks by real-time PCR.

    Science.gov (United States)

    Wiczk, Justyna; Westphal, Kinga; Rak, Janusz

    2016-09-05

    Real-time PCR (qPCR) - a modern methodology primarily used for studying gene expression has been employed for the quantitative assay of an important class of DNA damage - single strand breaks. These DNA lesions which may lead to highly cytotoxic double strand breaks were quantified in a model system where double stranded DNA was sensitized to UV photons by labeling with 5-bromo-2'-deoxyuridine. The amount of breaks formed due to irradiation with several doses of 320nm photons was assayed by two independent methods: LC-MS and qPCR. A very good agreement between the relative damage measured by the two completely different analytical tools proves the applicability of qPCR for the quantitative analysis of SSBs. Our results suggest that the popularity of the hitherto underestimated though accurate and site-specific technique of real-time PCR may increase in future DNA damage studies.

  19. Independence among People with Disabilities: II. Personal Independence Profile.

    Science.gov (United States)

    Nosek, Margaret A.; And Others

    1992-01-01

    Developed Personal Independence Profile (PIP) as an instrument to measure aspects of independence beyond physical and cognitive functioning in people with diverse disabilities. PIP was tested for reliability and validity with 185 subjects from 10 independent living centers. Findings suggest that the Personal Independence Profile measures the…

  20. Histone Methylation by Temozolomide; A Classic DNA Methylating Anticancer Drug

    Science.gov (United States)

    Pickard, Amanda J.; Diaz, Anthony Joseph; Mura, Hugo; Nyuwen, Lila; Coello, Daniel; Sheva, Saif; Maria, Nava; Gallo, James M.; Wang, Tieli

    2017-01-01

    Background/Aim The alkylating agent, temozolomide (TMZ), is considered the standard-of-care for high-grade astrocytomas –known as glioblastoma multiforme (GBM)– an aggressive type of tumor with poor prognosis. The therapeutic benefit of TMZ is attributed to formation of DNA adducts involving the methylation of purine bases in DNA. We investigated the effects of TMZ on arginine and lysine amino acids, histone H3 peptides and histone H3 proteins. Materials and Methods Chemical modification of amino acids, histone H3 peptide and protein by TMZ was performed in phosphate buffer at physiological pH. The reaction products were examined by mass spectrometry and western blot analysis. Results Our results showed that TMZ following conversion to a methylating cation, can methylate histone H3 peptide and histone H3 protein, suggesting that TMZ exerts its anticancer activity not only through its interaction with DNA, but also through alterations of protein post-translational modifications. Conclusion The possibility that TMZ can methylate histones involved with epigenetic regulation of protein indicates a potentially unique mechanism of action. The study will contribute to the understanding the anticancer activity of TMZ in order to develop novel targeted molecular strategies to advance the cancer treatment. PMID:27354585

  1. Methylation profiling of 48 candidate genes in tumor and matched normal tissues from breast cancer patients.

    Science.gov (United States)

    Li, Zibo; Guo, Xinwu; Wu, Yepeng; Li, Shengyun; Yan, Jinhua; Peng, Limin; Xiao, Zhi; Wang, Shouman; Deng, Zhongping; Dai, Lizhong; Yi, Wenjun; Xia, Kun; Tang, Lili; Wang, Jun

    2015-02-01

    Gene-specific methylation alterations in breast cancer have been suggested to occur early in tumorigenesis and have the potential to be used for early detection and prevention. The continuous increase in worldwide breast cancer incidences emphasizes the urgent need for identification of methylation biomarkers for early cancer detection and patient stratification. Using microfluidic PCR-based target enrichment and next-generation bisulfite sequencing technology, we analyzed methylation status of 48 candidate genes in paired tumor and normal tissues from 180 Chinese breast cancer patients. Analysis of the sequencing results showed 37 genes differentially methylated between tumor and matched normal tissues. Breast cancer samples with different clinicopathologic characteristics demonstrated distinct profiles of gene methylation. The methylation levels were significantly different between breast cancer subtypes, with basal-like and luminal B tumors having the lowest and the highest methylation levels, respectively. Six genes (ACADL, ADAMTSL1, CAV1, NPY, PTGS2, and RUNX3) showed significant differential methylation among the 4 breast cancer subtypes and also between the ER +/ER- tumors. Using unsupervised hierarchical clustering analysis, we identified a panel of 13 hypermethylated genes as candidate biomarkers that performed a high level of efficiency for cancer prediction. These 13 genes included CST6, DBC1, EGFR, GREM1, GSTP1, IGFBP3, PDGFRB, PPM1E, SFRP1, SFRP2, SOX17, TNFRSF10D, and WRN. Our results provide evidence that well-defined DNA methylation profiles enable breast cancer prediction and patient stratification. The novel gene panel might be a valuable biomarker for early detection of breast cancer.

  2. 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.

  3. Methylation status of the interferon-gamma gene promoter in chronic hepatitis B

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To evaluate the methylation status at CpG site -55 in the interferon-gamma (IFN-7) gene promoter and its effect on IFN-7 expression in chronic hepatitis B. Method The authors recruited 30 patients with UBeAg-positive chronic hepatitis B (CHB), 30 HBeAg-negative CHB patients, and 30 healthy blood donors. Pyrosequeneing was used to determine the methylation status at CpG site -55 in the IFN-γ gene promoter following bisulfite treatment of DNA in peripheral blood mononuclear cells (PBMCs). The expression of IFN-γ was analyzed by real-time RT-PCR and ELISA. HBV DNA in PBMCs was detected by nested PCR. Results The methylation level at CpG site -55 in the IFN-γ gene promoter was significantly increased, resulting in subsequent down-regulation of the expression of this cytoldne in CHB. The methylation level at CpG site -55 was significantly higher in HBeAg-positive patients than in HBeAg-negative ones (P<0.01) and was also significantly higher in PBMCs from HBV DNA-positive patients than from HBV DNA-negative ones (P<0.01) ; the methylation level at CpG site -55 was positively correlated with the amount of HBV DNA in serum (P<0.01). Oonclusion IFN-γ gene expression appears to be regulated by methylation of the IFN-γ gene promoter in CHB; the methylation level at CpG site -55 is associated with HBV infection.

  4. Mutation and methylation of hMLH1 in gastric carcinomas with microsatellite instability

    Institute of Scientific and Technical Information of China (English)

    Dian-Chun Fang; Rong-Quan Wang; Shi-Ming Yang; Jian-Ming Yang; Hai-Feng Liu; Gui-Yong Peng; Tian-Li Xiao; Yuan-Hui Luo

    2003-01-01

    AIM: To appraise the correlation of mutation and methylation of hMSH1 with microsatellite instability (MSI) in gastric cancers.METHODS: Mutation of hMLH1 was detected by Twodimensional electrophoresis (Two-D) and DNA sequencing;Methylation of hMLH1 promoter was measured with methylation-specific PCR; MST was analyzed by PCR-basecl methods.RESULTS: Sixty-eight cases of sporadic gastric carcinoma were studied for mutation and methylation of hMLH1 promoter and MSI. Three mutations were found, two of them were caused by a single bp substitution and one was caused by a 2 bp substitution, which displayed similar Two-D band pattern.Methylation of hMLH1 promoter was detected in 11(16.2%)gastric cancer. By using five MSI markers, MSI in at least one locus was detected in 17/68(25 %) of the tumors analyzed.Three hMLH1 mutations were all detected in MSI-H (≥2 loci,n=8), but no mutation was found in MSI-L (only one locus,n=9) or MSS (tumor lacking MSI or stable, n=51). Methylation frequency of hMLH1 in MSI-H (87.5 %, 7/8) was significantly higher than that in MSI-L (11.1%, 1/9) or MSS (5.9%, 3/51)(P<0.01-0.001), but no difference was found between MSI-L and MSS (P>0.05).CONCLUSION: Both mutation and methylation of hMLH1are involved in the MSI pathway but not related to the LOH pathway in gastric carcinogenesis.

  5. Dynamics of MBD2 deposition across methylated DNA regions during malignant transformation of human mammary epithelial cells.

    Science.gov (United States)

    Devailly, Guillaume; Grandin, Mélodie; Perriaud, Laury; Mathot, Pauline; Delcros, Jean-Guy; Bidet, Yannick; Morel, Anne-Pierre; Bignon, Jean-Yves; Puisieux, Alain; Mehlen, Patrick; Dante, Robert

    2015-07-13

    DNA methylation is thought to induce transcriptional silencing through the combination of two mechanisms: the repulsion of transcriptional activators unable to bind their target sites when methylated, and the recruitment of transcriptional repressors with specific affinity for methylated DNA. The Methyl CpG Binding Domain proteins MeCP2, MBD1 and MBD2 belong to the latter category. Here, we present MBD2 ChIPseq data obtained from the endogenous MBD2 in an isogenic cellular model of oncogenic transformation of human mammary cells. In immortalized (HMEC-hTERT) or transformed (HMLER) cells, MBD2 was found in a large proportion of methylated regions and associated with transcriptional silencing. A redistribution of MBD2 on methylated DNA occurred during oncogenic transformation, frequently independently of local DNA methylation changes. Genes downregulated during HMEC-hTERT transformation preferentially gained MBD2 on their promoter. Furthermore, depletion of MBD2 induced an upregulation of MBD2-bound genes methylated at their promoter regions, in HMLER cells. Among the 3,160 genes downregulated in transformed cells, 380 genes were methylated at their promoter regions in both cell lines, specifically associated by MBD2 in HMLER cells, and upregulated upon MBD2 depletion in HMLER. The transcriptional MBD2-dependent downregulation occurring during oncogenic transformation was also observed in two additional models of mammary cell transformation. Thus, the dynamics of MBD2 deposition across methylated DNA regions was associated with the oncogenic transformation of human mammary cells.

  6. Global DNA cytosine methylation as an evolving trait: phylogenetic signal and correlated evolution with genome size in Angiosperms

    Directory of Open Access Journals (Sweden)

    Conchita eAlonso

    2015-01-01

    Full Text Available DNA cytosine methylation is a widespread epigenetic mechanism in eukaryotes, and plant genomes commonly are densely methylated. Genomic methylation can be associated with functional consequences such as mutational events, genomic instability or altered gene expression, but little is known on interspecific variation in global cytosine methylation in plants. In this paper, we compare global cytosine methylation estimates obtained by HPLC and use a phylogenetically-informed analytical approach to test for significance of evolutionary signatures of this trait across 54 angiosperm species in 25 families. We evaluate whether interspecific variation in global cytosine methylation is statistically related to phylogenetic distance and also whether it is evolutionarily correlated with genome size (C-value. Global cytosine methylation varied widely between species, ranging between 5.3% (Arabidopsis and 39.2% (Narcissus. Differences between species were related to their evolutionary trajectories, as denoted by the strong phylogenetic signal underlying interspecific variation. Global cytosine methylation and genome size were evolutionarily correlated, as revealed by the significant relationship between the corresponding phylogenetically independent contrasts. On average, a ten-fold increase in genome size entailed an increase of about 10% in global cytosine methylation. Results show that global cytosine methylation is an evolving trait in angiosperms whose evolutionary trajectory is significantly linked to changes in genome size, and suggest that the evolutionary implications of epigenetic mechanisms are likely to vary between plant lineages.

  7. Anchored PCR (A-PCR):A new method for chromosome walking

    Institute of Scientific and Technical Information of China (English)

    CHEN Bojun; SUN Chao; WANG Yong; HU Yuanlei; LIN Zhongping

    2004-01-01

    @@ PCR-based techniques are most popular methods for isolation of DNA sequences flanking a known region.Such techniques published to date mainly include three types: inverse PCR (IPCR)[1-3], ligation-mediated PCR (LM-PCR)[4-9] and randomly primed PCR (RP-PCR)[10-12].IPCR was the first method developed for this kind of purpose. However, it is now rarely used because of the difficulty in finding suitable restriction sites in the target region or poor circularization of the template molecule.LM-PCR and RP-PCR are more frequently used nowadays, yet they also have some limitations. For example,LM-PCR depends on restriction sites within a reasonable distance in the flanking regions, while the amplified products of RP-PCR are generally small (<1 kb). Moreover, both methods often result in excessive amplification of non-specific molecules, which greatly reduces their efficiencies in obtaining sequences of interest. To resolve these problems, some new strategies have emerged in the past few years, such as Vectorette-PCR[6], biotin-capture PCR[7], TAIL-PCR[l2] and T-linker PCR[9]. These improved methods are more efficient than their old versions;however, most of them are still limited by restriction digestion or ligation. Although the intervening steps are avoided in TAIL-PCR, the amplified fragments are often small because of the use of random primers.

  8. Dynamics of nucleosome assembly and effects of DNA methylation.

    Science.gov (United States)

    Lee, Ju Yeon; Lee, Jaehyoun; Yue, Hongjun; Lee, Tae-Hee

    2015-02-13

    The nucleosome is the fundamental packing unit of the eukaryotic genome, and CpG methylation is an epigenetic modification associated with gene repression and silencing. We investigated nucleosome assembly mediated by histone chaperone Nap1 and the effects of CpG methylation based on three-color single molecule FRET measurements, which enabled direct monitoring of histone binding in the context of DNA wrapping. According to our observation, (H3-H4)2 tetramer incorporation must precede H2A-H2B dimer binding, which is independent of DNA termini wrapping. Upon CpG methylation, (H3-H4)2 tetramer incorporation and DNA termini wrapping are facilitated, whereas proper incorporation of H2A-H2B dimers is inhibited. We suggest that these changes are due to rigidified DNA and increased random binding of histones to DNA. According to the results, CpG methylation expedites nucleosome assembly in the presence of abundant DNA and histones, which may help facilitate gene packaging in chromatin. The results also indicate that the slowest steps in nucleosome assembly are DNA termini wrapping and tetramer positioning, both of which are affected heavily by changes in the physical properties of DNA.

  9. Genome-wide screen of genes imprinted in sorghum endosperm, and the roles of allelic differential cytosine methylation.

    Science.gov (United States)

    Zhang, Meishan; Li, Ning; He, Wenan; Zhang, Huakun; Yang, Wei; Liu, Bao

    2016-02-01

    Imprinting is an epigenetic phenomenon referring to allele-biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species-specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent-of-origin expression patterns in the endosperm of a pair of reciprocal F1 hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum-specific imprinted genes relative to these three plant species. Allele-biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty-six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT-PCR, and the majority of them showed endosperm-specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5' upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele-differential methylation.

  10. BRCA-1 methylation and TP53 mutation in triple-negative breast cancer patients without pathological complete response to taxane-based neoadjuvant chemotherapy.

    Science.gov (United States)

    Foedermayr, Mathilde; Sebesta, Miriam; Rudas, Margaretha; Berghoff, Anna S; Promberger, Regina; Preusser, Matthias; Dubsky, Peter; Fitzal, Florian; Gnant, Michael; Steger, Guenther G; Weltermann, Ansgar; Zielinski, Christoph C; Zach, Otto; Bartsch, Rupert

    2014-04-01

    Triple-negative breast cancer (TNBC) patients without pathological complete response (pCR) to neoadjuvant chemotherapy have an unfavourable prognosis. TNBC harbouring BRCA-1 germline mutations may be less responsive to taxanes, while sensitivity to DNA-damaging agents is retained. A similar effect was seen in tumours with epigenetic BRCA-1 silencing. Patients without pCR to neoadjuvant chemotherapy consisting of epirubicin plus docetaxel routinely received post-operative CMF at our centre. Here, we investigated the effect of adjuvant CMF in patients with or without BRCA-1 methylation or TP53 mutation. DNA was extracted from formalin-fixed paraffin-embedded tissue. For determining BRCA-1 methylation status, quantitative methylation-specific PCR was performed. For the investigation of TP53 mutation status, DNA was PCR amplified and sequenced by Sanger sequencing. Twenty-four patients were included; BRCA-1 methylation was present in 41.7 %, while TP53 mutations were observed in 66.7 %. At a median follow-up of 27.5 months, 20 % of patients with BRCA-1 methylation had a disease-free survival (DFS) event, as compared to 64.3 % in the non-methylated group (p = 0.0472). Median DFS in the non-methylated group was 16 months and was not reached in the methylated group (n.s.). No association TP53 mutation status with clinical outcome was observed. Adjuvant CMF is of limited activity in TNBC refractory to taxane-based neoadjuvant chemotherapy. In this population, BRCA-1 methylation was associated with a significant decrease in DFS events suggesting a better prognosis and potentially retained activity of DNA-damaging agents.

  11. 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 o

  12. Methyl 3-(4-methylbenzylidenecarbazate

    Directory of Open Access Journals (Sweden)

    Yu-Feng Li

    2010-06-01

    Full Text Available The title compound, C10H12N2O2, was prepared by the reaction of methyl carbazate and 4-methylbenzaldehyde. The dihedral angle between the benzene ring and the carbazate fragment is 20.86 (10°. In the crystal structure, molecules are linked by intermolecular N—H...O hydrogen bonds.

  13. 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.

  14. Water Column Methylation in Estuaries

    Science.gov (United States)

    Schartup, A. T.; Calder, R.; Soerensen, A. L.; Mason, R. P.; Balcom, P. H.; Sunderland, E. M.

    2014-12-01

    Methylmercury (MeHg) is a neurotoxin that bioaccumulates in aquatic food webs and affects humans and wildlife through fish consumption. Many studies have measured active methylation/demethylation in ocean margin sediments but few have reported similar rates for the marine water column. This presentation will review available evidence for water column methylation in estuaries, including new experimental measurements of methylation/demethylation rates from a deep subarctic fjord in Labrador Canada collected in Spring and Fall of 2012-2013. We used these and other data to construct a mass budget for MeHg in the estuary and show that water column methylation (with rates ranging from 1.5 to 2.8 % day-1), is the largest contributor, followed by inputs from rivers (4.9 mol year-1), to the in situ pool of MeHg available for uptake by biota. By contrast, the sediment in this system is a net sink for MeHg (-1.5 mol year-1). We discuss the relationship between observed MeHg and other ancillary environmental factors (organic carbon, sulfur and nutrients) as well as implications for the response time of fish to future changes in mercury inputs.

  15. Altered chromatin structure associated with methylation-induced gene silencing in cancer cells: correlation of accessibility, methylation, MeCP2 binding and acetylation

    Science.gov (United States)

    Nguyen, Carvell T.; Gonzales, Felicidad A.; Jones, Peter A.

    2001-01-01

    Silencing of tumor-suppressor genes by hypermethylation of promoter CpG islands is well documented in human cancer and may be mediated by methyl-CpG-binding proteins, like MeCP2, that are associated in vivo with chromatin modifiers and transcriptional repressors. However, the exact dynamic between methylation and chromatin structure in the regulation of gene expression is not well understood. In this study, we have analyzed the methylation status and chromatin structure of three CpG islands in the p14(ARF)/p16(INK4A) locus in a series of normal and cancer cell lines using methylation-sensitive digestion, MspI accessibility in intact nuclei and chromatin immunoprecipitation (ChIP) assays. We demonstrate the existence of an altered chromatin structure associated with the silencing of tumor-suppressor genes in human cancer cell lines involving CpG island methylation, chromatin condensation, histone deacetylation and MeCP2 binding. The data showed that MeCP2 could bind to methylated CpG islands in both promoters and exons; MeCP2 does not interfere with transcription when bound at an exon, suggesting a more generalized role for the protein beyond transcriptional repression. In the absence of methylation, it is demonstrated that CpG islands located in promoters versus exons display marked differences in the levels of acetylation of associated histone H3, suggesting that chromatin remodeling can be achieved by methylation-independent processes and perhaps explaining why non-promoter CpG islands are more susceptible to de novo methylation than promoter islands. PMID:11713309

  16. 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

  17. Application of Reverse Transcription-PCR and Real-Time PCR in Nanotoxicity Research

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2016-01-01

    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. PMID:22975959

  18. Dynamic Enhancer Methylation--A Previously Unrecognized Switch for Tissue-Type Plasminogen Activator Expression.

    Directory of Open Access Journals (Sweden)

    Mia Magnusson

    Full Text Available Tissue-type plasminogen activator (t-PA, which is synthesized in the endothelial cells lining the blood vessel walls, is a key player in the fibrinolytic system protecting the circulation against occluding thrombus formation. Although classical gene regulation has been quite extensively studied in order to understand the mechanisms behind t-PA regulation, epigenetics, including DNA methylation, still is a largely unexplored field. The aim of this study was to establish the methylation pattern in the t-PA promoter and enhancer in non-cultured compared to cultured human umbilical vein endothelial cells (HUVECs, and to simultaneously examine the level of t-PA gene expression. Bisulphite sequencing was used to evaluate the methylation status, and real-time RT-PCR to determine the gene expression level. While the t-PA promoter was stably unmethylated, we surprisingly observed a rapid reduction in the amount of methylation in the enhancer during cell culturing. This demethylation was in strong negative correlation with a pronounced (by a factor of approximately 25 increase in t-PA gene expression levels. In this study, we show that the methylation level in the t-PA enhancer appears to act as a previously unrecognized switch controlling t-PA expression. Our findings, which suggest that DNA methylation is quite dynamic, have implications also for the interpretation of cell culture experiments in general, as well as in a wider biological context.

  19. Increased methylation of interleukin 6 gene is associated with obesity in Korean women.

    Science.gov (United States)

    Na, Yeon Kyung; Hong, Hae Sook; Lee, Won Kee; Kim, Young Hun; Kim, Dong Sun

    2015-05-01

    Obesity is the fifth leading risk for death globally, and a significant challenge to global health. It is a common, complex, non-malignant disease and develops due to interactions between the genes and the environment. DNA methylation can act as a downstream effector of environmental signals; analysis of this process therefore holds substantial promise for identifying mechanisms through which genetic and environmental factors jointly contribute to disease risk. To assess the effects of excessive weight and obesity on gene-specific methylation levels of promoter regions, we determined the methylation status of four genes involved in inflammation and oxidative stress [interleukin 6 (IL6), tumor necrosis factor α (TNFα), mitochondrial transcription factor A (TFAM), and glucose transport 4 (GLUT4)] in blood cell-derived DNA from healthy women volunteers with a range of body mass indices (BMIs) by methylation-specific PCR. Interestingly, the samples from obese individuals (BMI ≥ 30 kg/m(2)) showed significantly increased hypermethylation for IL6 gene compared to normal weight (BMI genes between each group. These findings suggest that aberrant DNA methylation of IL6 gene promoter may play an important role in the etiology and pathogenesis of obesity and IL6 methylation could be used as molecular biomarker for obesity risk assessment. Further studies are required to elucidate the potential mechanisms underlying this relationship.

  20. 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.

  1. Adenine N6-methylation in diverse fungi.

    Science.gov (United States)

    Seidl, Michael F

    2017-05-26

    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 adenine methylation of transcriptionally active genes in early-diverging fungi that, together with recent other work, emphasizes the importance of adenine methylation in eukaryotes.

  2. Frame independent cosmological perturbations

    Energy Technology Data Exchange (ETDEWEB)

    Prokopec, Tomislav; Weenink, Jan, E-mail: t.prokopec@uu.nl, E-mail: j.g.weenink@uu.nl [Institute for Theoretical Physics and Spinoza Institute, Utrecht University, Leuvenlaan 4, 3585 CE Utrecht (Netherlands)

    2013-09-01

    We compute the third order gauge invariant action for scalar-graviton interactions in the Jordan frame. We demonstrate that the gauge invariant action for scalar and tensor perturbations on one physical hypersurface only differs from that on another physical hypersurface via terms proportional to the equation of motion and boundary terms, such that the evolution of non-Gaussianity may be called unique. Moreover, we demonstrate that the gauge invariant curvature perturbation and graviton on uniform field hypersurfaces in the Jordan frame are equal to their counterparts in the Einstein frame. These frame independent perturbations are therefore particularly useful in relating results in different frames at the perturbative level. On the other hand, the field perturbation and graviton on uniform curvature hypersurfaces in the Jordan and Einstein frame are non-linearly related, as are their corresponding actions and n-point functions.

  3. Emissivity independent optical pyrometer

    Science.gov (United States)

    Earl, Dennis Duncan; Kisner, Roger A.

    2017-04-04

    Disclosed herein are representative embodiments of methods, apparatus, and systems for determining the temperature of an object using an optical pyrometer. Certain embodiments of the disclosed technology allow for making optical temperature measurements that are independent of the surface emissivity of the object being sensed. In one of the exemplary embodiments disclosed herein, a plurality of spectral radiance measurements at a plurality of wavelengths is received from a surface of an object being measured. The plurality of the spectral radiance measurements is fit to a scaled version of a black body curve, the fitting comprising determining a temperature of the scaled version of the black body curve. The temperature is then output. The present disclosure is not to be construed as limiting and is instead directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone or in various combinations and subcombinations with one another.

  4. Emissivity independent optical pyrometer

    Energy Technology Data Exchange (ETDEWEB)

    Earl, Dennis Duncan; Kisner, Roger A.

    2017-04-04

    Disclosed herein are representative embodiments of methods, apparatus, and systems for determining the temperature of an object using an optical pyrometer. Certain embodiments of the disclosed technology allow for making optical temperature measurements that are independent of the surface emissivity of the object being sensed. In one of the exemplary embodiments disclosed herein, a plurality of spectral radiance measurements at a plurality of wavelengths is received from a surface of an object being measured. The plurality of the spectral radiance measurements is fit to a scaled version of a black body curve, the fitting comprising determining a temperature of the scaled version of the black body curve. The temperature is then output. The present disclosure is not to be construed as limiting and is instead directed toward all novel and nonobvious features and aspects of the various disclosed embodiments, alone or in various combinations and subcombinations with one another.

  5. Clinical significance of aberrant Wnt7a promoter methylation in human non-small cell lung cancer in Koreans.

    Science.gov (United States)

    Kim, Tae-Hyung; Moon, Ji-Yong; Kim, Sang-Heon; Paik, Seung Sam; Yoon, Ho Joo; Shin, Dong Ho; Park, Sung Soo; Sohn, Jang Won

    2015-02-01

    The Wnt signaling pathway has regulatory roles in cell proliferation, differentiation, and polarity. Aberrant Wnt pathway regulation can lead to abnormal cell proliferation and cancer, and loss of Wnt7a expression has been demonstrated in lung cancer cell lines. E-cadherin keeps intercellular integrity and prevents metastasis. Therefore, E-cadherin has been known as a prognostic factor in cancer. In the present study, we investigated the E-cadherin expression status by immunohistochemical stain and the Wnt7a promoter methylation status in human non-small cell lung carcinoma (NSCLC) by methylation-specific PCR. We also analyzed their correlations with clinicopathological factors. Methylation of the Wnt7a gene promoter was detected in the lung tissues of 32 of 121 (26.4%) patients with NSCLC. Wnt7a promoter methylation was correlated with advanced tumor stage (P = 0.036) and distant metastasis (P = 0.037). In addition, Wnt7a promoter methylation showed correlation with loss of E-cadherin expression (P promoter methylation was not closely related with gender, age, histological type, or smoking habit. Even though Wnt7a methylation could not show significant correlation with the long term survival of the patients with limited follow up data, these findings suggest that loss of the Wnt7a gene induced by promoter methylation might be another prognostic factor for NSCLC and that restoration of Wnt7a may be a promising treatment for NSCLC.

  6. [Analysis of the status of DACH1 gene promoter methylation in endometrial carcinoma and its clinical significance].

    Science.gov (United States)

    Deng, Xin-Chao; Li, Shao-Ru; Zhang, Qing; Zhou, Cheng-Jun; Yang, Qi-Feng; Jiang, Jie; Kong, Bei-Hua

    2012-04-01

    To analyze the status of DACH1 gene promoter methylation and explore its association with the expression of DACH1 gene promoter methylation and clinical significance of endometrium carcinoma (EC). From February 2004 to August 2008, a total of 80 EC tissue samples with comprehensive surgical pathology staging were collected and used for this study. Twenty normal endometrium tissues in 2008 were abstained from the fractional curettage because of dysfunctional uterine bleeding as control. All samples were confirmed pathologically. Methylation specific PCR (MSP) was performed to detect the promoter methylation of DACH1 gene, and analyze its influence on the expression of DACH1 and the relationship between DACH1 promoter methylation and clinicopathological factors in EC. DACH1 protein expression was detected by western blot. Chi-square test and Pearson test were used for statistical analysis. The rate of promoter methylation of DACH1 gene in the EC tissues was significantly higher than that in the normal endometrium issues (30% vs. 5%, P promoter methylation (r = -0.30, P 0.05). DACH1 gene promoter methylaion could lead to a decrease or absence in the DACH1 expression in EC. The promoter methylation of DACH1 gene may induce the inhibition of DACH1 expression, which might be one of the mechanisms of DACH1 gene inactivation in human EC.

  7. Aberrant methylation of the adenomatous polyposis coli (APC) gene promoter 1A in breast and lung carcinomas.

    Science.gov (United States)

    Virmani, A K; Rathi, A; Sathyanarayana, U G; Padar, A; Huang, C X; Cunnigham, H T; Farinas, A J; Milchgrub, S; Euhus, D M; Gilcrease, M; Herman, J; Minna, J D; Gazdar, A F

    2001-07-01

    The adenomatous polyposis coli (APC) gene is a tumor suppressor gene associated with both familial and sporadic cancer. Despite high rates of allelic loss in lung and breast cancers, point mutations of the APC gene are infrequent in these cancer types. Aberrant methylation of the APC promoter 1A occurs in some colorectal and gastric malignancies, and we investigated whether the same mechanism occurs in lung and breast cancers. The methylation status of the APC gene promoter 1A was analyzed in 77 breast, 50 small cell (SCLC), and 106 non-small cell (NSCLC) lung cancer tumors and cell lines and in 68 nonmalignant tissues by methylation-specific PCR. Expression of the APC promoter 1A transcript was examined in a subset of cell lines by reverse transcription-PCR, and loss of heterozygosity at the gene locus was analyzed by the use of 12 microsatellite and polymorphic markers. Statistical tests were two-sided. Promoter 1A was methylated in 34 of 77 breast cancer tumors and cell lines (44%), in 56 of 106 NSCLC tumors and cell lines (53%), in 13 of 50 SCLC cell lines (26%), and in 3 of 68 nonmalignant samples (4%). Most cell lines tested contained the unmethylated or methylated form exclusively. In 27 cell lines tested, there was complete concordance between promoter methylation and silencing of its transcript. Demethylation with 5-aza-2'-deoxycytidine treatment restored transcript 1A expression in all eight methylated cell lines tested. Loss of heterozygosity at the APC locus was observed in 85% of SCLCs, 83% of NSCLCs, and 63% of breast cancer cell lines. The frequency of methylation in breast cancers increased with tumor stage and size. In summary, aberrant methylation of the 1A promoter of the APC gene and loss of its specific transcript is frequently present in breast and NSCLC cancers and cell lines and, to a lesser extent, in SCLC cell lines. Our findings may be of biological and clinical importance.

  8. The polymerase chain reaction (PCR): general methods.

    Science.gov (United States)

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  9. The role of methylation in urological tumours

    NARCIS (Netherlands)

    Heijden, A.G. van der

    2013-01-01

    Alterations in DNA methylation have been described in human cancer for more than thirty years now. Since the last decade DNA methylation gets more and more important in cancer research. In this review the different alterations of DNA methylation are discussed in testicular germ cell tumours, Wilms't

  10. Quantitative DNA Methylation Profiling in Cancer.

    Science.gov (United States)

    Ammerpohl, Ole; Haake, Andrea; Kolarova, Julia; Siebert, Reiner

    2016-01-01

    Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.

  11. HPV16 oncogenes E6 or/and E7 may influence the methylation status of RASSFIA gene promoter region in cervical cancer cell line HT-3.

    Science.gov (United States)

    Yin, Fufen; Wang, Ning; Wang, Shanshan; Yu, Fengsheng; Sun, Xin; Yu, Xiao; Luo, Bing; Zhao, Chengquan; Wang, Yankui

    2017-04-01

    Both human papillomavirus (HPV) infection and the aberrant Ras associated domain family gene 1A (RASSF1A) promoter methylation status participate in the pathogenesis of cervical cancer. Some studies suggest that E6, and E7 are involved in the pathogenetic mechanisms of RASSF1A. We mainly explored a possible involvement of HPV16 oncogenes E6 or/and E7 in RASSF1A promoter methylation status and possible roles of RASSF1A gene methylation in cervical cancer. Bisulfite genomic sequencing (BGS) PCR combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation status of the RASSF1A gene promoter in HPV16/18-positive and HPV-negative cervical cancer cell lines; ectopically expressed HPV16 E6, E7 and E6/E7 cervical cancer cell lines; normal cervical and cervical cancer tissues. The mRNA and protein expression of RASSF1A was detected by RT-PCR and western blotting. Re-expression and downregulated promoter methylation status were detected in the ectopically expressed HPV16 E6 and E7 cervical cancer cell line HT-3. The methylation status and expression of RASSF1A could be downregulated or reactivated by 5-Aza-dc in HT-3 and C33A cells. Additionally, statistics showed significant hypermethylation of RASSF1A in cervical cancer samples compared to that in normal cervical samples (PE6 and/or E7 may be involved in aberrant methylation and expression of the RASSF1A gene. RASSF1A gene expression could be regulated by its promoter methylation status. Additionally, the false negativity of the HPV detection may contribute to the uncertain relationship between HPV infection and aberrant RASSF1A promoter methylation.

  12. Hypervirulent Clostridium difficile PCR-ribotypes exhibit resistance to widely used disinfectants.

    Directory of Open Access Journals (Sweden)

    Lisa F Dawson

    Full Text Available The increased prevalence of Clostridium difficile infection (CDI has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs, peroxygens, quaternary ammonium compounds (QAC and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings.

  13. Restriction endonucleases digesting DNA in PCR buffer

    Institute of Scientific and Technical Information of China (English)

    LIU Xue-dong; ZHENG Dong; ZHOU Yan-na; MAO Wei-wei; MA Jian-zhang

    2005-01-01

    Six commonly used restriction endonucleases (Res) (Acc I, Ban II, EcoR I, Hind III, Sac I, Sca I) were tested for their ability to directly digest DNA completely in the Polymerase Chain Reaction (PCR) buffers. The results showed that: with the requirement for additional magnesium supplemented as activator, Res, except EcoR I appeared star activity, completely digested unmethylated lambda DNA after overnight incubation in PCR buffer and functioned as equally well as in recommended Restriction Enzyme Buffer provided with each enzyme; all Res tested completely digested PCR products in PCR buffer, it implied digestion of PCR products may often be performed directly in the PCR tube without the requirement for any precipitation or purification steps; and the concentration of MgCl2 from 2.5 mmol·L-1 to 10 mmol·L-1 did not significantly affect activity of Res in PCR buffer. This simplified method for RE digestion of PCR products could have applications in restriction fragment length polymorphism (RFLP) analysis and single-stranded conformational polymorphism (SSCP) analysis of large PCR products. However, usage of this procedure for cloning applications needs further data.

  14. Real-time PCR in microfluidic devices

    Science.gov (United States)

    Becker, Holger; Hlawatsch, Nadine; Klemm, Richard; Moche, Christian; Hansen-Hagge, Thomas; Gärtner, Claudia

    2014-03-01

    A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In case of the "classical" PCR, detection is done by identification of DNA fragments electrophoretically separated in agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two main methods for on-chip PCRs are available: traditional "batch" PCR in chambers on a chip using thermal cycling, requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.

  15. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Science.gov (United States)

    Schrick, Livia; Nitsche, Andreas

    2016-01-01

    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.

  16. Detection of Eperythrozoon wenyoni by PCR assay

    Institute of Scientific and Technical Information of China (English)

    Jian WANG; Yutao ZHU; Jianhua QIN; Fumei ZHANG; Yuelan ZHAO

    2009-01-01

    The objective of this research was to develop a detection method for Eperythrozoon wenyoni infection using polymerase chain reaction (PCR) assay technique. A pair of primers was designed and synthesized according to the conservative sequence 16S rRNA. The PCR assay was performed with the primers. A 985-bp fragment was amplified by using PCR. The amplified fragments with the expected size were identified by EcoR I restriction digestion. The crossing-reaction, specific-reaction and duplicate-reaction indicated that the PCR method is a specific, sensitive, fast and effective method for diagnosing E. Wenyoni infection at group level.

  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. Strategies for discovery and validation of methylated and hydroxymethylated DNA biomarkers.

    Science.gov (United States)

    Olkhov-Mitsel, Ekaterina; Bapat, Bharati

    2012-10-01

    DNA methylation, consisting of the addition of a methyl group at the fifth-position of cytosine in a CpG dinucleotide, is one of the most well-studied epigenetic mechanisms in mammals with important functions in normal and disease biology. Disease-specific aberrant DNA methylation is a well-recognized hallmark of many complex diseases. Accordingly, various studies have focused on characterizing unique DNA methylation marks associated with distinct stages of disease development as they may serve as useful biomarkers for diagnosis, prognosis, prediction of response to therapy, or disease monitoring. Recently, novel CpG dinucleotide modifications with potential regulatory roles such as 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine have been described. These potential epigenetic marks cannot be distinguished from 5-methylcytosine by many current strategies and may potentially compromise assessment and interpretation of methylation data. A large number of strategies have been described for the discovery and validation of DNA methylation-based biomarkers, each with its own advantages and limitations. These strategies can be classified into three main categories: restriction enzyme digestion, affinity-based analysis, and bisulfite modification. In general, candidate biomarkers are discovered using large-scale, genome-wide, methylation sequencing, and/or microarray-based profiling strategies. Following discovery, biomarker performance is validated in large independent cohorts using highly targeted locus-specific assays. There are still many challenges to the effective implementation of DNA methylation-based biomarkers. Emerging innovative methylation and hydroxymethylation detection strategies are focused on addressing these gaps in the field of epigenetics. The development of DNA methylation- and hydroxymethylation-based biomarkers is an exciting and rapidly evolving area of research that holds promise for potential applications in diverse clinical

  19. 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

  20. 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

  1. Two-temperature LATE-PCR endpoint genotyping

    Science.gov (United States)

    Sanchez, J Aquiles; Abramowitz, Jessica D; Salk, Jesse J; Reis, Arthur H; Rice, John E; Pierce, Kenneth E; Wangh, Lawrence J

    2006-01-01

    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 2:1 and 1:2 ratios of the

  2. 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.

  3. DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

    Science.gov (United States)

    Cepek, Pavel; Zajacova, Marta; Kotrbova-Kozak, Anna; Silhova, Elena; Cerna, Marie

    2016-06-01

    Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time.

  4. 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

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

    Science.gov (United States)

    Marconi, Gianpiero; Pace, Roberta; Traini, Alessandra; Raggi, Lorenzo; Lutts, Stanley; Chiusano, Marialuisa; Guiducci, Marcello; Falcinelli, Mario; Benincasa, Paolo; Albertini, Emidio

    2013-01-01

    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-specific methylation differences

  6. Identification and quantification of ice nucleation active microorganisms by digital droplet PCR (ddPCR)

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    Linden, Martin; Pöschl, Ulrich; Fröhlich-Nowoisky, Janine

    2015-04-01

    Several bioaerosol types, including bacteria, fungi, pollen and lichen, have been identified as sources of biological ice nucleators (IN) which induce ice formation already at temperatures as high as -10 °C or above. Accordingly, they potentially contribute widely to environmental ice nucleation in the atmosphere and are of great interest in the study of natural heterogenous ice nucleation processes. Ice nucleation active microorganisms have been found and studied among bacteria (Proteobacteria) and fungi (phyla Basidiomycota and Ascomycota). The mechanisms enabling the microorganisms to ice nucleation are subject to ongoing research. While it has been demonstrated that whole cells can act as ice nucleators in the case of bacteria due to the presence of specific membrane proteins, cell-free ice nucleation active particles seem to be responsible for this phenomenon in fungi and lichen. The identification and quantification of these ice nucleation active microorganisms and their IN in atmospheric samples is crucial to understand their contribution to the pool of atmospheric IN. This is not a trivial task since the respective microorganisms are often prevalent in lowest concentrations and a variety of states, be it viable cells, spores or cell debris from dead cells. Molecular biology provides tools to identify and quantify ice nucleation active microorganisms independent of their state by detecting genetic markers specific for the organism of interest. Those methods are not without their drawbacks in terms of sample material concentration required or reliable standardization. Digital Droplet Polymerase Chain Reaction (ddPCR) was chosen for our demands as a more elegant, quick and specific method in the investigation of ice nucleation active microorganisms in atmospheric samples. The advantages of ddPCR lie in the simultaneous detection and quantification of genetic markers and their original copy numbers in a sample. This is facilitated by the fractionation of the

  7. Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells.

    Science.gov (United States)

    Irahara, Natsumi; Nosho, Katsuhiko; Baba, Yoshifumi; Shima, Kaori; Lindeman, Neal I; Hazra, Aditi; Schernhammer, Eva S; Hunter, David J; Fuchs, Charles S; Ogino, Shuji

    2010-03-01

    Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition, LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk, and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However, no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C + T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCR-pyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA, and may be useful in clinical and research settings.

  8. Heterogeneous DNA Methylation Patterns in the GSTP1 Promoter Lead to Discordant Results between Assay Technologies and Impede Its Implementation as Epigenetic Biomarkers in Breast Cancer.

    Science.gov (United States)

    Alnaes, Grethe I Grenaker; Ronneberg, Jo Anders; Kristensen, Vessela N; Tost, Jörg

    2015-09-17

    Altered DNA methylation patterns are found in many diseases, particularly in cancer, where the analysis of DNA methylation holds the promise to provide diagnostic, prognostic and predictive information of great clinical value. Methylation of the promoter-associated CpG island of GSTP1 occurs in many hormone-sensitive cancers, has been shown to be a biomarker for the early detection of cancerous lesions and has been associated with important clinical parameters, such as survival and response to treatment. In the current manuscript, we assessed the performance of several widely-used sodium bisulfite conversion-dependent methods (methylation-specific PCR, MethyLight, pyrosequencing and MALDI mass-spectrometry) for the analysis of DNA methylation patterns in the GSTP1 promoter. We observed large discordances between the results obtained by the different technologies. Cloning and sequencing of the investigated region resolved single-molecule DNA methylation patterns and identified heterogeneous DNA methylation patterns as the underlying cause of the differences. Heterogeneous DNA methylation patterns in the GSTP1 promoter constitute a major obstacle to the implementation of DNA methylation-based analysis of GSTP1 and might explain some of the contradictory findings in the analysis of the significance of GSTP1 promoter methylation in breast cancer.

  9. 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

  10. DNA methylation changes in atypical adenomatous hyperplasia, adenocarcinoma in situ, and lung adenocarcinoma.

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    Suhaida A Selamat

    Full Text Available BACKGROUND: Aberrant DNA methylation is common in lung adenocarcinoma, but its timing in the phases of tumor development is largely unknown. Delineating when abnormal DNA methylation arises may provide insight into the natural history of lung adenocarcinoma and the role that DNA methylation alterations play in tumor formation. METHODOLOGY/PRINCIPAL FINDINGS: We used MethyLight, a sensitive real-time PCR-based quantitative method, to analyze DNA methylation levels at 15 CpG islands that are frequently methylated in lung adenocarcinoma and that we had flagged as potential markers for non-invasive detection. We also used two repeat probes as indicators of global DNA hypomethylation. We examined DNA methylation in 249 tissue samples from 93 subjects, spanning the putative spectrum of peripheral lung adenocarcinoma development: histologically normal adjacent non-tumor lung, atypical adenomatous hyperplasia (AAH, adenocarcinoma in situ (AIS, formerly known as bronchioloalveolar carcinoma, and invasive lung adenocarcinoma. Comparison of DNA methylation levels between the lesion types suggests that DNA hypermethylation of distinct loci occurs at different time points during the development of lung adenocarcinoma. DNA methylation at CDKN2A ex2 and PTPRN2 is already significantly elevated in AAH, while CpG islands at 2C35, EYA4, HOXA1, HOXA11, NEUROD1, NEUROD2 and TMEFF2 are significantly hypermethylated in AIS. In contrast, hypermethylation at CDH13, CDX2, OPCML, RASSF1, SFRP1 and TWIST1 and global DNA hypomethylation appear to be present predominantly in invasive cancer. CONCLUSIONS/SIGNIFICANCE: The gradual increase in DNA methylation seen for numerous loci in progressively more transformed lesions supports the model in which AAH and AIS are sequential stages in the development of lung adenocarcinoma. The demarcation of DNA methylation changes characteristic for AAH, AIS and adenocarcinoma begins to lay out a possible roadmap for aberrant DNA

  11. Next-generation sequencing methylation profiling of subjects with obesity identifies novel gene changes.

    Science.gov (United States)

    Day, Samantha E; Coletta, Richard L; Kim, Joon Young; Campbell, Latoya E; Benjamin, Tonya R; Roust, Lori R; De Filippis, Elena A; Dinu, Valentin; Shaibi, Gabriel Q; Mandarino, Lawrence J; Coletta, Dawn K

    2016-01-01

    Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity. Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 ± 0.7 kg/m(2)) and obese (n = 10; BMI = 32.9 ± 0.7 kg/m(2)) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change -1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5'UTR (Chr.8:22,423,530-22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity. These results demonstrate that obesity alters the epigenome through DNA methylation and highlights

  12. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR.

    Science.gov (United States)

    Farrell, D J

    1999-02-01

    Certain strains of Neisseria subflava and Neisseria cinerea are known to produce false-positive results with the AMPLICOR Neisseria gonorrhoeae PCR (Roche Diagnostic Systems, Branchburg, N.J.). The analytical sensitivity and analytical specificity of three PCR tests were assessed with 3 geographically diverse N. gonorrhoeae strains and 30 non-N. gonorrhoeae Neisseria spp. The sensitivities of the in-house nested cppB gene and the 16S rRNA PCR methods were greater than that of the AMPLICOR N. gonorrhoeae PCR with purified DNA from all 3 N. gonorrhoeae strains. Six of 14 clinical strains of N. subflava (1 from a vaginal swab, 5 from respiratory sites) produced false-positive AMPLICOR N. gonorrhoeae PCR results and were negative by the two other PCR methods. When applied to 207 clinical specimens selected from a population with a high prevalence ( approximately 9%) of infection, the results for 15 of 96 (15.6%) AMPLICOR-positive specimens and 14 of 17 (82.3%) AMPLICOR-equivocal specimens were not confirmed by the more sensitive nested cppB PCR method. Only 2 of 94 (2.1%) of AMPLICOR N. gonorrhoeae PCR-negative specimens from the same population tested positive by the nested cppB method. These results suggest that for this population the AMPLICOR N. gonorrhoeae PCR test is suitable as a screening test only and all positive results should be confirmed by a PCR method that is more specific and at least as sensitive. This study also illustrates that caution should be used when introducing commercially available nucleic acid amplification-based diagnostic tests into the regimens of tests used for populations not previously tested with these products.

  13. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing.

    Science.gov (United States)

    Urich, Mark A; Nery, Joseph R; Lister, Ryan; Schmitz, Robert J; Ecker, Joseph R

    2015-03-01

    Current high-throughput DNA sequencing technologies enable acquisition of billions of data points through which myriad biological processes can be interrogated, including genetic variation, chromatin structure, gene expression patterns, small RNAs and protein-DNA interactions. Here we describe the MethylC-sequencing (MethylC-seq) library preparation method, a 2-d protocol that enables the genome-wide identification of cytosine DNA methylation states at single-base resolution. The technique involves fragmentation of genomic DNA followed by adapter ligation, bisulfite conversion and limited amplification using adapter-specific PCR primers in preparation for sequencing. To date, this protocol has been successfully applied to genomic DNA isolated from primary cell culture, sorted cells and fresh tissue from over a thousand plant and animal samples.

  14. 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.

  15. HIF3A DNA Methylation Is Associated with Childhood Obesity and ALT.

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    Shuo Wang

    Full Text Available Gene polymorphisms associated so far with body mass index (BMI can explain only 1.18-1.45% of observed variation in BMI. Recent studies suggest that epigenetic modifications, especially DNA methylation, could contribute to explain part of the missing heritability, and two epigenetic genome-wide analysis studies (EWAS have reported that Hypoxia Inducible Factor 3 Alpha Subunit (HIF3A methylation was associated with BMI or BMI change. We therefore assessed whether the HIF3A methylation is associated with obesity and other obesity-related phenotypes in Chinese children. The subjects included 110 severe obese cases aged 7-17y and 110 normal-weight controls matched by age and gender for measurement of blood DNA methylation levels at the HIF3A gene locus using the Sequenom's MassARRAY system. We observed significantly higher methylation levels in obese children than in controls at positions 46801642 and 46801699 in HIF3A gene (P<0.05, and found positive associations between methylation and alanine aminotransferase (ALT levels adjusted by gender, age and BMI at the position 46801699 (r = 0.226, P = 0.007. These results suggest that HIF3A DNA methylation is associated with childhood obesity, and has a BMI-independent association with ALT. The results provide evidence for identifying epigenetic factors of elivated ALT and may be useful for risk assessment and personalized medicine of liver diseases such as non-alcoholic fatty liver disease (NAFLD.

  16. Heritable genome-wide variation of gene expression and promoter methylation between wild and domesticated chickens

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    Nätt Daniel

    2012-02-01

    Full Text Available Abstract Background Variations in gene expression, mediated by epigenetic mechanisms, may cause broad phenotypic effects in animals. However, it has been debated to what extent expression variation and epigenetic modifications, such as patterns of DNA methylation, are transferred across generations, and therefore it is uncertain what role epigenetic variation may play in adaptation. Results In Red Junglefowl, ancestor of domestic chickens, gene expression and methylation profiles in thalamus/hypothalamus differed substantially from that of a domesticated egg laying breed. Expression as well as methylation differences were largely maintained in the offspring, demonstrating reliable inheritance of epigenetic variation. Some of the inherited methylation differences were tissue-specific, and the differential methylation at specific loci were little changed after eight generations of intercrossing between Red Junglefowl and domesticated laying hens. There was an over-representation of differentially expressed and methylated genes in selective sweep regions associated with chicken domestication. Conclusions Our results show that epigenetic variation is inherited in chickens, and we suggest that selection of favourable epigenomes, either by selection of genotypes affecting epigenetic states, or by selection of methylation states which are inherited independently of sequence differences, may have been an important aspect of chicken domestication.

  17. Prognosis value of MGMT promoter methylation for patients with lung cancer: a meta-analysis.

    Science.gov (United States)

    Chen, Chao; Hua, Haiqing; Han, Chenglong; Cheng, Yuan; Cheng, Yin; Wang, Zhen; Bao, Jutao

    2015-01-01

    The role of MGMT promoter methylation in lung cancer (LC) remains controversial. To clarify the association of MGMT promoter methylation with survival in LC, we performed a meta-analysis of the literature with meta-analysis. Trials were selected for further analysis if they provided an independent assessment of MGMT promoter methylation in LC and reported the survival data in the context of MGMT promoter methylation status. Subgroup analyses were conducted according to the study characteristic. A total of 9 trials, which comprised 859 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 1.27 [95% CI 0.88-1.82; test for heterogeneity P = 0.027] suggests that MGMT promoter methylation has none impact on patient survival. In Stage I-III or younger populations, a significant association was found for MGMT promoter methylation in the prognosis of LC. In addition, the heterogeneity disappeared when the analysis was restricted to Stage I-III LC. Our analysis indicates that MGMT promoter methylation in stage I-III or younger patients was significantly correlated with wore survival. Further study is needed to determine these specific subgroups of LC patients.

  18. Etiology matters - Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy.

    Science.gov (United States)

    Dębski, Konrad J; Pitkanen, Asla; Puhakka, Noora; Bot, Anna M; Khurana, Ishant; Harikrishnan, K N; Ziemann, Mark; Kaspi, Antony; El-Osta, Assam; Lukasiuk, Katarzyna; Kobow, Katja

    2016-05-09

    This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation, and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns.

  19. Etiology matters – Genomic DNA Methylation Patterns in Three Rat Models of Acquired Epilepsy

    Science.gov (United States)

    Dębski, Konrad J.; Pitkanen, Asla; Puhakka, Noora; Bot, Anna M.; Khurana, Ishant; Harikrishnan, KN; Ziemann, Mark; Kaspi, Antony; El-Osta, Assam; Lukasiuk, Katarzyna; Kobow, Katja

    2016-01-01

    This study tested the hypothesis that acquired epileptogenesis is accompanied by DNA methylation changes independent of etiology. We investigated DNA methylation and gene expression in the hippocampal CA3/dentate gyrus fields at 3 months following epileptogenic injury in three experimental models of epilepsy: focal amygdala stimulation, systemic pilocarpine injection, or lateral fluid-percussion induced traumatic brain injury (TBI) in rats. In the models studies, DNA methylation and gene expression profiles distinguished controls from injured animals. We observed consistent increased methylation in gene bodies and hypomethylation at non-genic regions. We did not find a common methylation signature in all three different models and few regions common to any two models. Our data provide evidence that genome-wide alteration of DNA methylation signatures is a general pathomechanism associated with epileptogenesis and epilepsy in experimental animal models, but the broad pathophysiological differences between models (i.e. pilocarpine, amygdala stimulation, and post-TBI) are reflected in distinct etiology-dependent DNA methylation patterns. PMID:27157830

  20. Methylation of the SLC6a2 gene promoter in major depression and panic disorder.

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    Richard Bayles

    Full Text Available Reduced function of the noradrenaline transporter (NET has been demonstrated in patients with major depressive disorder (MDD and panic disorder. Attempts to explain NET dysfunction in MDD and panic disorder by genetic variation in the NET gene SLC6a2 have been inconclusive. Transcriptional silencing of the SLC6a2 gene may be an alternative mechanism which can lead to NET dysfunction independent of DNA sequence. The objective of this study was to characterise the DNA methylation state of the SLC6a2 gene promoter in patients with MDD and panic disorder. SLC6a2 promoter methylation was also analysed before and after antidepressant treatment. This study was performed with DNA from blood, using bisulphite sequencing and EpiTYPER methylation analyses. Patients with MDD or panic disorder were not found to differ significantly from healthy controls in the pattern of methylation of the SLC6a2 gene promotor. While significant correlations between methylation levels at some CpG sites and physiological measures were identified, overall the variation in DNA methylation between patients was small, and the significance of this variation remains equivocal. No significant changes in SLC6a2 promoter methylation were observed in response to antidepressant treatment. Further in-depth analysis of alternative mechanisms of transcriptional regulation of the SLC6a2 gene in human health and disease would be of value.

  1. 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 (parsenic exposure: inorganic arsenic (iAs), 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; parsenic 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.

  2. Characterisation of DNA methylation status using spectroscopy (mid-IR versus Raman) with multivariate analysis.

    Science.gov (United States)

    Kelly, Jemma G; Najand, Ghazal M; Martin, Francis L

    2011-05-01

    Methylation status plays important roles in the regulation of gene expression and significantly influences the dynamics, bending and flexibility of DNA. The aim of this study was to determine whether attenuated total reflection Fourier-transform infrared (ATR-FTIR) or Raman spectroscopy with subsequent multivariate analysis could determine methylation patterning in oligonucleotides variously containing 5-methylcytosine, cytosine and guanine bases. Applied to Low-E reflective glass slides, 10 independent spectral acquisitions were acquired per oligonucleotide sample. Resultant spectra were baseline-corrected and vector normalised over the 1750 cm(-1) -760 cm(-1) (for ATR-FTIR spectroscopy) or the 1750 cm(-1) -600 cm(-1) (for Raman spectroscopy) regions. Data were then analysed using principal component analysis (PCA) coupled with linear discriminant analysis (LDA). Exploiting this approach, biomolecular signatures enabling sensitive and specific discrimination of methylation patterning were derived. For DNA sequence and methylation analysis, this approach has the potential to be an important tool, especially when material is scarce.

  3. 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

    Low birth weight (LBW) can have an impact on health outcomes in later life, especially in relation to pre-disposition to metabolic disease. Several studies suggest that LBW resulting from restricted intrauterine growth leaves a footprint on DNA methylation in utero, and this influence likely...... 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...... 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...

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

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    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.

  5. Disordered beta-catenin expression and E-cadherin/CDH1 promoter methylation in gastric carcinoma

    Institute of Scientific and Technical Information of China (English)

    Li Wang; Fan Zhang; Ping-Ping Wu; Xu-Cheng Jiang; Lin Zheng; Ying-Yan Yu

    2006-01-01

    AIM: To investigate the distribution of beta-catenin in nuclei or membrane/cytoplasm of gastric carcinoma cells,the relationship between E-cadherin gene methylation and its expression, and the role of beta-catenin and E-cadherin as potential molecular markers in predicting tumor infiltration.METHODS: Twenty-nine cases of gastric carcinoma,classified as diffuse and intestinal variants, were selected for study. Nuclear and cytoplasmic proteins were purified and beta-catenin content was detected by ELISA. DNA methylation of E-cadherin/CDH1 gene promoter was studied by methylation-specific PCR and compaired with E-cadherin expression detected by immunohistochemistry.RESULTS: In 27 cases of gastric carcinoma, the ratio of beta-catenin content between nuclei and membrane/cytoplasm was correlated with the T-classification (r =0.392, P = 0.043). The significance was present between T2 and T3 groups. No correlation was detected between diffuse and intestinal variants in terms of their betacatenin distribution. In 21 cases of diffuse variants of gastric carcinoma, there was a difference in E-cadherin expression between CDH1 gene-methylated group and non-methylated group (29 % vs 71%, P = 0.027).No correlation between CDH1 gene methylation and T-classification was found, neither was the significance between E-cadherin expression and tumor infiltration grade.CONCLJSION: Comparative analysis of nuclear and membrane/cytoplasmic beta-catenin can predict local tumor infiltration. E-cadherin/CDH1 gene methylation is an important cause for its gene silence in diffuse variant gastric carcinoma. Methylation of CDH1 gene in the absence of E-cadherin is an early event in gastric carcinogenesis.

  6. [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.

  7. Potential forensic application of DNA methylation profiling to body fluid identification.

    Science.gov (United States)

    Lee, Hwan Young; Park, Myung Jin; Choi, Ajin; An, Ja Hyun; Yang, Woo Ick; Shin, Kyoung-Jin

    2012-01-01

    DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not currently determine the type and origin of the biological material. Recent advances in whole genome epigenetic analysis indicate that chromosome pieces called tDMRs (tissue-specific differentially methylated regions) show different DNA methylation profiles according to the type of cell or tissue. We examined the potential of tissue-specific differential DNA methylation for body fluid identification. Five tDMRs for the genes DACT1, USP49, HOXA4, PFN3, and PRMT2 were selected, and DNA methylation profiles for these tDMRs were produced by bisulfite sequencing using pooled DNA from blood, saliva, semen, menstrual blood, and vaginal fluid. The tDMRs for DACT1 and USP49 showed semen-specific hypomethylation, and the tDMRs for HOXA4, PFN3, and PRMT2 displayed varying degrees of methylation according to the type of body fluid. Preliminary tests using methylation-specific PCR for the DACT1 and USP49 tDMRs showed that these two markers could be used successfully to identify semen samples including sperm cells. Body fluid-specific differential DNA methylation may be a promising indicator for body fluid identification. Because DNA methylation profiling uses the same biological source of DNA for individual identification profiling, the determination of more body fluid-specific tDMRs and the development of convenient tDMR analysis methods will facilitate the broad implementation of body fluid identification in forensic casework.

  8. Digital PCR dynamic range is approaching that of real-time quantitative PCR.

    Science.gov (United States)

    Jones, Gerwyn M; Busby, Eloise; Garson, Jeremy A; Grant, Paul R; Nastouli, Eleni; Devonshire, Alison S; Whale, Alexandra S

    2016-12-01

    Digital PCR (dPCR) has been reported to be more precise and sensitive than real-time quantitative PCR (qPCR) in a variety of models and applications. However, in the majority of commercially available dPCR platforms, the dynamic range is dependent on the number of partitions analysed and so is typically limited to four orders of magnitude; reduced compared with the typical seven orders achievable by qPCR. Using two different biological models (HIV DNA analysis and KRAS genotyping), we have demonstrated that the RainDrop Digital PCR System (RainDance Technologies) is capable of performing accurate and precise quantification over six orders of magnitude thereby approaching that achievable by qPCR.

  9. miRNAting control of DNA methylation

    Indian Academy of Sciences (India)

    Ashwani Jha; Ravi Shankar

    2014-06-01

    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 homologous proteins with siRNAs recruits the enzyme DRM2, which adds a methyl group at certain cytosine residues within the DNA sequence. In this study, it was found that de novo DNA methylation might be regulated by miRNAs through systematic targeting of the genes involved in DNA methylation. A comprehensive genome-wide and system-level study of miRNA targeting, transcription factors, DNA-methylation-causing genes and their target genes has provided a clear picture of an interconnected relationship of all these factors which regulate DNA methylation in Arabidopsis. The study has identified a DNA methylation system that is controlled by four different genes: IDN2, IDNl1, IDNl2 and DRM2. These four genes along with various critical transcription factors appear to be controlled by five different miRNAs. Altogether, DNA methylation appears to be a finely tuned process of opposite control systems of DNA-methylation-causing genes and certain miRNAs pitted against each other.

  10. 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

  11. Methods in DNA methylation profiling.

    Science.gov (United States)

    Zuo, Tao; Tycko, Benjamin; Liu, Ta-Ming; Lin, Juey-Jen L; Huang, Tim H-M

    2009-12-01

    Metastable and somatically heritable patterns of DNA methylation provide an important level of genomic regulation. In this article, we review methods for analyzing these genome-wide epigenetic patterns and offer a perspective on the ever-expanding literature, which we hope will be useful for investigators who are new to this area. The historical aspects that we cover will be helpful in interpreting this literature and we hope that our discussion of the newest analytical methods will stimulate future progress. We emphasize that no single approach can provide a complete view of the overall methylome, and that combinations of several modalities applied to the same sample set will give the clearest picture. Given the unexpected epigenomic patterns and new biological principles, as well as new disease markers, that have been uncovered in recent studies, it is likely that important discoveries will continue to be made using genome-wide DNA methylation profiling.

  12. DNA methylation in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Iris Tischoff; Andrea Tannapfel

    2008-01-01

    As for many other tumors, development of hepatocellular carcinoma (HCC) must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes (TSG). In the last decades, in addition to genetic alterations, epigenetic inactivation of (tumor suppressor) genes by promoter hypermethylation has been recognized as an important and alternative mechanism in tumorigenesis. In HCC, aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.

  13. 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.

  14. DNA methylation profiling of hematopoietic stem cells.

    Science.gov (United States)

    Begtrup, Amber Hogart

    2014-01-01

    DNA methylation is a key epigenetic mark that is essential for properly functioning hematopoietic stem cells. Determining where functionally relevant DNA methylation marks exist in the genome is crucial to understanding the role that methylation plays in hematopoiesis. This chapter describes a method to profile DNA methylation by selectively enriching methylated DNA sequences that are bound in vitro by methyl-binding domain (MBD) proteins. The MBD-pulldown approach selects for DNA sequences that have the potential to be "read" by the endogenous machinery involved in epigenetic regulation. Furthermore, this approach is feasible with very small quantities of DNA, and is compatible with the use of any downstream high-throughput sequencing approach. This technique offers a reliable, simple, and powerful tool for exploration of the role of DNA methylation in hematopoietic stem cells.

  15. 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...

  16. 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…

  17. 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 veri...

  18. 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…

  19. High-resolution melt analysis of DNA methylation to discriminate semen in biological stains.

    Science.gov (United States)

    Antunes, Joana; Silva, Deborah S B S; Balamurugan, Kuppareddi; Duncan, George; Alho, Clarice S; McCord, Bruce

    2016-02-01

    The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.

  20. CB1 and CB2 receptor expression and promoter methylation in patients with cannabis dependence.

    Science.gov (United States)

    Rotter, Andrea; Bayerlein, Kristina; Hansbauer, Max; Weiland, Judith; Sperling, Wolfgang; Kornhuber, Johannes; Biermann, Teresa

    2013-01-01

    CB1 and CB2 receptors are influenced via exogenous and endogenous cannabinoids. To date, little is known regarding changes in receptor expression and methylation in THC (tetrahydrocannabinol) dependence. Therefore, the CB1 and CB2 receptor mRNA expression levels and promoter methylation status in the peripheral blood cells of 77 subjects (36 with THC dependence, 21 cigarette smokers and 20 nonsmokers) were assessed by quantitative real-time PCR and methylation-specific PCR. There was a significant difference in CB1 receptor expression levels between the three groups (ANOVA, p CB1 receptor mRNA expression levels (Spearman's rho: r = -0.37; p = 0.002). Using a mixed general linear model, it was demonstrated that the CB1 mRNA expression (as the dependent variable) was associated with the satisfaction with life scale (SWLS) (r = 0.101; T = 2.8; p = 0.007), craving (as measured with the VAS; r = -0.023; T = -2.3; p = 0.023) and the WHO-Assist Subscale for Cannabis consumption (r = -0.068; T = -2.4; p = 0.02). CB1 receptor expression levels and methylation status appear to be altered in subjects with THC dependence.

  1. TET2 promoter DNA methylation and expression analysis in pediatric B-cell acute lymphoblastic leukemia

    Directory of Open Access Journals (Sweden)

    Ewa Musialik

    2014-03-01

    Full Text Available TET2 is a novel tumor suppressor gene involved in several hematological malignancies of myeloid and lymphoid origin. Besides loss-of-function mutations and deletions, hypermethylation of the CpG island at the TET2 promoter was found in human cancer. Previous analysis revealed no TET2 mutations in acute lymphoblastic leukemia (ALL. Since the TET2 promoter methylation status in pediatric ALL has not been reported, the aim of the present study was to determine if promoter hypermethylation may be a mechanism of TET2 inactivation in a group of pediatric ALL cases. Methylation of TET2 promoter region in one (1/45 ALL B-common patient was detected by methylation specific polymerase chain reaction (PCR and subsequently analyzed by bisulfite sequencing. We found no correlation between promoter methylation and gene expression, measured by quantitative reverse transcriptase-PCR, however the level of TET2 expression in ALL group was significantly decreased compared to children’s normal peripheral blood mononuclear cells and isolated B-cells. TET2 promoter hypermethylation seems to have limited clinical relevance in childhood B-cell ALL due to its low frequency.

  2. 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...

  3. Media independence and dividend policy

    DEFF Research Database (Denmark)

    Farooq, Omar; Dandoune, Salma

    2012-01-01

    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...... 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......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...

  4. Putting muscle in DNA methylation

    Institute of Scientific and Technical Information of China (English)

    James P Reddington; Richard R Meehan

    2011-01-01

    Over 25 years ago seminal experiments from the labs of Peter Jones and Harold Weintraub demonstrated that alteration in the DNA modification state underlie the myogenic conversion of fibroblast cell lines [1,2].This paved the way for the identification of myogenic helix-loop-helix (HLH) proteins in muscle differentiation,but the mechanism by which DNA methylation regulates muscle differentiation has remained elusive [3].

  5. Real-time PCR and PCR-tandem Mass Spectrometry for Biodetection

    Science.gov (United States)

    2005-10-01

    Real - time PCR and PCR- tandem mass spectrometry for biodetection Alvin Fox, University of South Carolina, School of Medicine Report Documentation...TYPE N/A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR and PCRtandem mass spectrometry for biodetection 5a. CONTRACT NUMBER 5b...interspace region Bacillus subtilis W23 standard Blank Barn dust House dust Cycle Real - time PCR (16s rRNA) - environmental samples Real - time

  6. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines1*

    Science.gov (United States)

    Wang, Yipeng; Yu, Qiuju; Cho, Ann H; Rondeau, Gaelle; Welsh, John; Adamson, Eileen; Mercola, Dan; McClelland, Michael

    2005-01-01

    Abstract DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and 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 and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors. PMID:16207477

  7. 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.

  8. DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project.

    Directory of Open Access Journals (Sweden)

    Vardhman K Rakyan

    2004-12-01

    Full Text Available The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine-guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated, tissue specificity, inter-individual variation, and correlation with independent gene expression data.

  9. Independent sets in chain cacti

    OpenAIRE

    Sedlar, Jelena

    2011-01-01

    In this paper chain cacti are considered. First, for two specific classes of chain cacti (orto-chains and meta-chains of cycles with h vertices) the recurrence relation for independence polynomial is derived. That recurrence relation is then used in deriving explicit expressions for independence number and number of maximum independent sets for such chains. Also, the recurrence relation for total number of independent sets for such graphs is derived. Finaly, the proof is provided that orto-ch...

  10. Addressing PCR Biases in Environmental Microbiology Studies

    Science.gov (United States)

    Sipos, Rita; Székely, Anna; Révész, Sára; Márialigeti, Károly

    Each step of a molecular environmental microbiology study is prone to errors, though the qualitative and quantitative biases of PCR amplification could result in the most serious biases. One has to be aware of this fact, and well-characterized PCR biases have to be avoided by using target-optimized PCR protocols. The most important tasks are primer and thermal profile optimization. We have shown that primer mismatches, even in the case of universal primers, can cause almost complete missing of common taxa from clone libraries, for example. Similarly high annealing temperatures can drastically distort community composition of the sample in the PCR product. Strategies of primer selection and PCR thermal profile design are discussed in detail.

  11. COLD-PCR: Applications and Advantages.

    Science.gov (United States)

    Zuo, Zhuang; Jabbar, Kausar J

    2016-01-01

    Co-amplification at lower denaturation temperature-based polymerase chain reaction (COLD-PCR) is a single-step amplification method that results in the enhancement of both known and unknown minority alleles during PCR, irrespective of mutation type and position. This method is based on exploitation of the critical temperature, Tc, at which mutation-containing DNA is preferentially melted over wild type. COLD-PCR can be a good strategy for mutation detection in specimens with high nonneoplastic cell content, small specimens in which neoplastic cells are difficult to micro-dissect and therefore enrich, and whenever a mutation is suspected to be present but is undetectable using conventional PCR and sequencing methods. We describe in this chapter our COLD-PCR-based pyrosequencing method for KRAS mutation detection in various clinical samples using DNA extracted from either fresh or fixed paraffin-embedded tissue specimens.

  12. Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

    Science.gov (United States)

    Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2015-12-01

    Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Studying the Independent School Library

    Science.gov (United States)

    Cahoy, Ellysa Stern; Williamson, Susan G.

    2008-01-01

    In 2005, the American Association of School Librarians' Independent Schools Section conducted a national survey of independent school libraries. This article analyzes the results of the survey, reporting specialized data and information regarding independent school library budgets, collections, services, facilities, and staffing. Additionally, the…

  14. 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

  15. Pre-PCR processing: strategies to generate PCR-compatible samples.

    Science.gov (United States)

    Rådström, Peter; Knutsson, Rickard; Wolffs, Petra; Lövenklev, Maria; Löfström, Charlotta

    2004-02-01

    Polymerase chain reaction (PCR) is recognized as a rapid, sensitive, and specific molecular diagnostic tool for the analysis of nucleic acids. However, the sensitivity and kinetics of diagnostic PCR may be dramatically reduced when applied directly to biological samples, such as blood and feces, owing to PCR-inhibitory components. As a result, pre-PCR processing procedures have been developed to remove or reduce the effects of PCR inhibitors. Pre-PCR processing comprises all steps prior to the detection of PCR products, that is, sampling, sample preparation, and deoxyribonucleic acid (DNA) amplification. The aim of pre-PCR processing is to convert a complex biological sample with its target nucleic acids/cells into PCR-amplifiable samples by combining sample preparation and amplification conditions. Several different pre-PCR processing strategies are used: (1) optimization of the DNA amplification conditions by the use of alternative DNA polymerases and/or amplification facilitators, (2) optimization of the sample preparation method, (3) optimization of the sampling method, and (4) combinations of the different strategies. This review describes different pre-PCR processing strategies to circumvent PCR inhibition to allow accurate and precise DNA amplification.

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

    NARCIS (Netherlands)

    Dijksman, J.F.; 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 adva

  17. First evidence of DNA methylation in insect Tribolium castaneum: environmental regulation of DNA methylation within heterochromatin.

    Science.gov (United States)

    Feliciello, Isidoro; Parazajder, Josip; Akrap, Ivana; Ugarković, Durđica

    2013-05-01

    DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.

  18. Microbial Pollution Tracking of Dairy Farm with a Combined PCR-DGGE and qPCR Approach.

    Science.gov (United States)

    Xi, Xiaoxia; Zhang, Jiachao; Kwok, Laiyu; Huo, Dongxue; Feng, Shuzhen; Zhang, Heping; Sun, Tiansong

    2015-12-01

    Animal husbandry is a traditional industry with regional characteristic in the Inner Mongolia of China. Recent years, animal breeding has been one of the main pollution sources in this area, followed by domestic sewage and industrial wastewater. The pollution of livestock farm feces may accelerate the development of pathogens and antibiotic resistance genes which pose health risks to humans and animals. In present research, culture-independent molecular ecological methods based on DGGE combined with qPCR were used to investigate the pollution to surrounding environment with different degrees of livestock farm. The cluster analysis of DGGE patterns showed that the livestock farm feces from point pollution source flowed with wastewater discharge has resulted in an impacted range of at least 3000 m, but it did not cause pollution to residential water delivered from upstream of sewage drain outlet. qPCR results revealed that 5 common pathogens (Escherichia coli, Enterococcus, Staphylococcus aureus, Shigella, and Salmonella) presented decreased trend as the sampled distance from point pollution source increased. Also, qPCR assays of 10 common antibiotic resistance genes (tetO, tetL, rpp, rpoB, sul2, sulA, floR, yidY, mphA, and ermC) which cause resistance to tetracycline, rifampicin, fluoroquinolone, quinolone, and erythromycin have been found in the environmental samples. This study clearly indicates the livestock farm discharge pollutants contaminated to the surrounding environment. Our data have provided important information to pollution control in the future.

  19. DNA methylation-based measures of biological age: Meta-analysis predicting time to death

    NARCIS (Netherlands)

    B.H. Chen (Brian); R.E. Marioni (Riccardo); Colicino, E. (Elena); M.J. Peters (Marjolein); C.K. Ward-Caviness (Cavin K.); Tsai, P.-C. (Pei-Chien); Roetker, N.S. (Nicholas S.); Just, A.C. (Allan C.); E.W. Demerath (Ellen); W. Guan (Weihua); J. Bressler (Jan); M. Fornage (Myriam); Studenski, S. (Stephanie); Vandiver, A.R. (Amy R.); Moore, A.Z. (Ann Zenobia); T. Tanaka (Toshiko); D.P. Kiel (Douglas P.); Liang, L. (Liming); Vokonas, P. (Pantel); Schwartz, J. (Joel); K.L. Lunetta (Kathryn); J. Murabito (Joanne); S. Bandinelli (Stefania); D.G. Hernandez (Dena); D. Melzer (David); M.A. Nalls (Michael); L.C. Pilling (Luke); Price, T.R. (Timothy R.); A. Singleton (Andrew); C. Gieger (Christian); R. Holle (Rolf); Kretschmer, A. (Anja); F. Kronenberg (Florian); Kunze, S. (Sonja); J. Linseisen (Jakob); Meisinger, C. (Christine); W. Rathmann (Wolfgang); M. Waldenberger (Melanie); P.M. Visscher (Peter); Shah, S. (Sonia); N.R. Wray (Naomi); A.F. McRae (Allan F.); O.H. Franco (Oscar); A. Hofman (Albert); A.G. Uitterlinden (André); D. Absher (Devin); T.L. Assimes (Themistocles); Levine, M.E. (Morgan E.); Lu, A.T. (Ake T.); Tsao, P.S. (Philip S.); Hou, L. (Lifang); J.E. Manson (Joann); C. Carty (Cara); LaCroix, A.Z. (Andrea Z.); A. Reiner (Alexander); T.D. Spector (Timothy); A.P. Feinberg (Andrew P.); D. Levy (Daniel); A.A. Baccarelli (Andrea A.); Meurs, J. (Joyce van); J.T. Bell (Jordana); A. Peters (Annette); I.J. Deary (Ian J.); J.S. Pankow (James); L. Ferrucci (Luigi); S. Horvath (Steve)

    2016-01-01

    textabstractEstimates of biological age based on DNA methylation patterns, often referred to as "epigenetic age", "DNAm age", have been shown to be robust biomarkers of age in humans. We previously demonstrated that independent of chronological age, epigenetic age assessed in blood predicted all-cau

  20. Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens.

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    Edith T Zemanick

    Full Text Available The cystic fibrosis (CF airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2 rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.

  1. Independent sets in chain cacti

    CERN Document Server

    Sedlar, Jelena

    2011-01-01

    In this paper chain cacti are considered. First, for two specific classes of chain cacti (orto-chains and meta-chains of cycles with h vertices) the recurrence relation for independence polynomial is derived. That recurrence relation is then used in deriving explicit expressions for independence number and number of maximum independent sets for such chains. Also, the recurrence relation for total number of independent sets for such graphs is derived. Finaly, the proof is provided that orto-chains and meta-chains are the only extremal chain cacti with respect to total number of independent sets (orto-chains minimal and meta-chains maximal).

  2. USE OF BACTEROIDES PCR-BASED METHODS TO EXAMINE FECAL CONTAMINATION SOURCES IN TROPICAL COASTAL WATERS

    Science.gov (United States)

    Several library independent Microbial Source Tracking methods have been developed to rapidly determine the source of fecal contamination. Thus far, none of these methods have been tested in tropical marine waters. In this study, we used a Bacteroides 16S rDNA PCR-based...

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

    Directory of Open Access Journals (Sweden)

    Recillas-Targa Félix

    2011-06-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusions 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.

  4. Methylation analysis and HPV genotyping of self-collected cervical samples from women not responding to screening invitation and review of the literature

    Science.gov (United States)

    Del Mistro, Annarosa; Frayle, Helena; Rizzi, Martina; Fantin, Gianpiero; Ferro, Antonio; Angeletti, Paolo Matteo; Giorgi Rossi, Paolo; Altobelli, Emma

    2017-01-01

    Aim of the study To assess the feasibility of partial HPV genotyping and methylation analysis of CADM1, MAL, and miR124-2 genes as triage tests in assaying self-collected cervical samples positive for high-risk HPV on primary screening, and to review the literature regarding host cellular gene methylation analysis of self-collected cervical samples. Material and methods Women residing in North-East Italy who had failed to respond to the invitation to participate in an organized population-based program were invited to provide a self-sample. Their stored baseline (self-collected) and follow-up (clinician-collected) cervical samples were included in the study. DNA was extracted from HPV-positive (Qiagen’s Hybrid Capture 2, HC2) samples. Partial genotyping with separate detection of HPV types 16 and 18 was performed with a hybrid capture-based method and a quantitative PCR assay. Methylation was assayed with a quantitative methylation-specific PCR. Results High-risk HPV infection was detected in 48% of baseline and 71% of follow-up HC2-positive samples. Methylation was demonstrated respectively in 15% and 23.5% of baseline and follow-up samples and chiefly involved a single gene (miR124-2). Invalid quantitative PCR results were recorded in 5% of self-collected samples. The specificity of miR124-1, MAL, and CADM1 methylation was 84%, 94%, and 98%, respectively, and the specificity of the three markers combined was 84%. Sensitivity was not estimated due to the lack of CIN2+ samples. The systematic review showed that different methylation assays yield different accuracy values. Conclusion Self-collected samples are suitable for methylation assays included in reflex triage testing. The reproducibility and accuracy of the methylation tests described in the literature should be improved. PMID:28263992

  5. 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.

  6. Real-Time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei

    Science.gov (United States)

    2005-10-01

    1 Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B. pseudomallei Vipin K. Rastogi1, Tu-chen Cheng1, Lisa Collins1 and Jennifer Bagley2 1...A 3. DATES COVERED - 4. TITLE AND SUBTITLE Real - time PCR (RT-PCR) Assays for Burkholderia mallei and B.pseudomallei 5a. CONTRACT NUMBER 5b...risk. There is currently no real - time PCR assay for detection of both of these pathogens. Primers and probes corresponding to specific genomic regions

  7. Local genotype influences DNA methylation at two asthma-associated regions, 5q31 and 17q21, in a founder effect population.

    Science.gov (United States)

    Al Tuwaijri, Abeer; Gagné-Ouellet, Valérie; Madore, Anne-Marie; Laprise, Catherine; Naumova, Anna K

    2016-04-01

    Two asthma-associated regions 17q12-q21 and 5q31.1 harbour genes that show strong effect of genotype on expression levels. DNA methylation has an important role in gene regulation; therefore, we examined DNA methylation at promoters of 12 genes from 5q31 and 17q12-q21 regions. Our goal was to determine whether DNA methylation was associated with predisposition to asthma and whether such a relationship was independent from genetic association. Using sodium bisulfite sequencing and pyrosequencing methylation assays, we examined the effect of genotype on DNA methylation in peripheral blood cells from individuals from the Saguenay-Lac-Saint-Jean asthma familial collection and lymphoblastoid cell lines. The local genotype influenced methylation levels of solute carrier family 22 (organic 3 cation/carnitine transporter) member 5 (SLC22A5), zona pellucida binding protein 2 (ZPBP2) and gasdermin A (GSDMA) promoter regions. The genotype had a dominant effect on ZPBP2 and GSDMA methylation with lower methylation levels in individuals that carry the asthma-predisposing alleles. Males also had lower methylation at the ZPBP2 promoter than females. We did not observe an effect of asthma status that would be