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Sample records for methylation requires specific

  1. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

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

  2. Wp specific methylation of highly proliferated LCLs

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  3. TWRSview system requirements specification

    International Nuclear Information System (INIS)

    Caldwell, J.A.; Lee, A.K.

    1995-12-01

    This document provides the system requirements specification for the TWRSview software system. The TWRSview software system is being developed to integrate electronic data supporting the development of the TWRS technical baseline

  4. Allele specific expression and methylation in the bumblebee, Bombus terrestris

    Directory of Open Access Journals (Sweden)

    Zoë Lonsdale

    2017-09-01

    Full Text Available The social hymenoptera are emerging as models for epigenetics. DNA methylation, the addition of a methyl group, is a common epigenetic marker. In mammals and flowering plants methylation affects allele specific expression. There is contradictory evidence for the role of methylation on allele specific expression in social insects. The aim of this paper is to investigate allele specific expression and monoallelic methylation in the bumblebee, Bombus terrestris. We found nineteen genes that were both monoallelically methylated and monoallelically expressed in a single bee. Fourteen of these genes express the hypermethylated allele, while the other five express the hypomethylated allele. We also searched for allele specific expression in twenty-nine published RNA-seq libraries. We found 555 loci with allele-specific expression. We discuss our results with reference to the functional role of methylation in gene expression in insects and in the as yet unquantified role of genetic cis effects in insect allele specific methylation and expression.

  5. ENVIRONMENTAL SPECIFICATION REQUIREMENTS

    International Nuclear Information System (INIS)

    TIFFT, S.R.

    2003-01-01

    Through regulations, permitting or binding negotiations, Regulators establish requirements, limits, permit conditions and Notice of Construction (NOC) conditions with which the Office of River Protection (ORP) and the Tank Farm Contractor (TFC) must comply. Operating Specifications are technical limits which are set on a process to prevent injury to personnel, or damage to the facility or environment. The main purpose of this document is to provide specification limits and recovery actions for the TFC Environmental Surveillance Program at the Hanford Site. Specification limits are given for monitoring frequencies and permissible variation of readings from an established baseline or previous reading. The requirements in this document are driven by environmental considerations and data analysis issues, rather than facility design or personnel safety issues. This document is applicable to all single-shell tank (SST) and double-shell tank (DST) waste tanks, and the associated catch tanks and receiver tanks, and transfer systems. This Tank Farm Environmental Specifications Document (ESD) implements environmental-regulatory limits on the configuration and operation of the Hanford Tank Farms facility that have been established by Regulators. This ESD contains specific field operational limits and recovery actions for compliance with airborne effluent regulations and agreements, liquid effluents regulations and agreements, and environmental tank system requirements. The scope of this ESD is limited to conditions that have direct impact on Operations Projects or that Operations/Projects have direct impact upon. This document does not supercede or replace any DOE Orders, regulatory permits, notices of construction, or Regulatory agency agreements binding on the ORP or the TFC. Refer to the appropriate regulation, permit, or NOC for an inclusive listing of requirements

  6. ENVIRONMENTAL SPECIFICATION REQUIREMENTS

    International Nuclear Information System (INIS)

    TIFFT, S.R.

    2003-01-01

    Through regulations, permitting or binding negotiations, Regulators establish requirements, limits, permit conditions and Notice of Construction (NOC) conditions with which the Office of River Protection (ORP) and the Tank Farm Contractor (TFC) must comply. Operating Specifications are technical limits which are set on a process to prevent injury to personnel, or damage to the facility or environment. The main purpose of this document is to provide specification limits and recovery actions for the TFC Environmental Surveillance Program at the Hanford Site. Specification limits are given for monitoring frequencies and permissible variation of readings from an established baseline or previous reading. The requirements in this document are driven by environmental considerations and data analysis issues, rather than facility design or personnel safety issues. This document is applicable to all SST and DST waste tanks, and the associated catch tanks and receiver tanks, and transfer systems. This Tank Farm ESD implements environmental-regulatory limits on the configuration and operation of the Hanford Tank Farms facility that have been established by Regulators. This ESD contains specific field operational limits and recovery actions for compliance with airborne effluent regulations and agreements, liquid effluents regulations and agreements, and environmental tank system requirements. The scope of this ESD is limited to conditions that have direct impact on Operations/Projects or that Operations/Projects have direct impact upon. This document does not supercede or replace any DOE Orders, regulatory permits, notices of construction, or Regulatory agency agreements binding on the ORP or the TFC. Refer to the appropriate regulation, permit, or NOC for an inclusive listing of requirements

  7. Allele-Specific DNA Methylation Detection by Pyrosequencing®

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  8. 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

  9. ROS signalling - specificity is required

    DEFF Research Database (Denmark)

    Møller, Ian M; Sweetlove, Lee J

    2010-01-01

    Reactive oxygen species (ROS) production increases in plants under stress. ROS can damage cellular components, but they can also act in signal transduction to help the cell counteract the oxidative damage in the stressed compartment. H2O2 might induce a general stress response, but it does not have...... the required specificity to selectively regulate nuclear genes required for dealing with localized stress, e.g. in chloroplasts or mitochondria. Here we argue that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS messengers...... and regulate source-specific genes and in this way contribute to retrograde ROS signalling during oxidative stress. Likewise, unmodified peptides deriving from the breakdown of redundant proteins could help coordinate organellar and nuclear gene expression...

  10. Infant sex-specific placental cadmium and DNA methylation associations

    Energy Technology Data Exchange (ETDEWEB)

    Mohanty, April F., E-mail: april.mohanty@va.gov [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Farin, Fred M., E-mail: freddy@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Bammler, Theo K., E-mail: tbammler@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); MacDonald, James W., E-mail: jmacdon@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Afsharinejad, Zahra, E-mail: zafshari@u.washington.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, 4225 Roosevelt Way N.E., Suite #100, Seattle, WA 98105 (United States); Burbacher, Thomas M., E-mail: tmb@uw.edu [Department of Environmental and Occupational Health Sciences, School of Public Health, University of Washington, Box: 357234, 1705 N.E. Pacific Street, Seattle, WA 98195 (United States); Siscovick, David S., E-mail: dsiscovick@nyam.org [Cardiovascular Health Research Unit, University of Washington, 1730 Minor Ave, Seattle, WA 98101 (United States); Department of Epidemiology, School of Public Health, University of Washington, Seattle, WA (United States); Department of Medicine, University of Washington, Seattle, WA (United States); and others

    2015-04-15

    Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

  11. Infant sex-specific placental cadmium and DNA methylation associations

    International Nuclear Information System (INIS)

    Mohanty, April F.; Farin, Fred M.; Bammler, Theo K.; MacDonald, James W.; Afsharinejad, Zahra; Burbacher, Thomas M.; Siscovick, David S.

    2015-01-01

    Background: Recent evidence suggests that maternal cadmium (Cd) burden and fetal growth associations may vary by fetal sex. However, mechanisms contributing to these differences are unknown. Objectives: Among 24 maternal-infant pairs, we investigated infant sex-specific associations between placental Cd and placental genome-wide DNA methylation. Methods: We used ANOVA models to examine sex-stratified associations of placental Cd (dichotomized into high/low Cd using sex-specific Cd median cutoffs) with DNA methylation at each cytosine-phosphate-guanine site or region. Statistical significance was defined using a false discovery rate cutoff (<0.10). Results: Medians of placental Cd among females and males were 5 and 2 ng/g, respectively. Among females, three sites (near ADP-ribosylation factor-like 9 (ARL9), siah E3 ubiquitin protein ligase family member 3 (SIAH3), and heparin sulfate (glucosamine) 3-O-sulfotransferase 4 (HS3ST4) and one region on chromosome 7 (including carnitine O-octanoyltransferase (CROT) and TP5S target 1 (TP53TG1)) were hypomethylated in high Cd placentas. Among males, high placental Cd was associated with methylation of three sites, two (hypomethylated) near MDS1 and EVI1 complex locus (MECOM) and one (hypermethylated) near spalt-like transcription factor 1 (SALL1), and two regions (both hypomethylated, one on chromosome 3 including MECOM and another on chromosome 8 including rho guanine nucleotide exchange factor (GEF) 10 (ARHGEF10). Differentially methylated sites were at or close to transcription start sites of genes involved in cell damage response (SIAH3, HS3ST4, TP53TG1) in females and cell differentiation, angiogenesis and organ development (MECOM, SALL1) in males. Conclusions: Our preliminary study supports infant sex-specific placental Cd-DNA methylation associations, possibly accounting for previously reported differences in Cd-fetal growth associations across fetal sex. Larger studies are needed to replicate and extend these

  12. [Specific features of 2-methyl hydroxybenzene and 3-methyl hydroxybenzene distribution in the organism of warm-blooded animals].

    Science.gov (United States)

    Shormanov, B K; Grishenko, V K; Astashkina, A P; Elizarova, M K

    2013-01-01

    The present work was designed to study the specific features of 2-methyl hydroxybezene and 3-methyl hydroxybenzene distribution after intragastric administration of these toxicants to warm-blooded animals (rats). They were detected in the unmetabolized form in the internal organs and blood of the animals. The levels of 2-methyl hydroxybezene were especially high in the stomach and blood whereas the maximum content of 3-methyl hydroxybenzene was found in brain, blood, small intestines of the poisoned rats.

  13. Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1).

    Science.gov (United States)

    Nwaobi, Sinifunanya E; Olsen, Michelle L

    2015-09-26

    DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

  14. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.

    2010-05-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  15. A Protein Complex Required for Polymerase V Transcripts and RNA- Directed DNA Methylation in Arabidopsis

    KAUST Repository

    Law, Julie A.; Ausí n, Israel; Johnson, Lianna M.; Vashisht, Ajay  A Amar; Zhu, Jian-Kang; Wohlschlegel, James  A A.; Jacobsen, Steven E.

    2010-01-01

    DNA methylation is an epigenetic modification associated with gene silencing. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), which is targeted by small interfering RNAs through a pathway termed RNA-directed DNA methylation (RdDM) [1, 2]. Recently, RdDM was shown to require intergenic noncoding (IGN) transcripts that are dependent on the Pol V polymerase. These transcripts are proposed to function as scaffolds for the recruitment of downstream RdDM proteins, including DRM2, to loci that produce both siRNAs and IGN transcripts [3]. However, the mechanism(s) through which Pol V is targeted to specific genomic loci remains largely unknown. Through affinity purification of two known RdDM components, DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1 (DRD1) [4] and DEFECTIVE IN MERISTEM SILENCING 3 (DMS3) [5, 6], we found that they copurify with each other and with a novel protein, RNA-DIRECTED DNA METHYLATION 1 (RDM1), forming a complex we term DDR. We also found that DRD1 copurified with Pol V subunits and that RDM1, like DRD1 [3] and DMS3 [7], is required for the production of Pol V-dependent transcripts. These results suggest that the DDR complex acts in RdDM at a step upstream of the recruitment or activation of Pol V. © 2010 Elsevier Ltd. All rights reserved.

  16. 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.

  17. 40 CFR 180.1189 - Methyl salicylate; exemption from the requirement of a tolerance.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 23 2010-07-01 2010-07-01 false Methyl salicylate; exemption from the... Exemptions From Tolerances § 180.1189 Methyl salicylate; exemption from the requirement of a tolerance. The biochemical pesticide methyl salicylate is exempt from the requirement of a tolerance for residues in or on...

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  1. Monoclonal antibodies specific for the organophosphate pesticide azinphos-methyl

    NARCIS (Netherlands)

    Jones, WT; Harvey, D; Jones, SD; Ryan, GB; Wynberg, H; TenHoeve, W; Reynolds, PHS

    1995-01-01

    2-(2-Mercapto-5-methyl-1,3,2-dioxaphosphorinan-5-yl,2-sulphide) methoxyacetic acid has been synthesized and used to prepare an azinphos hapten and protein conjugates. Monoclonal antibodies of high affinity against the pesticide azinphos-methyl were prepared from mice immunized with the

  2. Blood as a surrogate marker for tissue-specific DNA methylation and changes due to folate depletion in post-partum female mice.

    Science.gov (United States)

    McKay, Jill A; Xie, Long; Harris, Sarah; Wong, Yi K; Ford, Dianne; Mathers, John C

    2011-07-01

    DNA methylation patterns are tissue specific and may influence tissue-specific gene regulation. Human studies investigating DNA methylation in relation to environmental factors primarily use blood-derived DNA as a surrogate for DNA from target tissues. It is therefore important to know if DNA methylation changes in blood in response to environmental changes reflect those in target tissues. Folate intake can influence DNA methylation, via altered methyl donor supply. Previously, manipulations of maternal folate intake during pregnancy altered the patterns of DNA methylation in offspring but, to our knowledge, the consequences for maternal DNA methylation are unknown. Given the increased requirement for folate during pregnancy, mothers may be susceptible to aberrant DNA methylation due to folate depletion. Female mice were fed folate-adequate (2 mg folic acid/kg diet) or folate-deplete (0.4 mg folic acid/kg diet) diets prior to mating and during pregnancy and lactation. Following weaning, dams were killed and DNA methylation was assessed by pyrosequencing® in blood, liver, and kidney at the Esr1, Igf2 differentially methylated region (DMR)1, Igf2 DMR2, Slc39a4CGI1, and Slc39a4CGI2 loci. We observed tissue-specific differences in methylation at all loci. Folate depletion reduced Igf2 DMR1 and Slc39a4CGI1 methylation across all tissues and altered Igf2 DMR2 methylation in a tissue-specific manner (pmethylation measurements may not always reflect methylation within other tissues. Further measurements of blood-derived and tissue-specific methylation patterns are warranted to understand the complexity of tissue-specific responses to altered nutritional exposure. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. DNMT1-interacting RNAs block gene specific DNA methylation

    Science.gov (United States)

    Di Ruscio, Annalisa; Ebralidze, Alexander K.; Benoukraf, Touati; Amabile, Giovanni; Goff, Loyal A.; Terragni, Joylon; Figueroa, Maria Eugenia; De Figureido Pontes, Lorena Lobo; Alberich-Jorda, Meritxell; Zhang, Pu; Wu, Mengchu; D’Alò, Francesco; Melnick, Ari; Leone, Giuseppe; Ebralidze, Konstantin K.; Pradhan, Sriharsa; Rinn, John L.; Tenen, Daniel G.

    2013-01-01

    Summary DNA methylation was described almost a century ago. However, the rules governing its establishment and maintenance remain elusive. Here, we present data demonstrating that active transcription regulates levels of genomic methylation. We identified a novel RNA arising from the CEBPA gene locus critical in regulating the local DNA methylation profile. This RNA binds to DNMT1 and prevents CEBPA gene locus methylation. Deep sequencing of transcripts associated with DNMT1 combined with genome-scale methylation and expression profiling extended the generality of this finding to numerous gene loci. Collectively, these results delineate the nature of DNMT1-RNA interactions and suggest strategies for gene selective demethylation of therapeutic targets in disease. PMID:24107992

  4. Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells

    OpenAIRE

    Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q.

    2013-01-01

    DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells...

  5. Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

    2011-11-15

    Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to {approx}1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

  6. Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

    Directory of Open Access Journals (Sweden)

    Xiang Gao

    Full Text Available Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3 and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

  7. Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

    Science.gov (United States)

    Gao, Xiang; Cao, Donghui; Liu, Jie; Wang, Xiaoping; Geng, Shujuan; Liu, Bao; Shi, Decheng

    2013-01-01

    Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3) and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

  8. Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells.

    Science.gov (United States)

    Guo, Weilong; Chung, Wen-Yu; Qian, Minping; Pellegrini, Matteo; Zhang, Michael Q

    2014-03-01

    DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.

  9. Exploiting E. coli auxotrophs for leucine, valine, and threonine specific methyl labeling of large proteins for NMR applications

    Energy Technology Data Exchange (ETDEWEB)

    Monneau, Yoan R. [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Ishida, Yojiro [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Rossi, Paolo; Saio, Tomohide; Tzeng, Shiou-Ru [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States); Inouye, Masayori, E-mail: inouye@cabm.rutgers.edu [Rutgers University, Center for Advanced Biotechnology and Medicine (United States); Kalodimos, Charalampos G., E-mail: ckalodim@umn.edu [Rutgers University, Center for Integrative Proteomics Research and Department of Chemistry and Chemical Biology (United States)

    2016-06-15

    A simple and cost effective method to independently and stereo-specifically incorporate [{sup 1}H,{sup 13}C]-methyls in Leu and Val in proteins is presented. Recombinant proteins for NMR studies are produced using a tailored set of auxotrophic E. coli strains. NMR active isotopes are routed to either Leu or Val methyl groups from the commercially available and scrambling-free precursors α-ketoisovalerate and acetolactate. The engineered strains produce deuterated proteins with stereospecific [{sup 1}H,{sup 13}C]-methyl labeling separately at Leu or Val amino acids. This is the first method that achieves Leu-specific stereospecific [{sup 1}H,{sup 13}C]-methyl labeling of proteins and scramble-free Val-specific labeling. Use of auxotrophs drastically decreases the amount of labeled precursor required for expression without impacting the yield. The concept is extended to Thr methyl labeling by means of a Thr-specific auxotroph that provides enhanced efficiency for use with the costly L-[4-{sup 13}C,2,3-{sup 2}H{sub 2},{sup 15}N]-Thr reagent. The Thr-specific strain allows for the production of Thr-[{sup 13}CH{sub 3}]{sup γ2} labeled protein with an optimal isotope incorporation using up to 50 % less labeled Thr than the traditional E. coli strain without the need for {sup 2}H-glycine to prevent scrambling.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  11. Synthesis of specific labelled [methyl-14C]sarin

    International Nuclear Information System (INIS)

    Sin-Ren, A.C.; Riggio, G.; Hopff, W.H.; Waser, P.G.

    1988-01-01

    The synthesis of [methyl- 14 C]sarin ([ 14 C]methylphosphono-fluoridic acid 1-methylethyl ester) was accomplished by another approach as for nonlabelled sarin in a tele-conducted reaction vessel. The purity was estimated by IR, GC, and GC-MS and the stability in different media was examined. (author)

  12. DNMT1-interacting RNAs block gene-specific DNA methylation

    Czech Academy of Sciences Publication Activity Database

    Di Ruscio, A.; Ebralidze, A.; Benoukraf, T.; Amabile, G.; Goff, L.A.; Terragni, J.; Figueroa, M.E.; Pontes, L.L.D.; Alberich-Jorda, Meritxell; Zhang, P.; Wu, M.C.; D´Alo, F.; Melnick, A.; Leone, G.; Ebralidze, K.K.; Pradhan, S.; Rinn, J.L.; Tenen, D.G.

    2013-01-01

    Roč. 503, č. 7476 (2013), s. 371-376 ISSN 0028-0836 R&D Projects: GA MŠk LK21307 Institutional support: RVO:68378050 Keywords : DNA methylation * non-coding RNA * DNMT1 Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 42.351, year: 2013

  13. Critical threshold levels of DNA methyltransferase 1 are required to maintain DNA methylation across the genome in human cancer cells.

    Science.gov (United States)

    Cai, Yi; Tsai, Hsing-Chen; Yen, Ray-Whay Chiu; Zhang, Yang W; Kong, Xiangqian; Wang, Wei; Xia, Limin; Baylin, Stephen B

    2017-04-01

    Reversing DNA methylation abnormalities and associated gene silencing, through inhibiting DNA methyltransferases (DNMTs) is an important potential cancer therapy paradigm. Maximizing this potential requires defining precisely how these enzymes maintain genome-wide, cancer-specific DNA methylation. To date, there is incomplete understanding of precisely how the three DNMTs, 1, 3A, and 3B, interact for maintaining DNA methylation abnormalities in cancer. By combining genetic and shRNA depletion strategies, we define not only a dominant role for DNA methyltransferase 1 (DNMT1) but also distinct roles of 3A and 3B in genome-wide DNA methylation maintenance. Lowering DNMT1 below a threshold level is required for maximal loss of DNA methylation at all genomic regions, including gene body and enhancer regions, and for maximally reversing abnormal promoter DNA hypermethylation and associated gene silencing to reexpress key genes. It is difficult to reach this threshold with patient-tolerable doses of current DNMT inhibitors (DNMTIs). We show that new approaches, like decreasing the DNMT targeting protein, UHRF1, can augment the DNA demethylation capacities of existing DNA methylation inhibitors for fully realizing their therapeutic potential. © 2017 Cai et al.; Published by Cold Spring Harbor Laboratory Press.

  14. A Requirement Specification Language for AADL

    Science.gov (United States)

    2016-06-01

    models. The objective of ReqSpec is to support the elicitation , definition, and modeling of requirements for real-time embedded systems in an iterative...A Requirement Specification Language for AADL Peter H. Feiler Julien Delange Lutz Wrage June 2016 TECHNICAL REPORT CMU/SEI-2016-TR-008...is required for any other external and/or commercial use. Requests for permission should be directed to the Software En- gineering Institute at

  15. Methods for measuring specific rates of mercury methylation and degradation and their use in determining factors controlling net rates of mercury methylation

    International Nuclear Information System (INIS)

    Ramlal, P.S.; Rudd, J.W.M.; Hecky, R.E.

    1986-01-01

    A method was developed to estimate specific rates of demethylation of methyl mercury in aquatic samples by measuring the volatile 14 C end products of 14 CH 3 HgI demethylation. This method was used in conjuction with a 203 Hg 2+ radiochemical method which determines specific rates of mercury methylation. Together, these methods enabled us to examine some factors controlling the net rate of mercury methylation. The methodologies were field tested, using lake sediment samples from a recently flooded reservoir in the Southern Indian Lake system which had developed a mercury contamination problem in fish. Ratios of the specific rates of methylation/demethylation were calculated. The highest ratios of methylation/demethylation were calculated. The highest ratios of methylation/demethylation occurred in the flooded shorelines of Southern Indian Lake. These results provide an explanation for the observed increases in the methyl mercury concentrations in fish after flooding

  16. CaMV-35S promoter sequence-specific DNA methylation in lettuce.

    Science.gov (United States)

    Okumura, Azusa; Shimada, Asahi; Yamasaki, Satoshi; Horino, Takuya; Iwata, Yuji; Koizumi, Nozomu; Nishihara, Masahiro; Mishiba, Kei-ichiro

    2016-01-01

    We found 35S promoter sequence-specific DNA methylation in lettuce. Additionally, transgenic lettuce plants having a modified 35S promoter lost methylation, suggesting the modified sequence is subjected to the methylation machinery. We previously reported that cauliflower mosaic virus 35S promoter-specific DNA methylation in transgenic gentian (Gentiana triflora × G. scabra) plants occurs irrespective of the copy number and the genomic location of T-DNA, and causes strong gene silencing. To confirm whether 35S-specific methylation can occur in other plant species, transgenic lettuce (Lactuca sativa L.) plants with a single copy of the 35S promoter-driven sGFP gene were produced and analyzed. Among 10 lines of transgenic plants, 3, 4, and 3 lines showed strong, weak, and no expression of sGFP mRNA, respectively. Bisulfite genomic sequencing of the 35S promoter region showed hypermethylation at CpG and CpWpG (where W is A or T) sites in 9 of 10 lines. Gentian-type de novo methylation pattern, consisting of methylated cytosines at CpHpH (where H is A, C, or T) sites, was also observed in the transgenic lettuce lines, suggesting that lettuce and gentian share similar methylation machinery. Four of five transgenic lettuce lines having a single copy of a modified 35S promoter, which was modified in the proposed core target of de novo methylation in gentian, exhibited 35S hypomethylation, indicating that the modified sequence may be the target of the 35S-specific methylation machinery.

  17. ROS signalling – Specificity is required

    DEFF Research Database (Denmark)

    Møller, Ian Max; Sweetlove, Lee J

    2011-01-01

    The production of reactive oxygen species (ROS) increases in plants under stress. ROS can damage cellular components, but they can also act in signal transduction to help the cell counteract the oxidative damage in the stressed compartment. H2O2 may induce a general stress response, but it does...... messengers and regulate source-specific genes and in this way contribute to retrograde ROS signalling during oxidative stress. (This is a new project funded by FNU) References: Møller, I.M. & Sweetlove, L.J. 2010. ROS signalling – Specificity is required. Trends Plant Sci. 15: 370-374...... not have the required specificity to selectively regulate nuclear genes required for dealing with localized stress, e.g., in chloroplasts or mitochondria. We here argue that peptides deriving from proteolytic breakdown of oxidatively damaged proteins have the requisite specificity to act as secondary ROS...

  18. Improvements to Technical Specifications surveillance requirements

    International Nuclear Information System (INIS)

    Lobel, R.; Tjader, T.R.

    1992-12-01

    In August 1983 an NRC task group was formed to investigate problems with surveillance testing required by Technical Specifications, and to recommend approaches to effect improvements. NUREG-1024 (''Technical Specifications-Enhancing Safety Impact'') resulted, and it contained recommendations to review the basis for test frequencies; to ensure that the tests promote safety and do not degrade equipment; and to review surveillance tests so that they do not unnecessarily burden personnel. The Technical Specifications Improvement Program (TSIP) was established in December 1984 to provide the framework for rewriting and improving the Technical Specifications. As an element of the TSIP, all Technical Specifications surveillance requirements were comprehensively examined as recommended in NUREG-1024. The results of that effort are presented in this report. The study found that while some testing at power is essential to verify equipment and system operability, safety can be improved, equipment degradation decreased, and unnecessary personnel burden relaxed by reducing the amount of testing at power

  19. Identification of body fluid-specific DNA methylation markers for use in forensic science.

    Science.gov (United States)

    Park, Jong-Lyul; Kwon, Oh-Hyung; Kim, Jong Hwan; Yoo, Hyang-Sook; Lee, Han-Chul; Woo, Kwang-Man; Kim, Seon-Young; Lee, Seung-Hwan; Kim, Yong Sung

    2014-11-01

    DNA methylation, which occurs at the 5'-position of the cytosine in CpG dinucleotides, has great potential for forensic identification of body fluids, because tissue-specific patterns of DNA methylation have been demonstrated, and DNA is less prone to degradation than proteins or RNA. Previous studies have reported several body fluid-specific DNA methylation markers, but DNA methylation differences are sometimes low in saliva and vaginal secretions. Moreover, specific DNA methylation markers in four types of body fluids (blood, saliva, semen, and vaginal secretions) have not been investigated with genome-wide profiling. Here, we investigated novel DNA methylation markers for identification of body fluids for use in forensic science using the Illumina HumanMethylation 450K bead array, which contains over 450,000 CpG sites. Using methylome data from 16 samples of blood, saliva, semen, and vaginal secretions, we first selected 2986 hypermethylated or hypomethylated regions that were specific for each type of body fluid. We then selected eight CpG sites as novel, forensically relevant DNA methylation markers: cg06379435 and cg08792630 for blood, cg26107890 and cg20691722 for saliva, cg23521140 and cg17610929 for semen, and cg01774894 and cg14991487 for vaginal secretions. These eight selected markers were evaluated in 80 body fluid samples using pyrosequencing, and all showed high sensitivity and specificity for identification of the target body fluid. We suggest that these eight DNA methylation markers may be good candidates for developing an effective molecular assay for identification of body fluids in forensic science. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  20. 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

  1. CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci.

    Science.gov (United States)

    Kawasaki, Takako; Ohnishi, Mutsuko; Nosho, Katsuhiko; Suemoto, Yuko; Kirkner, Gregory J; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

    2008-03-01

    The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct phenotype in colorectal cancer. However, the concept of CIMP-low with less extensive CpG island methylation is still evolving. Our aim is to examine whether density of methylation in individual CpG islands was different between CIMP-low and CIMP-high tumors. Utilizing MethyLight technology and 889 population-based colorectal cancers, we quantified DNA methylation (methylation index, percentage of methylated reference) at 14 CpG islands, including 8 CIMP-high-specific loci (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). Methylation positivity in each locus was defined as methylation index>4. Low-level methylation (methylation index>0, CIMP-high-specific locus was significantly more common in 340 CIMP-low tumors (1/8-5/8 methylation-positive loci) than 133 CIMP-high tumors (> or =6/8 methylation-positive loci) and 416 CIMP-0 tumors (0/8 methylation-positive loci) (PCIMP-high, low-level methylation, was not persistently more prevalent in CIMP-low tumors. In conclusion, compared to CIMP-high and CIMP-0 tumors, CIMP-low colorectal cancers show not only few methylated CIMP-high-specific CpG islands, but also more frequent low-level methylation at individual loci. Our data may provide supporting evidence for a difference in pathogenesis of DNA methylation between CIMP-low and CIMP-high tumors.

  2. The impact of endurance exercise on global and AMPK gene-specific DNA methylation

    Energy Technology Data Exchange (ETDEWEB)

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Resch, Eduard [Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Peil, Johannes [Sports Clinic, Bad Nauheim, MCI GmbH, In der Aue 30-32, 61231, Bad Nauheim (Germany); Geisslinger, Gerd [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany); Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group for Translational Medicine & Pharmacology (TMP), 60596, Frankfurt/Main (Germany); Niederberger, Ellen, E-mail: e.niederberger@em.uni-frankfurt.de [pharmazentrum frankfurt/ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590, Frankfurt am Main (Germany)

    2016-05-27

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

  3. The impact of endurance exercise on global and AMPK gene-specific DNA methylation

    International Nuclear Information System (INIS)

    King-Himmelreich, Tanya S.; Schramm, Stefanie; Wolters, Miriam C.; Schmetzer, Julia; Möser, Christine V.; Knothe, Claudia; Resch, Eduard; Peil, Johannes; Geisslinger, Gerd; Niederberger, Ellen

    2016-01-01

    Alterations in gene expression as a consequence of physical exercise are frequently described. The mechanism of these regulations might depend on epigenetic changes in global or gene-specific DNA methylation levels. The AMP-activated protein kinase (AMPK) plays a key role in maintenance of energy homeostasis and is activated by increases in the AMP/ATP ratio as occurring in skeletal muscles after sporting activity. To analyze whether exercise has an impact on the methylation status of the AMPK promoter, we determined the AMPK methylation status in human blood samples from patients before and after sporting activity in the context of rehabilitation as well as in skeletal muscles of trained and untrained mice. Further, we examined long interspersed nuclear element 1 (LINE-1) as indicator of global DNA methylation changes. Our results revealed that light sporting activity in mice and humans does not alter global DNA methylation but has an effect on methylation of specific CpG sites in the AMPKα2 gene. These regulations were associated with a reduced AMPKα2 mRNA and protein expression in muscle tissue, pointing at a contribution of the methylation status to AMPK expression. Taken together, these results suggest that exercise influences AMPKα2 gene methylation in human blood and eminently in the skeletal muscle of mice and therefore might repress AMPKα2 gene expression. -- Highlights: •AMPK gene methylation increases after moderate endurance exercise in humans and mice. •AMPKα mRNA and protein decrease after moderate endurance exercise in mice. •Global DNA methylation is not affected under the same conditions.

  4. Technical specifications requirements: Automated reasoning applications

    International Nuclear Information System (INIS)

    Lidsky, L.M.; Dobrzeniecki, A.B.

    1990-03-01

    Several software systems were developed and tested to determine what advantages could be gained from explicitly translating complicated regulatory requirements into computerized relationships. The Technical Specifications for US nuclear power plants were chosen as the test-bed application domain, and two analysis systems were developed to monitor plant compliance with operational limits, and track and schedule equipment test and maintenance activities mandated by Technical Specifications. Choosing PROLOG as the computer language to represent these regulatory requirements resulted in a natural match between the semantic structure of the written specifications and the corollary coded rules. Additional research results affirmed the utility of declarative programming styles, explicit management of problem complexity, and attention to the robustness and flexibility of the overall software systems. 5 refs., 2 figs

  5. Cell-Specific PEAR1 Methylation Studies Reveal a Locus that Coordinates Expression of Multiple Genes

    Directory of Open Access Journals (Sweden)

    Benedetta Izzi

    2018-04-01

    Full Text Available Chromosomal interactions connect distant enhancers and promoters on the same chromosome, activating or repressing gene expression. PEAR1 encodes the Platelet-Endothelial Aggregation Receptor 1, a contact receptor involved in platelet function and megakaryocyte and endothelial cell proliferation. PEAR1 expression during megakaryocyte differentiation is controlled by DNA methylation at its first CpG island. We identified a PEAR1 cell-specific methylation sensitive region in endothelial cells and megakaryocytes that showed strong chromosomal interactions with ISGL20L2, RRNAD1, MRLP24, HDGF and PRCC, using available promoter capture Hi-C datasets. These genes are involved in ribosome processing, protein synthesis, cell cycle and cell proliferation. We next studied the methylation and expression profile of these five genes in Human Umbilical Vein Endothelial Cells (HUVECs and megakaryocyte precursors. While cell-specific PEAR1 methylation corresponded to variability in expression for four out of five genes, no methylation change was observed in their promoter regions across cell types. Our data suggest that PEAR1 cell-type specific methylation changes may control long distance interactions with other genes. Further studies are needed to show whether such interaction data might be relevant for the genome-wide association data that showed a role for non-coding PEAR1 variants in the same region and platelet function, platelet count and cardiovascular risk.

  6. Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

    Science.gov (United States)

    Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

    2016-01-01

    DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

  7. DNA methylation profiling reveals the presence of population-specific signatures correlating with phenotypic characteristics.

    Science.gov (United States)

    Giri, Anil K; Bharadwaj, Soham; Banerjee, Priyanka; Chakraborty, Shraddha; Parekatt, Vaisak; Rajashekar, Donaka; Tomar, Abhishek; Ravindran, Aarthi; Basu, Analabha; Tandon, Nikhil; Bharadwaj, Dwaipayan

    2017-06-01

    Phenotypic characteristics are known to vary substantially among different ethnicities around the globe. These variations are mediated by number of stochastic events and cannot be attributed to genetic architecture alone. DNA methylation is a well-established mechanism that sculpts our epigenome influencing phenotypic variation including disease manifestation. Since DNA methylation is an important determinant for health issues of a population, it demands a thorough investigation of the natural differences in genome wide DNA methylation patterns across different ethnic groups. This study is based on comparative analyses of methylome from five different ethnicities with major focus on Indian subjects. The current study uses hierarchical clustering approaches, principal component analysis and locus specific differential methylation analysis on Illumina 450K methylation data to compare methylome of different ethnic subjects. Our data indicates that the variations in DNA methylation patterns of Indians are less among themselves compared to other global population. It empirically correlated with dietary, cultural and demographical divergences across different ethnic groups. Our work further suggests that Indians included in this study, despite their genetic similarity with the Caucasian population, are in close proximity with Japanese in terms of their methylation signatures.

  8. Genome-wide screen of ovary-specific DNA methylation in polycystic ovary syndrome.

    Science.gov (United States)

    Yu, Ying-Ying; Sun, Cui-Xiang; Liu, Yin-Kun; Li, Yan; Wang, Li; Zhang, Wei

    2015-07-01

    To compare genome-wide DNA methylation profiles in ovary tissue from women with polycystic ovary syndrome (PCOS) and healthy controls. Case-control study matched for age and body mass index. University-affiliated hospital. Ten women with PCOS who underwent ovarian drilling to induce ovulation and 10 healthy women who were undergoing laparoscopic sterilization, hysterectomy for benign conditions, diagnostic laparoscopy for pelvic pain, or oophorectomy for nonovarian indications. None. Genome-wide DNA methylation patterns determined by immunoprecipitation and microarray (MeDIP-chip) analysis. The methylation levels were statistically significantly higher in CpG island shores (CGI shores), which lie outside of core promoter regions, and lower within gene bodies in women with PCOS relative to the controls. In addition, high CpG content promoters were the most frequently hypermethylated promoters in PCOS ovaries but were more often hypomethylated in controls. Second, 872 CGIs, specifically methylated in PCOS, represented 342 genes that could be associated with various molecular functions, including protein binding, hormone activity, and transcription regulator activity. Finally, methylation differences were validated in seven genes by methylation-specific polymerase chain reaction. These genes correlated to several functional families related to the pathogenesis of PCOS and may be potential biomarkers for this disease. Our results demonstrated that epigenetic modification differs between PCOS and normal ovaries, which may help to further understand the pathophysiology of this disease. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  9. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Science.gov (United States)

    Demarre, Gaëlle; Chattoraj, Dhruba K

    2010-05-06

    DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated) sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII) of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  10. DNA adenine methylation is required to replicate both Vibrio cholerae chromosomes once per cell cycle.

    Directory of Open Access Journals (Sweden)

    Gaëlle Demarre

    2010-05-01

    Full Text Available DNA adenine methylation is widely used to control many DNA transactions, including replication. In Escherichia coli, methylation serves to silence newly synthesized (hemimethylated sister origins. SeqA, a protein that binds to hemimethylated DNA, mediates the silencing, and this is necessary to restrict replication to once per cell cycle. The methylation, however, is not essential for replication initiation per se but appeared so when the origins (oriI and oriII of the two Vibrio cholerae chromosomes were used to drive plasmid replication in E. coli. Here we show that, as in the case of E. coli, methylation is not essential for oriI when it drives chromosomal replication and is needed for once-per-cell-cycle replication in a SeqA-dependent fashion. We found that oriII also needs SeqA for once-per-cell-cycle replication and, additionally, full methylation for efficient initiator binding. The requirement for initiator binding might suffice to make methylation an essential function in V. cholerae. The structure of oriII suggests that it originated from a plasmid, but unlike plasmids, oriII makes use of methylation for once-per-cell-cycle replication, the norm for chromosomal but not plasmid replication.

  11. Human active X-specific DNA methylation events showing stability across time and tissues

    Science.gov (United States)

    Joo, Jihoon Eric; Novakovic, Boris; Cruickshank, Mark; Doyle, Lex W; Craig, Jeffrey M; Saffery, Richard

    2014-01-01

    The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork' of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena. PMID:24713664

  12. Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

    Science.gov (United States)

    Ruiz-García, Leonor; Cabezas, Jose Antonio; de María, Nuria; Cervera, María-Teresa

    2010-01-01

    Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

  13. Identification of sex-specific DNA methylation changes driven by specific chemicals in cord blood in a Faroese birth cohort

    DEFF Research Database (Denmark)

    Leung, Yuet-Kin; Ouyang, Bin; Niu, Liang

    2018-01-01

    Faroe islanders consume marine foods contaminated with methylmercury (MeHg), polychlorinated biphenyls (PCBs), and other toxicants associated with chronic disease risks. Differential DNA methylation at specific CpG sites in cord blood may serve as a surrogate biomarker of health impacts from...... chemical exposures. We aimed to identify key environmental chemicals in cord blood associated with DNA methylation changes in a population with elevated exposure to chemical mixtures. We studied 72 participants of a Faroese birth cohort recruited between 1986 and 1987 and followed until adulthood. The cord...... blood DNA methylome was profiled using Infinium HumanMethylation450 BeadChips. We determined the associations of CpG site changes with concentrations of MeHg, major PCBs, other organochlorine compounds [hexachlorobenzene (HCB), p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) and p...

  14. DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination.

    Science.gov (United States)

    Qin, Weihua; Wolf, Patricia; Liu, Nan; Link, Stephanie; Smets, Martha; La Mastra, Federica; Forné, Ignasi; Pichler, Garwin; Hörl, David; Fellinger, Karin; Spada, Fabio; Bonapace, Ian Marc; Imhof, Axel; Harz, Hartmann; Leonhardt, Heinrich

    2015-08-01

    DNMT1 is recruited by PCNA and UHRF1 to maintain DNA methylation after replication. UHRF1 recognizes hemimethylated DNA substrates via the SRA domain, but also repressive H3K9me3 histone marks with its TTD. With systematic mutagenesis and functional assays, we could show that chromatin binding further involved UHRF1 PHD binding to unmodified H3R2. These complementation assays clearly demonstrated that the ubiquitin ligase activity of the UHRF1 RING domain is required for maintenance DNA methylation. Mass spectrometry of UHRF1-deficient cells revealed H3K18 as a novel ubiquitination target of UHRF1 in mammalian cells. With bioinformatics and mutational analyses, we identified a ubiquitin interacting motif (UIM) in the N-terminal regulatory domain of DNMT1 that binds to ubiquitinated H3 tails and is essential for DNA methylation in vivo. H3 ubiquitination and subsequent DNA methylation required UHRF1 PHD binding to H3R2. These results show the manifold regulatory mechanisms controlling DNMT1 activity that require the reading and writing of epigenetic marks by UHRF1 and illustrate the multifaceted interplay between DNA and histone modifications. The identification and functional characterization of the DNMT1 UIM suggests a novel regulatory principle and we speculate that histone H2AK119 ubiquitination might also lead to UIM-dependent recruitment of DNMT1 and DNA methylation beyond classic maintenance.

  15. Multiple Arginine Residues Are Methylated in Drosophila Mre11 and Required for Survival Following Ionizing Radiation.

    Science.gov (United States)

    Yuan, Qing; Tian, Ran; Zhao, Haiying; Li, Lijuan; Bi, Xiaolin

    2018-05-31

    Mre11 is a key player for DNA double strand break repair. Previous studies have shown that mammalian Mre11 is methylated at multiple arginines in its C-terminal Glycine-Arginine-Rich motif (GAR) by protein arginine methyltransferase PRMT1. Here, we found that the Drosophila Mre11 is methylated at arginines 559, 563, 565, and 569 in the GAR motif by DART1, the Drosophila homolog of PRMT1. Mre11 interacts with DART1 in S2 cells, and this interaction does not require the GAR motif. Arginines methylated Mre11 localizes exclusively in the nucleus as soluble nuclear protein or chromatin-binding protein. To study the in vivo functions of methylation, we generated the single Arg-Ala and all Arginines mutated flies. We found these mutants were sensitive to ionizing radiation. Furthermore, Arg-Ala mutated flies had no irradiation induced G2/M checkpoint defect in wing disc and eye disc. Thus, we provided evidence that arginines in Drosophila Mre11 are methylated by DART1 methytransferase and flies loss of arginine methylation are sensitive to irradiation. Copyright © 2018 Yuan et al.

  16. Sequence-specific protection of duplex DNA against restriction and methylation enzymes by pseudocomplementary PNAs

    DEFF Research Database (Denmark)

    Izvolsky, K I; Demidov, V V; Nielsen, P E

    2000-01-01

    I restriction endonuclease and dam methylase. The pcPNA-assisted protection against enzymatic methylation is more efficient when the PNA-binding site embodies the methylase-recognition site rather than overlaps it. We conclude that pcPNAs may provide the robust tools allowing to sequence-specifically manipulate...... DNA duplexes in a virtually sequence-unrestricted manner....

  17. BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

    NARCIS (Netherlands)

    E.H.J. van Roon (Eddy); A. Boot (Arnoud); A.A. Dihal (Ashwin); R.F. Ernst (Robert); T. van Wezel (Tom); H. Morreau (Hans); J.M. Boer (Judith)

    2013-01-01

    textabstractBackground: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have

  18. Technical Specification action statements requiring shutdown

    International Nuclear Information System (INIS)

    Mankamo, T.; Kim, I.S.; Samanta, P.K.

    1993-11-01

    When safety systems fail during power operation, the limiting conditions for operation (LCOs) and associated action statements of technical specifications typically require that the plant be shut down within the limits of allowed outage time (AOT). However, when a system needed to remove decay heat, such as the residual heat removal (RHR) system, is inoperable or degraded, shutting down the plant may not necessarily be preferable, from a risk perspective, to continuing power operation over a usual repair time, giving priority to the repairs. The risk impact of the basic operational alternatives, i.e., continued operation or shutdown, was evaluated for failures in the RHR and standby service water (SSW) systems of a boiling-water reactor (BWR) nuclear power plant. A complete or partial failure of the SSW system fails or degrades not only the RHR system but other front-line safety systems supported by the SSW system. This report presents the methodology to evaluate the risk impact of LCOs and associated AOT; the results of risk evaluation from its application to the RHR and SSW systems of a BWR; the findings from the risk-sensitivity analyses to identify alternative operational policies; and the major insights and recommendations to improve the technical specifications action statements

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

    Science.gov (United States)

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

    2016-01-01

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

  20. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Directory of Open Access Journals (Sweden)

    Jason D Thompson

    Full Text Available Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  1. Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment.

    Science.gov (United States)

    Thompson, Jason D; Shibahara, Gosuke; Rajan, Sweta; Pel, Joel; Marziali, Andre

    2012-01-01

    Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.

  2. A novel CpG island set identifies tissue-specific methylation at developmental gene loci.

    Directory of Open Access Journals (Sweden)

    Robert Illingworth

    2008-01-01

    Full Text Available CpG islands (CGIs are dense clusters of CpG sequences that punctuate the CpG-deficient human genome and associate with many gene promoters. As CGIs also differ from bulk chromosomal DNA by their frequent lack of cytosine methylation, we devised a CGI enrichment method based on nonmethylated CpG affinity chromatography. The resulting library was sequenced to define a novel human blood CGI set that includes many that are not detected by current algorithms. Approximately half of CGIs were associated with annotated gene transcription start sites, the remainder being intra- or intergenic. Using an array representing over 17,000 CGIs, we established that 6%-8% of CGIs are methylated in genomic DNA of human blood, brain, muscle, and spleen. Inter- and intragenic CGIs are preferentially susceptible to methylation. CGIs showing tissue-specific methylation were overrepresented at numerous genetic loci that are essential for development, including HOX and PAX family members. The findings enable a comprehensive analysis of the roles played by CGI methylation in normal and diseased human tissues.

  3. Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a

    International Nuclear Information System (INIS)

    Miranda, Tina Branscombe; Webb, Kristofor J.; Edberg, Dale D.; Reeves, Raymond; Clarke, Steven

    2005-01-01

    The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation

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

    Directory of Open Access Journals (Sweden)

    Mohamed I Husseiny

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

  5. Requirements and specifications for a particle database

    International Nuclear Information System (INIS)

    2015-01-01

    simple as possible, the database will include unique names (or 'id's) for every particle that it stores. Reaction databases can then refer to a specific particle either by id. Where possible, particle ids should be made descriptive to assist human readers in understanding the contents of reaction and particle databases. The particle database is being primarily developed to store nuclei and nuclear states, but it must also be capable of storing some atomic and molecular properties. In particular the database must support atomic electron configurations since they can play an important role in nuclear reactions and decays. Excited nuclear states sometimes decay via internal conversion, 'kicking out' an electron from an inner shell and leaving the remaining electrons to de-excite (emitting x-rays) to fill the new vacancy. Excited atomic states can also be populated through photo-atomic reactions Adding electronic configurations to the database has the potential to drastically increase its size: if unique ids must be given not only to each nucleus and excited nuclear level but also to every possible electron configuration for each nucleus and level, the database will quickly grow to an unmaintainable size. In order to get around this problem and support atomic and molecular properties without drastically increasing the number of particles, the database will allow the use of 'qualifiers' that can be added to a particle to modify its properties. This document describes the requirements and specifications for a particle data hierarchy, including documentation, bibliography information, particle qualifiers as well as particle families and particle groups. This document uses the XML meta-language to illustrate examples of how particle data will be stored. However, like other tasks under SG38, it should be possible to store the particle database in other meta-languages (that is, languages that define a general syntax that can be used to define more specific languages) that support

  6. Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

    Science.gov (United States)

    Paliwal, Anupam; Temkin, Alexis M; Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

    2013-08-01

    Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.

  7. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    Energy Technology Data Exchange (ETDEWEB)

    Jobert, Laure; Argentini, Manuela [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France); Tora, Laszlo, E-mail: laszlo@igbmc.u-strasbg.fr [Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U 596, Universite Louis Pasteur de Strasbourg, BP 10142 - 67404 Illkirch Cedex, CU de Strasbourg (France)

    2009-04-15

    TAF15 (formerly TAF{sub II}68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

  8. PRMT1 mediated methylation of TAF15 is required for its positive gene regulatory function

    International Nuclear Information System (INIS)

    Jobert, Laure; Argentini, Manuela; Tora, Laszlo

    2009-01-01

    TAF15 (formerly TAF II 68) is a nuclear RNA-binding protein that is associated with a distinct population of TFIID and RNA polymerase II complexes. TAF15 harbours an N-terminal activation domain, an RNA recognition motif (RRM) and many Arg-Gly-Gly (RGG) repeats at its C-terminal end. The N-terminus of TAF15 serves as an essential transforming domain in the fusion oncoprotein created by chromosomal translocation in certain human chondrosarcomas. Post-transcriptional modifications (PTMs) of proteins are known to regulate their activity, however, nothing is known on how PTMs affect TAF15 function. Here we demonstrate that endogenous human TAF15 is methylated in vivo at its numerous RGG repeats. Furthermore, we identify protein arginine N-methyltransferase 1 (PRMT1) as a TAF15 interactor and the major PRMT responsible for its methylation. In addition, the RGG repeat-containing C-terminus of TAF15 is responsible for the shuttling between the nucleus and the cytoplasm and the methylation of RGG repeats affects the subcellular localization of TAF15. The methylation of TAF15 by PRMT1 is required for the ability of TAF15 to positively regulate the expression of the studied endogenous TAF15-target genes. Our findings demonstrate that arginine methylation of TAF15 by PRMT1 is a crucial event determining its proper localization and gene regulatory function.

  9. Disposal of Radioactive Waste. Specific Safety Requirements

    International Nuclear Information System (INIS)

    2011-01-01

    This publication establishes requirements applicable to all types of radioactive waste disposal facility. It is linked to the fundamental safety principles for each disposal option and establishes a set of strategic requirements that must be in place before facilities are developed. Consideration is also given to the safety of existing facilities developed prior to the establishment of present day standards. The requirements will be complemented by Safety Guides that will provide guidance on good practice for meeting the requirements for different types of waste disposal facility. Contents: 1. Introduction; 2. Protection of people and the environment; 3. Safety requirements for planning for the disposal of radioactive waste; 4. Requirements for the development, operation and closure of a disposal facility; 5. Assurance of safety; 6. Existing disposal facilities; Appendices.

  10. Synergy of Technical Specification, functional specifications and scenarios in requirements specifications

    NARCIS (Netherlands)

    Miedema, J.; van der Voort, Mascha C.; Lutters, Diederick; van Houten, Frederikus J.A.M.; Krause, Frank-Lothar

    2007-01-01

    In the (mechanical) design process, the requirements specification is a formal registration of the conditions that are imposed on a new or altered product design, both preceding as well as during the corresponding product development cycle. For a long time, the use of technical specifications has

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

    Directory of Open Access Journals (Sweden)

    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&#8211;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.

  12. Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

    Science.gov (United States)

    Schaefer, Jeffra K.; Rocks, Sara S.; Zheng, Wang; Liang, Liyuan; Gu, Baohua; Morel, François M. M.

    2011-01-01

    The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron- and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments. PMID:21555571

  13. Global differences in specific histone H3 methylation are associated with overweight and type 2 diabetes

    OpenAIRE

    Jufvas, ?sa; Sj?din, Simon; Lundqvist, Kim; Amin, Risul; Vener, Alexander V; Str?lfors, Peter

    2013-01-01

    BACKGROUND: Epidemiological evidence indicates yet unknown epigenetic mechanisms underlying a propensity for overweight and type 2 diabetes. We analyzed the extent of methylation at lysine 4 and lysine 9 of histone H3 in primary human adipocytes from 43 subjects using modification-specific antibodies. RESULTS: The level of lysine 9 dimethylation was stable, while adipocytes from type 2 diabetic and non-diabetic overweight subjects exhibited about 40% lower levels of lysine 4 dimethylation com...

  14. Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data

    OpenAIRE

    Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

    2013-01-01

    Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multip...

  15. 78 FR 32157 - Methyl 5-(dimethylamino)-2-methyl-5-oxopentanoate; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2013-05-29

    ... study showed no treatment- related effects on mating or fertility. There were no treatment-related... a petition to EPA under the Federal Food, Drug, and Cosmetic Act (FFDCA), requesting establishment... received and the nature of the adverse effects caused by methyl 5-(dimethylamino)-2-methyl-5- oxopentanoate...

  16. Depleted uranium induces sex- and tissue-specific methylation patterns in adult zebrafish

    International Nuclear Information System (INIS)

    Gombeau, Kewin; Pereira, Sandrine; Ravanat, Jean-Luc; Camilleri, Virginie; Cavalie, Isabelle; Bourdineaud, Jean-Paul; Adam-Guillermin, Christelle

    2016-01-01

    We examined the effects of chronic exposure to different concentrations (2 and 20 μg L"−"1) of environmentally relevant waterborne depleted uranium (DU) on the DNA methylation patterns both at HpaII restriction sites (5′-CCGG-3′) and across the whole genome in the zebrafish brain, gonads, and eyes. We first identified sex-dependent differences in the methylation level of HpaII sites after exposure. In males, these effects were present as early as 7 days after exposure to 20 μg L"−"1 DU, and were even more pronounced in the brain, gonads, and eyes after 24 days. However, in females, hypomethylation was only observed in the gonads after exposure to 20 μg L"−"1 DU for 24 days. Sex-specific effects of DU were also apparent at the whole-genome level, because in males, exposure to 20 μg L"−"1 DU for 24 days resulted in cytosine hypermethylation in the brain and eyes and hypomethylation in the gonads. In contrast, in females, hypermethylation was observed in the brain after exposure to both concentrations of DU for 7 days. Based on our current knowledge of uranium toxicity, several hypotheses are proposed to explain these findings, including the involvement of oxidative stress, alteration of demethylation enzymes and the calcium signaling pathway. This study reports, for the first time, the sex- and tissue-specific epigenetic changes that occur in a nonhuman organism after exposure to environmentally relevant concentrations of uranium, which could induce transgenerational epigenetic effects. - Highlights: • This study demonstrates a sex-related effect of DU exposure on DNA methylation patterns. • Impacts on DNA methylation patterns revealed a tissue-specific effect of DU exposure. • The MS–AFLP and HPLC–MS/MS sensitively and complementarily demonstrated the responses to environmental concentrations of DU.

  17. Species-specific isotopic fractionation of mercury during methylation by bacteria

    International Nuclear Information System (INIS)

    Rodriguez-Gonzalez, P.; Epov, V.N.; Bridou, R.; Tessier, E.; Monperrus, M.; Guyoneaud, R.; Amouroux, D.

    2009-01-01

    Full text: The environmental reactivity of Hg is extremely dependent on its chemical form. In fact, Hg bioaccumulation is due to the greater trophic transfer efficiency of methylmercury which is formed as a result of biotic or abiotic transformations caused by specific redox gradients and bacterial activity. The study of stable isotope biogeochemistry of Hg may provide a powerful tool to track and understand its cycle and pathways in the environment. This work presents the measurement of species-specific Hg isotopic composition by GC-MCICPMS during Hg methylation experiments using cultures of pure bacterial strains incubated with Hg (II) standard NIST 3133. (author)

  18. Light duty utility arm software requirements specification

    International Nuclear Information System (INIS)

    Kiebel, G.R.

    1995-01-01

    This document defines the software requirements for the integrated control and data acquisition system of the Light Duty Utility Arm (LDUA) System. It is intended to be used to guide the design of the application software, to be a basis for assessing the application software design, and to establish what is to be tested in the finished application software product

  19. PDSS/IMC requirements and functional specifications

    Science.gov (United States)

    1983-01-01

    The system (software and hardware) requirements for the Payload Development Support System (PDSS)/Image Motion Compensator (IMC) are provided. The PDSS/IMC system provides the capability for performing Image Motion Compensator Electronics (IMCE) flight software test, checkout, and verification and provides the capability for monitoring the IMC flight computer system during qualification testing for fault detection and fault isolation.

  20. Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

    Directory of Open Access Journals (Sweden)

    Anupam Paliwal

    2013-08-01

    Full Text Available Allele-specific DNA methylation (ASM is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons, one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs, each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS peaks near CTCF binding sites with ASM.

  1. Suppression of prolactin gene expression in GH cells correlates with site-specific DNA methylation.

    Science.gov (United States)

    Zhang, Z X; Kumar, V; Rivera, R T; Pasion, S G; Chisholm, J; Biswas, D K

    1989-10-01

    Prolactin- (PRL) producing and nonproducing subclones of the GH line of (rat) pituitary tumor cells have been compared to elucidate the regulatory mechanisms of PRL gene expression. Particular emphasis was placed on delineating the molecular basis of the suppressed state of the PRL gene in the prolactin-nonproducing (PRL-) GH subclone (GH(1)2C1). We examined six methylatable cytosine residues (5, -CCGG- and 1, -GCGC-) within the 30-kb region of the PRL gene in these subclones. This analysis revealed that -CCGG-sequences of the transcribed region, and specifically, one in the fourth exon of the PRL gene, were heavily methylated in the PRL-, GH(1)2C1 cells. Furthermore, the inhibition of PRL gene expression in GH(1)2C1 was reversed by short-term treatment of the cells with a sublethal concentration of azacytidine (AzaC), an inhibitor of DNA methylation. The reversion of PRL gene expression by AzaC was correlated with the concurrent demethylation of the same -CCGG- sequences in the transcribed region of PRL gene. An inverse correlation between PRL gene expression and the level of methylation of the internal -C- residues in the specific -CCGG-sequence of the transcribed region of the PRL gene was demonstrated. The DNase I sensitivity of these regions of the PRL gene in PRL+, PRL-, and AzaC-treated cells was also consistent with an inverse relationship between methylation state, a higher order of structural modification, and gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Simulation Data Management - Requirements and Design Specification

    Energy Technology Data Exchange (ETDEWEB)

    Clay, Robert L. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Friedman-Hill, Ernest J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Gibson, Marcus J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Hoffman, Edward L. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Olson, Kevin H. [Science Applications International Corporation (SAIC), Reston, VA (United States); Laney, Daniel [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2017-11-01

    Simulation Data Management (SDM), the ability to securely organize, archive, and share analysis models and the artifacts used to create them, is a fundamental requirement for modern engineering analysis based on computational simulation. We have worked separately to provide secure, network SDM services to engineers and scientists at our respective laboratories for over a decade. We propose to leverage our experience and lessons learned to help develop and deploy a next-generation SDM service as part of a multi-laboratory team. This service will be portable across multiple sites and platforms, and will be accessible via a range of command-line tools and well-documented APIs. In this document, we’ll review our high-level and low-level requirements for such a system, review one existing system, and briefly discuss our proposed implementation.

  3. Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl–methyl nuclear overhauser enhancement spectroscopy

    International Nuclear Information System (INIS)

    Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius

    2011-01-01

    Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ∼1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl–methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

  4. LHCb electronics - requirements, specifications and implementation

    CERN Document Server

    Bibby, J

    2001-01-01

    This document is intended to serve as a requirements document, a design manual and an implementation reference for the RICH electronics systems. At the current time, the electronics design is under active consideration and this is reflected in this document which represents a working proposal as regards both the functional model and physical implementation. Comments on installation, commissioning, and maintenance are included. For convenience a description of the proposed RICH data formats is appended.

  5. Smart grid standards specifications, requirements, and technologies

    CERN Document Server

    Sato, Takuro; Duan, Bin; Macuha, Martin; Zhou, Zhenyu; Wu, Jun; Tariq, Muhammad; Asfaw, Solomon A

    2015-01-01

    With numerous existing Smart Grid standards, it is clear that governments and industrial organizations world-wide have understood and acknowledged the critical role they play. This timely book is a useful guide for Smart Grid professionals in easily classifying fundamental Smart Grid standards, and to quickly find the building blocks they need to analyse and implement a Smart Grid system. The standards are organized in a systematic manner that aids identification, according to grid requirements. It also covers broader Smart Grid areas including, but not limited to, the following: A fully c

  6. Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease.

    Science.gov (United States)

    Chiba, Hirofumi; Kakuta, Yoichi; Kinouchi, Yoshitaka; Kawai, Yosuke; Watanabe, Kazuhiro; Nagao, Munenori; Naito, Takeo; Onodera, Motoyuki; Moroi, Rintaro; Kuroha, Masatake; Kanazawa, Yoshitake; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Negoro, Kenichi; Nagasaki, Masao; Unno, Michiaki; Shimosegawa, Tooru

    2018-01-01

    Inflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions. CD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score ([Formula: see text]) >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing [Formula: see text] >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed. We extracted six candidate ASM SNPs around IBD susceptibility genes. The top of [Formula: see text] (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 ([Formula: see text] = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016). We confirmed the existence of cis-regulated ASM around

  7. Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

    Science.gov (United States)

    Chiba, Hirofumi; Kakuta, Yoichi; Kinouchi, Yoshitaka; Kawai, Yosuke; Watanabe, Kazuhiro; Nagao, Munenori; Naito, Takeo; Onodera, Motoyuki; Moroi, Rintaro; Kuroha, Masatake; Kanazawa, Yoshitake; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Negoro, Kenichi; Nagasaki, Masao; Unno, Michiaki; Shimosegawa, Tooru

    2018-01-01

    Background Inflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions. Methods CD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score (ΔRAS¯) >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing ΔRAS¯ >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed. Results We extracted six candidate ASM SNPs around IBD susceptibility genes. The top of ΔRAS¯ (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 (ΔRAS¯ = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016). Conclusions We confirmed the existence of cis-regulated ASM around IBD

  8. FLECH PowerMax Service Requirement Specification

    DEFF Research Database (Denmark)

    It is expected that in the future, ancillary services will be required at distribution level. This work describes how one of these ancillary services, the PowerMax service, must be spec ified within a market framework. The basis of this work is the Flexibility Clearing House (FLE CH) platform...... and the Distribution System Operator (DSO) services defined in [1]. The PowerMax service has been further discussed in [2], and this technical report seeks to clarify the implementation details first discussed in that paper. The aim of the PowerMax service it to relieve congestion issue s in the distribution system...... by establishing a maximum power that a cluster of consumer devices may draw from the syste m. The consumers will receive a payment for providing this service, and the DSO will be able to postpone grid reinfo rcement...

  9. Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Hagen Jeffrey A

    2007-10-01

    Full Text Available Abstract Background Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients. Results We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value Conclusion The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

  10. Region-specific RNA m6A methylation represents a new layer of control in the gene regulatory network in the mouse brain.

    Science.gov (United States)

    Chang, Mengqi; Lv, Hongyi; Zhang, Weilong; Ma, Chunhui; He, Xue; Zhao, Shunli; Zhang, Zhi-Wei; Zeng, Yi-Xin; Song, Shuhui; Niu, Yamei; Tong, Wei-Min

    2017-09-01

    N 6 -methyladenosine (m 6 A) is the most abundant epitranscriptomic mark found on mRNA and has important roles in various physiological processes. Despite the relatively high m 6 A levels in the brain, its potential functions in the brain remain largely unexplored. We performed a transcriptome-wide methylation analysis using the mouse brain to depict its region-specific methylation profile. RNA methylation levels in mouse cerebellum are generally higher than those in the cerebral cortex. Heterogeneity of RNA methylation exists across different brain regions and different types of neural cells including the mRNAs to be methylated, their methylation levels and methylation site selection. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. In addition, high methylation levels of fragile X mental retardation protein (FMRP) target mRNAs suggest that m 6 A methylation is likely to be used for selective recognition of target mRNAs by FMRP in the synapse. Overall, we provide a region-specific map of RNA m 6 A methylation and characterize the distinct features of specific and common methylation in mouse cerebellum and cerebral cortex. Our results imply that RNA m 6 A methylation is a newly identified element in the region-specific gene regulatory network in the mouse brain. © 2017 The Authors.

  11. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. [Haemophilus influenzae, Escherichia coli, Paramecium aurelia

    Energy Technology Data Exchange (ETDEWEB)

    Lacks, S.; Greenberg, B.

    1977-01-01

    Restriction endonucleases Dpn I and Dpn II are produced by two distinct strains of Diplococcus pneumoniae. The two enzymes show complementary specificity with respect to methylation of sites in DNA. From the identity of its cleavage site with that of Mbo I, it appears that Dpn II cleaves at the unmodified sequence 5'-G-A-T-C-3'. Dpn I cleaves at the same sequence when the adenine residue is methylated. Both enzymes produce only double-strand breaks in susceptible DNA. Their susceptibility to Dpn I and not Dpn II shows that essentially all the G-A-T-C sequences are methylated in DNA from the pneumococcal strain that produces Dpn II as well as in DNA from Hemophilus influenzae and Escherichia coli. In the dam-3 mutant of E. coli none of these sequences appear to be methylated. Residual adenine methylation in the dam-3 mutant DNA most likely occurs at different sites. Different but characteristic degrees of methylation at G-A-T-C sites are found in the DNA of bacterial viruses grown in E. coli. DNAs from mammalian cells and viruses are not methylated at this sequence. Mitochondrial DNA from Paramecium aurelia is not methylated, but a small proportion of G-A-T-C sequences in the macronuclear DNA of this eukaryote appear to be methylated. Possible roles of sequence-specific methylation in the accommodation of plasmids, in the replication of DNA, in the regulation of gene function and in the restriction of viral infection are discussed.

  12. Structuring Formal Requirements Specifications for Reuse and Product Families

    Science.gov (United States)

    Heimdahl, Mats P. E.

    2001-01-01

    In this project we have investigated how formal specifications should be structured to allow for requirements reuse, product family engineering, and ease of requirements change, The contributions of this work include (1) a requirements specification methodology specifically targeted for critical avionics applications, (2) guidelines for how to structure state-based specifications to facilitate ease of change and reuse, and (3) examples from the avionics domain demonstrating the proposed approach.

  13. Introducing a game approach towards IS requirements specification

    DEFF Research Database (Denmark)

    Jensen, Mika Yasuoka; Kadoya, Kyoichi; Niwa, Takashi

    2014-01-01

    Devising a system requirements specification is a challenging task. Even after several decades of system development research, specifications for large-scale, widely-used systems remain difficult. In this paper, we suggest a first step toward a requirements specification through a stakeholder inv...

  14. Sex-Specific Muscular Maturation Responses Following Prenatal Exposure to Methylation-Related Micronutrients in Pigs

    Directory of Open Access Journals (Sweden)

    Michael Oster

    2017-01-01

    Full Text Available Supplementation of micronutrients involved in DNA methylation, particularly during pregnancy, is recommended because of its impacts on human health, but further evidence is needed regarding the effects of over-supplementation and differences between sexes. Here, a porcine model was used to assess effects of maternal supplementation with one-carbon-cycle compounds during prenatal and postnatal stages on offspring muscle development. Sows received either a standard diet (CON or a standard diet supplemented with folate, B6, B12, methionine, choline, and zinc (MET throughout gestation. Myogenesis-, growth-, and nutrient utilization-related transcript expression was assessed using quantitative PCR. Organismal phenotype and gene expression effects differed significantly between males and females. Male MET-offspring showed increased fetal weight during late pregnancy but decreased live weight postnatally, with compensatory transcriptional responses comprising myogenic key drivers (Pax7, MyoD1, myogenin. In contrast, female weights were unaffected by diet, and mRNA abundances corresponded to a phenotype of cellular reorganization via FABP3, FABP4, SPP1 and Insulin-like Growth Factor-signaling. These findings in an animal model suggest that supplementation during pregnancy with methylation-related micronutrients can promote sex-specific myogenic maturation processes related to organismal growth and muscle metabolism. The usage of maternal dietary supplements should be more carefully considered regarding its ability to promote fetal and postnatal health.

  15. 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.

  16. Requirement of RIZ1 for cancer prevention by methyl-balanced diet

    OpenAIRE

    Wenyun Zhou; Sergio Alonso; Daisaku Takai; Shelly C. Lu; Fumiichiro Yamamoto; Manuel Perucho; Shi Huang

    2008-01-01

    Background The typical Western diet is not balanced in methyl nutrients that regulate the level of the methyl donor S-adenosylmethionine (SAM) and its derivative metabolite S-adenosylhomocysteine (SAH), which in turn may control the activity of certain methyltransferases. Feeding rodents with amino acid defined and methyl-imbalanced diet decreases hepatic SAM and causes liver cancers. RIZ1 (PRDM2 or KMT8) is a tumor suppressor and functions in transcriptional repression by methylating histone...

  17. Screening for SNPs with Allele-Specific Methylation based on Next-Generation Sequencing Data.

    Science.gov (United States)

    Hu, Bo; Ji, Yuan; Xu, Yaomin; Ting, Angela H

    2013-05-01

    Allele-specific methylation (ASM) has long been studied but mainly documented in the context of genomic imprinting and X chromosome inactivation. Taking advantage of the next-generation sequencing technology, we conduct a high-throughput sequencing experiment with four prostate cell lines to survey the whole genome and identify single nucleotide polymorphisms (SNPs) with ASM. A Bayesian approach is proposed to model the counts of short reads for each SNP conditional on its genotypes of multiple subjects, leading to a posterior probability of ASM. We flag SNPs with high posterior probabilities of ASM by accounting for multiple comparisons based on posterior false discovery rates. Applying the Bayesian approach to the in-house prostate cell line data, we identify 269 SNPs as candidates of ASM. A simulation study is carried out to demonstrate the quantitative performance of the proposed approach.

  18. Integrated genetic and epigenetic analysis identifies haplotype-specific methylation in the FTO type 2 diabetes and obesity susceptibility locus.

    Directory of Open Access Journals (Sweden)

    Christopher G Bell

    2010-11-01

    Full Text Available Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D, focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip and absolute methylation values were estimated using a Bayesian algorithm (BATMAN. Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10(-4, permutation p = 1.0×10(-3. Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10(-7. Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM, encapsulates a Highly Conserved Non-Coding Element (HCNE that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

  19. [Analysis of tissue-specific differentially methylated genes with differential gene expression in non-small cell lung cancer].

    Science.gov (United States)

    Yin, L G; Zou, Z Q; Zhao, H Y; Zhang, C L; Shen, J G; Qi, L; Qi, M; Xue, Z Q

    2014-01-01

    Adenocarcinoma (ADC) and squamous cell carcinomas (SCC) are two subtypes of non-small cell lung carcinomas which are regarded as the leading cause of cancer-related malignancy worldwide. The aim of this study is to detect the differentially methylated loci (DMLs) and differentially methylated genes (DMGs) of these two tumor sets, and then to illustrate the different expression level of specific methylated genes. Using TCGA database and Illumina HumanMethylation 27 arrays, we first screened the DMGs and DMLs in tumor samples. Then, we explored the BiologicalProcess terms of hypermethylated and hypomethylated genes using Functional Gene Ontology (GO) catalogues. Hypermethylation intensively occurred in CpG-island, whereas hypomethylation was located in non-CpG-island. Most SCC and ADC hypermethylated genes involved GO function of DNA dependenit regulation of transcription, and hypomethylated genes mainly 'enriched in the term of immune responses. Additionally, the expression level of specific differentially methylated genesis distinctbetween ADC and SCC. It is concluded that ADC and SCC have different methylated status that might play an important role in carcinogenesis.

  20. A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

    Science.gov (United States)

    Brendler, T; Abeles, A; Austin, S

    1995-08-15

    The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.

  1. Obesity is associated with depot-specific alterations in adipocyte DNA methylation and gene expression

    DEFF Research Database (Denmark)

    Sonne, Si Brask; Yadav, Rachita; Yin, Guangliang

    2017-01-01

    The present study aimed to identify genes exhibiting concomitant obesity-dependent changes in DNA methylation and gene expression in adipose tissues in the mouse using diet-induced obese (DIO) C57BL/6J and genetically obese ob/ob mice as models. Mature adipocytes were isolated from epididymal...... and inguinal adipose tissues of ob/ob and DIO C57BL/6J mice. DNA methylation was analyzed by MeDIP-sequencing and gene expression by microarray analysis. The majority of differentially methylated regions (DMRs) were hypomethylated in obese mice. Global methylation of long interspersed elements indicated......57BL/6J mice occurred primarily in exons, whereas inguinal adipocytes of ob/ob mice exhibited a higher enrichment of DMRs in promoter regions than in other regions of the genome, suggesting an influence of leptin on DNA methylation in inguinal adipocytes. We observed altered methylation...

  2. Association of Tissue-Specific DNA Methylation Alterations with α-Thalassemia Southeast Asian Deletion

    Directory of Open Access Journals (Sweden)

    Tanapat Pangeson

    2017-11-01

    Full Text Available In the wild-type allele, DNA methylation levels of 10 consecutive CpG sites adjacent to the upstream 5′-breakpoint of α-thalassemia Southeast Asian (SEA deletion are not different between placenta and leukocytes. However, no previous study has reported the map of DNA methylation in the SEA allele. This report aims to show that the SEA mutation is associated with DNA methylation changes, resulting in differential methylation between placenta and leukocytes. Methylation-sensitive high-resolution analysis was used to compare DNA methylation among placenta, leukocytes, and unmethylated control DNA. The result indicates that the DNA methylation between placenta and leukocyte DNA is different and shows that the CpG status of both is not fully unmethylated. Mapping of individual CpG sites was performed by targeted bisulfite sequencing. The DNA methylation level of the 10 consecutive CpG sites was different between placenta and leukocyte DNA. When the 10th CpG of the mutation allele was considered as a hallmark for comparing DNA methylation level, it was totally different from the unmethylated 10th CpG of the wild-type allele. Finally, the distinct DNA methylation patterns between both DNA were extracted. In total, 24 patterns were found in leukocyte samples and 9 patterns were found in placenta samples. This report shows that the large deletion is associated with DNA methylation change. In further studies for clinical application, the distinct DNA methylation pattern might be a potential marker for detecting cell-free fetal DNA.

  3. Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

    Directory of Open Access Journals (Sweden)

    Weis Robert M

    2005-03-01

    Full Text Available Abstract Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr, which contributes to an increase in the steady-state (adapted methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA and an adaptor protein (CheW, but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493. The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively differed from those found previously for the aspartate receptor (Tar, which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four. This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of

  4. Identified adjustability dimensions when generating a product specific requirements specification by requirements reuse

    DEFF Research Database (Denmark)

    Hauksdóttir, Dagný; Mortensen, Niels Henrik; Nielsen, Poul Erik

    2014-01-01

    . An extensive state of the art is included to introduce the presented methods related to each adjustability dimensions. The options for implementing each adjustability dimensions in a requirement reuse approach are illustrated along with a discussion regarding the benefits and issues resulting from each option....... This discussion should help practitioners to better understand the possible methods that can be implemented and to design a user friendly and sustainable approach. A case study, describing how the dimensions are incorporated in two requirements reuse approaches, for Danfoss Solar Inverters (SI) and Danfoss...

  5. Requirement of RIZ1 for cancer prevention by methyl-balanced diet.

    Science.gov (United States)

    Zhou, Wenyun; Alonso, Sergio; Takai, Daisaku; Lu, Shelly C; Yamamoto, Fumiichiro; Perucho, Manuel; Huang, Shi

    2008-01-01

    The typical Western diet is not balanced in methyl nutrients that regulate the level of the methyl donor S-adenosylmethionine (SAM) and its derivative metabolite S-adenosylhomocysteine (SAH), which in turn may control the activity of certain methyltransferases. Feeding rodents with amino acid defined and methyl-imbalanced diet decreases hepatic SAM and causes liver cancers. RIZ1 (PRDM2 or KMT8) is a tumor suppressor and functions in transcriptional repression by methylating histone H3 lysine 9. Here we show that a methyl-balanced diet conferred additional survival benefits compared to a tumor-inducing methyl-imbalanced diet only in mice with wild type RIZ1 but not in mice deficient in RIZ1. While absence of RIZ1 was tumorigenic in mice fed the balanced diet, its presence did not prevent tumor formation in mice fed the imbalanced diet. Microarray and gene expression analysis showed that, unlike most of its related enzymes, RIZ1 was upregulated by methyl-balanced diet. Methyl-balanced diet did not fully repress oncogenes such as c-Jun in the absence of RIZ1. Higher RIZ1 activity was associated with greater H3 lysine 9 methylation in RIZ1 target genes as shown by chromatin immunoprecipitation analysis. The data identify RIZ1 as a critical target of methyl-balanced diet in cancer prevention. The molecular understanding of dietary carcinogenesis may help people make informed choices on diet, which may greatly reduce the incidence of cancer.

  6. SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants.

    Science.gov (United States)

    Tripathi, Diwaker; Jiang, Yu-Lin; Kumar, Dhirendra

    2010-08-04

    Tobacco SABP2, a 29kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance. Copyright (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins

    Energy Technology Data Exchange (ETDEWEB)

    Mas, Guillaume; Crublet, Elodie [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France); Hamelin, Olivier [CNRS (France); Gans, Pierre; Boisbouvier, Jérôme, E-mail: jerome.boisbouvier@ibs.fr [Univ. Grenoble Alpes, Institut de Biologie Structurale (IBS) (France)

    2013-09-28

    The specific protonation of valine and leucine methyl groups in proteins is typically achieved by overexpressing proteins in M9/D{sub 2}O medium supplemented with either labeled α-ketoisovalerate for the labeling of the four prochiral methyl groups or with 2-acetolactate for the stereospecific labeling of the valine and leucine side chains. However, when these labeling schemes are applied to large protein assemblies, significant overlap between the correlations of the valine and leucine methyl groups occurs, hampering the analysis of 2D methyl-TROSY spectra. Analysis of the leucine and valine biosynthesis pathways revealed that the incorporation of labeled precursors in the leucine pathway can be inhibited by the addition of exogenous l-leucine-d{sub 10}. We exploited this property to label stereospecifically the pro-R and pro-S methyl groups of valine with minimal scrambling to the leucine residues. This new labeling protocol was applied to the 468 kDa homododecameric peptidase TET2 to decrease the complexity of its NMR spectra. All of the pro-S valine methyl resonances of TET2 were assigned by combining mutagenesis with this innovative labeling approach. The assignments were transferred to the pro-R groups using an optimally labeled sample and a set of triple resonance experiments. This improved labeling scheme enables us to overcome the main limitation of overcrowding in the NMR spectra of prochiral methyl groups, which is a prerequisite for the site-specific measurement of the structural and dynamic parameters or for the study of interactions in very large protein assemblies.

  8. Solid Waste Information and Tracking System (SWITS) Software Requirements Specification

    International Nuclear Information System (INIS)

    MAY, D.L.

    2000-01-01

    This document is the primary document establishing requirements for the Solid Waste Information and Tracking System (SWITS) as it is converted to a client-server architecture. The purpose is to provide the customer and the performing organizations with the requirements for the SWITS in the new environment. This Software Requirement Specification (SRS) describes the system requirements for the SWITS Project, and follows the PHMC Engineering Requirements, HNF-PRO-1819, and Computer Software Qualify Assurance Requirements, HNF-PRO-309, policies. This SRS includes sections on general description, specific requirements, references, appendices, and index. The SWITS system defined in this document stores information about the solid waste inventory on the Hanford site. Waste is tracked as it is generated, analyzed, shipped, stored, and treated. In addition to inventory reports a number of reports for regulatory agencies are produced

  9. Solid Waste Information and Tracking System (SWITS) Software Requirements Specification

    Energy Technology Data Exchange (ETDEWEB)

    MAY, D.L.

    2000-03-22

    This document is the primary document establishing requirements for the Solid Waste Information and Tracking System (SWITS) as it is converted to a client-server architecture. The purpose is to provide the customer and the performing organizations with the requirements for the SWITS in the new environment. This Software Requirement Specification (SRS) describes the system requirements for the SWITS Project, and follows the PHMC Engineering Requirements, HNF-PRO-1819, and Computer Software Qualify Assurance Requirements, HNF-PRO-309, policies. This SRS includes sections on general description, specific requirements, references, appendices, and index. The SWITS system defined in this document stores information about the solid waste inventory on the Hanford site. Waste is tracked as it is generated, analyzed, shipped, stored, and treated. In addition to inventory reports a number of reports for regulatory agencies are produced.

  10. DNA methyltransferase homologue TRDMT1 in Plasmodium falciparum specifically methylates endogenous aspartic acid tRNA.

    Science.gov (United States)

    Govindaraju, Gayathri; Jabeena, C A; Sethumadhavan, Devadathan Valiyamangalath; Rajaram, Nivethika; Rajavelu, Arumugam

    2017-10-01

    In eukaryotes, cytosine methylation regulates diverse biological processes such as gene expression, development and maintenance of genomic integrity. However, cytosine methylation and its functions in pathogenic apicomplexan protozoans remain enigmatic. To address this, here we investigated the presence of cytosine methylation in the nucleic acids of the protozoan Plasmodium falciparum. Interestingly, P. falciparum has TRDMT1, a conserved homologue of DNA methyltransferase DNMT2. However, we found that TRDMT1 did not methylate DNA, in vitro. We demonstrate that TRDMT1 methylates cytosine in the endogenous aspartic acid tRNA of P. falciparum. Through RNA bisulfite sequencing, we mapped the position of 5-methyl cytosine in aspartic acid tRNA and found methylation only at C38 position. P. falciparum proteome has significantly higher aspartic acid content and a higher proportion of proteins with poly aspartic acid repeats than other apicomplexan pathogenic protozoans. Proteins with such repeats are functionally important, with significant roles in host-pathogen interactions. Therefore, TRDMT1 mediated C38 methylation of aspartic acid tRNA might play a critical role by translational regulation of important proteins and modulate the pathogenicity of the malarial parasite. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Detection of Turner syndrome using X-chromosome inactivation specific differentially methylated CpG sites: A pilot study.

    Science.gov (United States)

    Zhang, Qiang; Guo, Xiaohong; Tian, Tian; Wang, Teng; Li, Qiaoli; Wang, Lei; Liu, Yun; Xing, Qinghe; He, Lin; Zhao, Xinzhi

    2017-05-01

    Early diagnosis of Turner syndrome (TS) may improve preventive measures and treatment. X-chromosome inactivation specific differentially methylated CpG sites (XIDMSs) that are high methylated in inactive X chromosomes (Xi) and unmethylated in active X chromosomes (Xa) may be potential makers for TS detection. The candidate XIDMSs were screened from 9 male and 12 female DNA samples with normal karyotypes using the Illumina 450k array and validated by bisulfite sequencing PCR and pyrosequencing assay. X chromosome dosage was calculated according to the methylation level of multiple XIDMSs. Overall, 108 candidate XIDMSs were screened by the 450k array. Validations indicated that XIDMSs gathered and formed the X-chromosome inactivation specific differentially methylated regions (XIDMRs). Using 3 XIDMRs at SAT1, UXT and UTP14A loci, 36 TS, 22 normal female and 6 male samples were analyzed. Methylation levels of the 20 XIDMSs in the XIDMRs could distinguish between TS and normal female DNA samples, the X chromosome dosage was consistent with karyotyping data. Analyzing samples of 2 triple X syndrome and 3 Klinefelter syndrome patients suggested that this method could be used to detect X chromosome aneuploids other than TS. XIDMSs are widely spread along the X chromosome and might be effective markers for detection of TS and other X chromosome aneuploids. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

    Directory of Open Access Journals (Sweden)

    Kiwako S Araki

    Full Text Available In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals. We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers. We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms

  13. Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

    Science.gov (United States)

    Araki, Kiwako S; Kubo, Takuya; Kudoh, Hiroshi

    2017-01-01

    In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed) and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals). We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers). We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP) loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms, particularly for

  14. Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall.

    Science.gov (United States)

    Amsbury, Sam; Hunt, Lee; Elhaddad, Nagat; Baillie, Alice; Lundgren, Marjorie; Verhertbruggen, Yves; Scheller, Henrik V; Knox, J Paul; Fleming, Andrew J; Gray, Julie E

    2016-11-07

    Stomatal opening and closure depends on changes in turgor pressure acting within guard cells to alter cell shape [1]. The extent of these shape changes is limited by the mechanical properties of the cells, which will be largely dependent on the structure of the cell walls. Although it has long been observed that guard cells are anisotropic due to differential thickening and the orientation of cellulose microfibrils [2], our understanding of the composition of the cell wall that allows them to undergo repeated swelling and deflation remains surprisingly poor. Here, we show that the walls of guard cells are rich in un-esterified pectins. We identify a pectin methylesterase gene, PME6, which is highly expressed in guard cells and required for stomatal function. pme6-1 mutant guard cells have walls enriched in methyl-esterified pectin and show a decreased dynamic range in response to triggers of stomatal opening/closure, including elevated osmoticum, suggesting that abrogation of stomatal function reflects a mechanical change in the guard cell wall. Altered stomatal function leads to increased conductance and evaporative cooling, as well as decreased plant growth. The growth defect of the pme6-1 mutant is rescued by maintaining the plants in elevated CO 2 , substantiating gas exchange analyses, indicating that the mutant stomata can bestow an improved assimilation rate. Restoration of PME6 rescues guard cell wall pectin methyl-esterification status, stomatal function, and plant growth. Our results establish a link between gene expression in guard cells and their cell wall properties, with a corresponding effect on stomatal function and plant physiology. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. SHH1, a homeodomain protein required for DNA methylation, as well as RDR2, RDM4, and chromatin remodeling factors, associate with RNA polymerase IV.

    Directory of Open Access Journals (Sweden)

    Julie A Law

    2011-07-01

    Full Text Available DNA methylation is an evolutionarily conserved epigenetic modification that is critical for gene silencing and the maintenance of genome integrity. In Arabidopsis thaliana, the de novo DNA methyltransferase, domains rearranged methyltransferase 2 (DRM2, is targeted to specific genomic loci by 24 nt small interfering RNAs (siRNAs through a pathway termed RNA-directed DNA methylation (RdDM. Biogenesis of the targeting siRNAs is thought to be initiated by the activity of the plant-specific RNA polymerase IV (Pol-IV. However, the mechanism through which Pol-IV is targeted to specific genomic loci and whether factors other than the core Pol-IV machinery are required for Pol-IV activity remain unknown. Through the affinity purification of nuclear RNA polymerase D1 (NRPD1, the largest subunit of the Pol-IV polymerase, we found that several previously identified RdDM components co-purify with Pol-IV, namely RNA-dependent RNA polymerase 2 (RDR2, CLASSY1 (CLSY1, and RNA-directed DNA methylation 4 (RDM4, suggesting that the upstream siRNA generating portion of the RdDM pathway may be more physically coupled than previously envisioned. A homeodomain protein, SAWADEE homeodomain homolog 1 (SHH1, was also found to co-purify with NRPD1; and we demonstrate that SHH1 is required for de novo and maintenance DNA methylation, as well as for the accumulation of siRNAs at specific loci, confirming it is a bonafide component of the RdDM pathway.

  16. Quantitative global and gene-specific promoter methylation in relation to biological properties of neuroblastomas

    Directory of Open Access Journals (Sweden)

    Kiss Nimrod B

    2012-09-01

    Full Text Available Abstract Background In this study we aimed to quantify tumor suppressor gene (TSG promoter methylation densities levels in primary neuroblastoma tumors and cell lines. A subset of these TSGs is associated with a CpG island methylator phenotype (CIMP in other tumor types. Methods The study panel consisted of 38 primary tumors, 7 established cell lines and 4 healthy references. Promoter methylation was determined by bisulphate Pyrosequencing for 14 TSGs; and LINE-1 repeat element methylation was used as an indicator of global methylation levels. Results Overall mean TSG Z-scores were significantly increased in cases with adverse outcome, but were unrelated to global LINE-1 methylation. CIMP with hypermethylation of three or more gene promoters was observed in 6/38 tumors and 7/7 cell lines. Hypermethylation of one or more TSG (comprising TSGs BLU, CASP8, DCR2, CDH1, RASSF1A and RASSF2 was evident in 30/38 tumors. By contrast only very low levels of promoter methylation were recorded for APC, DAPK1, NORE1A, P14, P16, TP73, PTEN and RARB. Similar involvements of methylation instability were revealed between cell line models and neuroblastoma tumors. Separate analysis of two proposed CASP8 regulatory regions revealed frequent and significant involvement of CpG sites between exon 4 and 5, but modest involvement of the exon 1 region. Conclusions/significance The results highlight the involvement of TSG methylation instability in neuroblastoma tumors and cell lines using quantitative methods, support the use of DNA methylation analyses as a prognostic tool for this tumor type, and underscore the relevance of developing demethylating therapies for its treatment.

  17. Planning is not sufficient - Reliable computers need good requirements specifications

    International Nuclear Information System (INIS)

    Matras, J.R.

    1992-01-01

    Computer system reliability is the assurance that a computer system will perform its functions when required to do so. To ensure such reliability, it is important to plan the activities needed for computer system development. These development activities, in turn, require a Computer Quality Assurance Plan (CQAP) that provides the following: a Configuration Management Plan, a Verification and Validation (V and V) Plan, documentation requirements, a defined life cycle, review requirements, and organizational responsibilities. These items are necessary for system reliability; ultimately, however, they are not enough. Development of a reliable system is dependent on the requirements specification. This paper discusses how to use existing industry standards to develop a CQAP. In particular, the paper emphasizes the importance of the requirements specification and of methods for establishing reliability goals. The paper also describes how the revision of ANSI/IEE-ANS-7-4.3.2, Application Criteria for Digital Computer Systems of Nuclear Power Generating Stations, has addressed these issues

  18. 47 CFR 76.75 - Specific EEO program requirements.

    Science.gov (United States)

    2010-10-01

    ... MULTICHANNEL VIDEO AND CABLE TELEVISION SERVICE Equal Employment Opportunity Requirements § 76.75 Specific EEO... or sex is prohibited and that they may notify the Equal Employment Opportunity Commission, the... necessary. Nothing in this section shall be interpreted to require a multichannel video programming...

  19. Human-Specific Histone Methylation Signatures at Transcription Start Sites in Prefrontal Neurons

    Science.gov (United States)

    Cheung, Iris; Bharadwaj, Rahul; Chou, Hsin-Jung; Houston, Isaac B.; Peter, Cyril J.; Mitchell, Amanda C.; Yao, Wei-Dong; Myers, Richard H.; Chen, Jiang-fan; Preuss, Todd M.; Rogaev, Evgeny I.; Jensen, Jeffrey D.; Weng, Zhiping; Akbarian, Schahram

    2012-01-01

    Cognitive abilities and disorders unique to humans are thought to result from adaptively driven changes in brain transcriptomes, but little is known about the role of cis-regulatory changes affecting transcription start sites (TSS). Here, we mapped in human, chimpanzee, and macaque prefrontal cortex the genome-wide distribution of histone H3 trimethylated at lysine 4 (H3K4me3), an epigenetic mark sharply regulated at TSS, and identified 471 sequences with human-specific enrichment or depletion. Among these were 33 loci selectively methylated in neuronal but not non-neuronal chromatin from children and adults, including TSS at DPP10 (2q14.1), CNTN4 and CHL1 (3p26.3), and other neuropsychiatric susceptibility genes. Regulatory sequences at DPP10 and additional loci carried a strong footprint of hominid adaptation, including elevated nucleotide substitution rates and regulatory motifs absent in other primates (including archaic hominins), with evidence for selective pressures during more recent evolution and adaptive fixations in modern populations. Chromosome conformation capture at two neurodevelopmental disease loci, 2q14.1 and 16p11.2, revealed higher order chromatin structures resulting in physical contact of multiple human-specific H3K4me3 peaks spaced 0.5–1 Mb apart, in conjunction with a novel cis-bound antisense RNA linked to Polycomb repressor proteins and downregulated DPP10 expression. Therefore, coordinated epigenetic regulation via newly derived TSS chromatin could play an important role in the emergence of human-specific gene expression networks in brain that contribute to cognitive functions and neurological disease susceptibility in modern day humans. PMID:23185133

  20. DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

    Directory of Open Access Journals (Sweden)

    Shimrat Mamrut

    Full Text Available Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

  1. Dietary selenomethionine increases exon-specific DNA methylation of the p53 gene in rat liver and colon mucosa.

    Science.gov (United States)

    Zeng, Huawei; Yan, Lin; Cheng, Wen-Hsing; Uthus, Eric O

    2011-08-01

    The regulation of site-specific DNA methylation of tumor suppressor genes has been considered as a leading mechanism by which certain nutrients exert their anticancer property. This study was to investigate whether selenium (Se) affects the methylation of globe genomic DNA and the exon-specific p53 gene. Three groups of rats (n = 6-7/group) were fed the AIN-93G basal diet supplemented with 0 [Se deficient (D)], 0.15 [Se adequate (A)], or 4 mg [Se supranutritional (S)] (Se as l-selenomethionine)/kg diet for 104 d, respectively. Rats fed the A or S diet had greater plasma and liver glutathione peroxidase activity, liver thioredoxin reductase activity, and plasma homocysteine concentration than those fed the D diet. However, compared with the A diet, rats fed the S diet did not further increase these Se-dependent enzyme activities or homocysteine concentration. In contrast, Se concentrations in kidney, liver, gastrocnemius muscle, and plasma were increased in a Se-dose-dependent manner. Interestingly, rats fed the S diet had significantly less global liver genomic DNA methylation than those fed the D diet. However, the S diet significantly increased the methylation of the p53 gene (exons 5-8) but not the β-actin gene (exons 2-3) DNA in liver and colon mucosa compared with those fed the D diet. Taken together, long-term Se consumption not only affects selenoprotein enzyme activities, homocysteine, tissue Se concentrations, and global genomic DNA methylation but also increases exon-specific DNA methylation of the p53 gene in a Se-dose-dependent manner in rat liver and colon mucosa.

  2. Defining differentially methylated regions specific for the acquisition of pluripotency and maintenance in human pluripotent stem cells via microarray.

    Directory of Open Access Journals (Sweden)

    WenYin He

    Full Text Available Epigenetic regulation is critical for the maintenance of human pluripotent stem cells. It has been shown that pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, appear to have a hypermethylated status compared with differentiated cells. However, the epigenetic differences in genes that maintain stemness and regulate reprogramming between embryonic stem cells and induced pluripotent stem cells remain unclear. Additionally, differential methylation patterns of induced pluripotent stem cells generated using diverse methods require further study.Here, we determined the DNA methylation profiles of 10 human cell lines, including 2 ESC lines, 4 virally derived iPSC lines, 2 episomally derived iPSC lines, and the 2 parental cell lines from which the iPSCs were derived using Illumina's Infinium HumanMethylation450 BeadChip. The iPSCs exhibited a hypermethylation status similar to that of ESCs but with distinct differences from the parental cells. Genes with a common methylation pattern between iPSCs and ESCs were classified as critical factors for stemness, whereas differences between iPSCs and ESCs suggested that iPSCs partly retained the parental characteristics and gained de novo methylation aberrances during cellular reprogramming. No significant differences were identified between virally and episomally derived iPSCs. This study determined in detail the de novo differential methylation signatures of particular stem cell lines.This study describes the DNA methylation profiles of human iPSCs generated using both viral and episomal methods, the corresponding somatic cells, and hESCs. Series of ss-DMRs and ES-iPS-DMRs were defined with high resolution. Knowledge of this type of epigenetic information could be used as a signature for stemness and self-renewal and provides a potential method for selecting optimal pluripotent stem cells for human regenerative medicine.

  3. CONCERNING CHAIN GROWTH SPECIFIC REACTION RATE AS A PART OF THE PROCESS OF METHYL METHACRYLATE MASS RADICAL POLYMERIZATION

    Directory of Open Access Journals (Sweden)

    A. A. Sultanova

    2017-02-01

    Full Text Available It is the chain growth specific reaction rate that was determined for the process of methyl methacrylate mass radical polymerization within the temperature range of 40–900 С in quasi-steady approximation by means of Monte Carlo method. The theoretical model of radical polymerization was developed taking the gel effect into account. Computer software was developed that enables to imitate radical polymerization process taking gel effect into account within the minimum run time. The programme was tested on asymptotic examples as well as was applied for methyl methacrylate mass radical polymerization. The programme makes it possible to calculate monomer conversion, molecular mass variation, molecular-mass distribution, etc.

  4. Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes

    Directory of Open Access Journals (Sweden)

    Josh Lewis Stern

    2017-12-01

    Full Text Available A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2 on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival.

  5. Allele-Specific DNA Methylation and Its Interplay with Repressive Histone Marks at Promoter-Mutant TERT Genes.

    Science.gov (United States)

    Stern, Josh Lewis; Paucek, Richard D; Huang, Franklin W; Ghandi, Mahmoud; Nwumeh, Ronald; Costello, James C; Cech, Thomas R

    2017-12-26

    A mutation in the promoter of the Telomerase Reverse Transcriptase (TERT) gene is the most frequent noncoding mutation in cancer. The mutation drives unusual monoallelic expression of TERT, allowing immortalization. Here, we find that DNA methylation of the TERT CpG island (CGI) is also allele-specific in multiple cancers. The expressed allele is hypomethylated, which is opposite to cancers without TERT promoter mutations. The continued presence of Polycomb repressive complex 2 (PRC2) on the inactive allele suggests that histone marks of repressed chromatin may be causally linked to high DNA methylation. Consistent with this hypothesis, TERT promoter DNA containing 5-methyl-CpG has much increased affinity for PRC2 in vitro. Thus, CpG methylation and histone marks appear to collaborate to maintain the two TERT alleles in different epigenetic states in TERT promoter mutant cancers. Finally, in several cancers, DNA methylation levels at the TERT CGI correlate with altered patient survival. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Requirement of RIZ1 for Cancer Prevention by Methyl-Balanced Diet

    Science.gov (United States)

    Zhou, Wenyun; Alonso, Sergio; Takai, Daisaku; Lu, Shelly C.; Yamamoto, Fumiichiro; Perucho, Manuel; Huang, Shi

    2008-01-01

    Background The typical Western diet is not balanced in methyl nutrients that regulate the level of the methyl donor S-adenosylmethionine (SAM) and its derivative metabolite S-adenosylhomocysteine (SAH), which in turn may control the activity of certain methyltransferases. Feeding rodents with amino acid defined and methyl-imbalanced diet decreases hepatic SAM and causes liver cancers. RIZ1 (PRDM2 or KMT8) is a tumor suppressor and functions in transcriptional repression by methylating histone H3 lysine 9. Methodology/Principal Findings Here we show that a methyl-balanced diet conferred additional survival benefits compared to a tumor-inducing methyl-imbalanced diet only in mice with wild type RIZ1 but not in mice deficient in RIZ1. While absence of RIZ1 was tumorigenic in mice fed the balanced diet, its presence did not prevent tumor formation in mice fed the imbalanced diet. Microarray and gene expression analysis showed that, unlike most of its related enzymes, RIZ1 was upregulated by methyl-balanced diet. Methyl-balanced diet did not fully repress oncogenes such as c-Jun in the absence of RIZ1. Higher RIZ1 activity was associated with greater H3 lysine 9 methylation in RIZ1 target genes as shown by chromatin immunoprecipiation analysis. Conclusions/Significance The data identify RIZ1 as a critical target of methyl-balanced diet in cancer prevention. The molecular understanding of dietary carcinogenesis may help people make informed choices on diet, which may greatly reduce the incidence of cancer. PMID:18852888

  7. A Framework for RFID Survivability Requirement Analysis and Specification

    Science.gov (United States)

    Zuo, Yanjun; Pimple, Malvika; Lande, Suhas

    Many industries are becoming dependent on Radio Frequency Identification (RFID) technology for inventory management and asset tracking. The data collected about tagged objects though RFID is used in various high level business operations. The RFID system should hence be highly available, reliable, and dependable and secure. In addition, this system should be able to resist attacks and perform recovery in case of security incidents. Together these requirements give rise to the notion of a survivable RFID system. The main goal of this paper is to analyze and specify the requirements for an RFID system to become survivable. These requirements, if utilized, can assist the system in resisting against devastating attacks and recovering quickly from damages. This paper proposes the techniques and approaches for RFID survivability requirements analysis and specification. From the perspective of system acquisition and engineering, survivability requirement is the important first step in survivability specification, compliance formulation, and proof verification.

  8. Safety of Nuclear Power Plants: Design. Specific Safety Requirements

    International Nuclear Information System (INIS)

    2012-01-01

    On the basis of the principles included in the Fundamental Safety Principles, IAEA Safety Standards Series No. SF-1, this Safety Requirements publication establishes requirements applicable to the design of nuclear power plants. It covers the design phase and provides input for the safe operation of the power plant. It elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  9. Epigenome-wide analysis of DNA methylation reveals a rectal cancer-specific epigenomic signature

    Czech Academy of Sciences Publication Activity Database

    Vymetálková, Veronika; Vodička, Pavel; Pardini, Barbara; Rosa, F.; Levý, M.; Schneiderová, M.; Liška, V.; Vodičková, Ludmila; Nilsson, T.K.; Farkas, S. A.

    2016-01-01

    Roč. 8, č. 9 (2016), s. 1193-1207 ISSN 1750-1911 R&D Projects: GA MZd(CZ) NT13424; GA MZd(CZ) NV15-27580A; GA ČR(CZ) GA15-08239S; GA MŠk(CZ) LD14050 Institutional support: RVO:68378041 Keywords : DNA methylation * Illumina Human Methylation 450 BeadChip * rectal cancer Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.541, year: 2016

  10. 75 FR 51388 - 2-methyl-1,3-propanediol; Exemption from the Requirement of a Tolerance

    Science.gov (United States)

    2010-08-20

    ... dermal sensitization study in guinea pigs, 2- methyl-1,3-propanediol was determined to have mild... entities may include, but are not limited to: Crop production (NAICS code 111). Animal production (NAICS...

  11. Transforming Multidisciplinary Customer Requirements to Product Design Specifications

    Science.gov (United States)

    Ma, Xiao-Jie; Ding, Guo-Fu; Qin, Sheng-Feng; Li, Rong; Yan, Kai-Yin; Xiao, Shou-Ne; Yang, Guang-Wu

    2017-09-01

    With the increasing of complexity of complex mechatronic products, it is necessary to involve multidisciplinary design teams, thus, the traditional customer requirements modeling for a single discipline team becomes difficult to be applied in a multidisciplinary team and project since team members with various disciplinary backgrounds may have different interpretations of the customers' requirements. A new synthesized multidisciplinary customer requirements modeling method is provided for obtaining and describing the common understanding of customer requirements (CRs) and more importantly transferring them into a detailed and accurate product design specifications (PDS) to interact with different team members effectively. A case study of designing a high speed train verifies the rationality and feasibility of the proposed multidisciplinary requirement modeling method for complex mechatronic product development. This proposed research offersthe instruction to realize the customer-driven personalized customization of complex mechatronic product.

  12. Functional requirement specification in the packaging development chain

    NARCIS (Netherlands)

    Lutters, Diederick; ten Klooster, Roland

    2008-01-01

    As it is clear that the full packaging life cycle – at least partially – coincides with the product life cycle, both cycles are interwoven. Each has a network of functional requirements, with specific hierarchic propensities. These networks overlap, with prevailing hierarchies playing important

  13. Information Requirements Specification II: Brainstorming Collective Decision-Making Technique.

    Science.gov (United States)

    Telem, Moshe

    1988-01-01

    Information requirements specification (IRS) constitutes an Achilles heel in the system life cycle of management information systems. This article establishes a systematic overall IRS technique applicable to organizations of all types and sizes. The technique's integration of brainstorming and theory Z principles creates an effective, stimulating,…

  14. 49 CFR 179.401-1 - Individual specification requirements.

    Science.gov (United States)

    2010-10-01

    ...) § 179.400-5(d). Standard heat transfer rate. (Btu per day per lb. of water capacity, max.) (see § 179... FOR TANK CARS Specification for Cryogenic Liquid Tank Car Tanks and Seamless Steel Tanks (Classes DOT... restrictions § 179.400-20 179.400-20. Transfer line insulation § 179.400-17 Not required. [Amdt. 179-32, 48 FR...

  15. DNA Methylation in Skeletal Muscle Stem Cell Specification, Proliferation, and Differentiation

    Directory of Open Access Journals (Sweden)

    Rhianna C. Laker

    2016-01-01

    Full Text Available An unresolved and critically important question in skeletal muscle biology is how muscle stem cells initiate and regulate the genetic program during muscle development. Epigenetic dynamics are essential for cellular development and organogenesis in early life and it is becoming increasingly clear that epigenetic remodeling may also be responsible for the cellular adaptations that occur in later life. DNA methylation of cytosine bases within CpG dinucleotide pairs is an important epigenetic modification that reduces gene expression when located within a promoter or enhancer region. Recent advances in the field suggest that epigenetic regulation is essential for skeletal muscle stem cell identity and subsequent cell development. This review summarizes what is currently known about how skeletal muscle stem cells regulate the myogenic program through DNA methylation, discusses a novel role for metabolism in this process, and addresses DNA methylation dynamics in adult skeletal muscle in response to physical activity.

  16. DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

    Science.gov (United States)

    Spada, Fabio; Haemmer, Andrea; Kuch, David; Rothbauer, Ulrich; Schermelleh, Lothar; Kremmer, Elisabeth; Carell, Thomas; Längst, Gernot; Leonhardt, Heinrich

    2007-01-01

    DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required. PMID:17312023

  17. Draft Geologic Disposal Requirements Basis for STAD Specification

    Energy Technology Data Exchange (ETDEWEB)

    Ilgen, Anastasia G. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Bryan, Charles R. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Hardin, Ernest [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

    2015-03-25

    This document provides the basis for requirements in the current version of Performance Specification for Standardized Transportation, Aging, and Disposal Canister Systems, (FCRD-NFST-2014-0000579) that are driven by storage and geologic disposal considerations. Performance requirements for the Standardized Transportation, Aging, and Disposal (STAD) canister are given in Section 3.1 of that report. Here, the requirements are reviewed and the rationale for each provided. Note that, while FCRD-NFST-2014-0000579 provides performance specifications for other components of the STAD storage system (e.g. storage overpack, transfer and transportation casks, and others), these have no impact on the canister performance during disposal, and are not discussed here.

  18. Anti-N-Methyl-d-Aspartate Receptor Encephalitis in Adult Patients Requiring Intensive Care.

    Science.gov (United States)

    de Montmollin, Etienne; Demeret, Sophie; Brulé, Noëlle; Conrad, Marie; Dailler, Frédéric; Lerolle, Nicolas; Navellou, Jean-Christophe; Schwebel, Carole; Alves, Mikaël; Cour, Martin; Engrand, Nicolas; Tonnelier, Jean-Marie; Maury, Eric; Ruckly, Stéphane; Picard, Géraldine; Rogemond, Véronique; Magalhaes, Éric; Sharshar, Tarek; Timsit, Jean-François; Honnorat, Jérôme; Sonneville, Romain

    2017-02-15

    Encephalitis caused by anti-N-methyl-d-aspartate receptor (NMDAR) antibodies is the leading cause of immune-mediated encephalitis. There are limited data on intensive care unit (ICU) management of these patients. To identify prognostic factors of good neurologic outcome in patients admitted to an ICU with anti-NMDAR encephalitis. This was an observational multicenter study of all consecutive adult patients diagnosed with anti-NMDAR encephalitis at the French National Reference Centre, admitted to an ICU between 2008 and 2014. The primary outcome was a good neurologic outcome at 6 months after ICU admission, defined by a modified Rankin Scale score of 0-2. Seventy-seven patients were included from 52 ICUs. First-line immunotherapy consisted of steroids (n = 61/74; 82%), intravenous immunoglobulins (n = 71/74; 96%), and plasmapheresis (n = 17/74; 23%). Forty-five (61%) patients received second-line immunotherapy (cyclophosphamide, rituximab, or both). At 6 months, 57% of patients had a good neurologic outcome. Independent factors of good neurologic outcome were early (≤8 d after ICU admission) immunotherapy (odds ratio, 16.16; 95% confidence interval, 3.32-78.64; for combined first-line immunotherapy with steroids and intravenous immunoglobulins vs. late immunotherapy), and a low white blood cell count on the first cerebrospinal examination (odds ratio, 9.83 for 50 cells/mm 3 ; 95% confidence interval, 1.07-90.65). Presence of nonneurologic organ failures at ICU admission and occurrence of status epilepticus during ICU stay were not associated with neurologic outcome. The prognosis of adult patients with anti-NMDAR encephalitis requiring intensive care is good, especially when immunotherapy is initiated early, advocating for prompt diagnosis and early aggressive treatment.

  19. Romanian regulatory requirements on nuclear field specific education needs

    International Nuclear Information System (INIS)

    Biro, L.; Velicu, O.

    2004-01-01

    This work is intended as a general presentation of the educational system and research field, with reference to nuclear sciences, and the legal system, with reference to requirements established by the regulatory body for the professional qualification and periodic training of personnel involved in different activities in the nuclear field. Thus, part 2 and 3 of the work present only public information regarding the education in nuclear sciences and nuclear research in Romania; in part 4 the CNCAN requirements for the personnel training, specific to nuclear activities are slightly detailed; part 5 consists of few words about the public information activities in Romania; and part 6 tries to draw a conclusion. (authors)

  20. 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.

  1. Risk impact of BWR technical specifications requirements during shutdown

    International Nuclear Information System (INIS)

    Staple, B.D.; Kirk, H.K.; Yakle, J.

    1994-10-01

    This report presents an application of probabilistic models and risk based criteria for determining the risk impact of the Limiting Conditions of Operations (LCOs) in the Technical Specifications (TSs) of a boiling water reactor during shutdown. This analysis studied the risk impact of the current requirements of Allowed Outage Times (AOTs) and Surveillance Test Intervals (STIs) in eight Plant Operational States (POSs) which encompass power operations, shutdown, and refueling. This report also discusses insights concerning TS action statements

  2. 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

  3. Modeling of requirement specification for safety critical real time computer system using formal mathematical specifications

    International Nuclear Information System (INIS)

    Sankar, Bindu; Sasidhar Rao, B.; Ilango Sambasivam, S.; Swaminathan, P.

    2002-01-01

    Full text: Real time computer systems are increasingly used for safety critical supervision and control of nuclear reactors. Typical application areas are supervision of reactor core against coolant flow blockage, supervision of clad hot spot, supervision of undesirable power excursion, power control and control logic for fuel handling systems. The most frequent cause of fault in safety critical real time computer system is traced to fuzziness in requirement specification. To ensure the specified safety, it is necessary to model the requirement specification of safety critical real time computer systems using formal mathematical methods. Modeling eliminates the fuzziness in the requirement specification and also helps to prepare the verification and validation schemes. Test data can be easily designed from the model of the requirement specification. Z and B are the popular languages used for modeling the requirement specification. A typical safety critical real time computer system for supervising the reactor core of prototype fast breeder reactor (PFBR) against flow blockage is taken as case study. Modeling techniques and the actual model are explained in detail. The advantages of modeling for ensuring the safety are summarized

  4. 78 FR 22789 - Methyl Jasmonate; Exemption From the Requirement of a Tolerance

    Science.gov (United States)

    2013-04-17

    ... present in most fruits, with especially high concentrations in apples and strawberries. As such, it is... consumed fruits as apples and strawberries (Memorandum from Miachel Rexrode, Ph.D., July 19, 2012). Data... methyl jasmonate because it naturally occurs in apples, strawberries and mangos (Lalel et al., 2003...

  5. Tumor-specific histone signature and DNA methylation in multiple myeloma and leukemia cells

    Czech Academy of Sciences Publication Activity Database

    Foltánková, Veronika; Legartová, Soňa; Kozubek, Stanislav; Bártová, Eva

    2012-01-01

    Roč. 59, č. 4 (2012), s. 450-462 ISSN 0028-2685 R&D Projects: GA ČR(CZ) GAP302/10/1022; GA ČR GBP302/12/G157 Institutional research plan: CEZ:AV0Z50040702 Keywords : ChIP * histones * DNA methylation Subject RIV: BO - Biophysics Impact factor: 1.574, year: 2012

  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. Prediction of disease-related genes based on weighted tissue-specific networks by using DNA methylation.

    Science.gov (United States)

    Li, Min; Zhang, Jiayi; Liu, Qing; Wang, Jianxin; Wu, Fang-Xiang

    2014-01-01

    Predicting disease-related genes is one of the most important tasks in bioinformatics and systems biology. With the advances in high-throughput techniques, a large number of protein-protein interactions are available, which make it possible to identify disease-related genes at the network level. However, network-based identification of disease-related genes is still a challenge as the considerable false-positives are still existed in the current available protein interaction networks (PIN). Considering the fact that the majority of genetic disorders tend to manifest only in a single or a few tissues, we constructed tissue-specific networks (TSN) by integrating PIN and tissue-specific data. We further weighed the constructed tissue-specific network (WTSN) by using DNA methylation as it plays an irreplaceable role in the development of complex diseases. A PageRank-based method was developed to identify disease-related genes from the constructed networks. To validate the effectiveness of the proposed method, we constructed PIN, weighted PIN (WPIN), TSN, WTSN for colon cancer and leukemia, respectively. The experimental results on colon cancer and leukemia show that the combination of tissue-specific data and DNA methylation can help to identify disease-related genes more accurately. Moreover, the PageRank-based method was effective to predict disease-related genes on the case studies of colon cancer and leukemia. Tissue-specific data and DNA methylation are two important factors to the study of human diseases. The same method implemented on the WTSN can achieve better results compared to those being implemented on original PIN, WPIN, or TSN. The PageRank-based method outperforms degree centrality-based method for identifying disease-related genes from WTSN.

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

    Directory of Open Access Journals (Sweden)

    Michael P Trimarchi

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

  9. Accurate CpG and non-CpG cytosine methylation analysis by high-throughput locus-specific pyrosequencing in plants.

    Science.gov (United States)

    How-Kit, Alexandre; Daunay, Antoine; Mazaleyrat, Nicolas; Busato, Florence; Daviaud, Christian; Teyssier, Emeline; Deleuze, Jean-François; Gallusci, Philippe; Tost, Jörg

    2015-07-01

    Pyrosequencing permits accurate quantification of DNA methylation of specific regions where the proportions of the C/T polymorphism induced by sodium bisulfite treatment of DNA reflects the DNA methylation level. The commercially available high-throughput locus-specific pyrosequencing instruments allow for the simultaneous analysis of 96 samples, but restrict the DNA methylation analysis to CpG dinucleotide sites, which can be limiting in many biological systems. In contrast to mammals where DNA methylation occurs nearly exclusively on CpG dinucleotides, plants genomes harbor DNA methylation also in other sequence contexts including CHG and CHH motives, which cannot be evaluated by these pyrosequencing instruments due to software limitations. Here, we present a complete pipeline for accurate CpG and non-CpG cytosine methylation analysis at single base-resolution using high-throughput locus-specific pyrosequencing. The devised approach includes the design and validation of PCR amplification on bisulfite-treated DNA and pyrosequencing assays as well as the quantification of the methylation level at every cytosine from the raw peak intensities of the Pyrograms by two newly developed Visual Basic Applications. Our method presents accurate and reproducible results as exemplified by the cytosine methylation analysis of the promoter regions of two Tomato genes (NOR and CNR) encoding transcription regulators of fruit ripening during different stages of fruit development. Our results confirmed a significant and temporally coordinated loss of DNA methylation on specific cytosines during the early stages of fruit development in both promoters as previously shown by WGBS. The manuscript describes thus the first high-throughput locus-specific DNA methylation analysis in plants using pyrosequencing.

  10. Genetic and non-genetic influences during pregnancy on infant global and site specific DNA methylation: role for folate gene variants and vitamin B12.

    Directory of Open Access Journals (Sweden)

    Jill A McKay

    Full Text Available Inter-individual variation in patterns of DNA methylation at birth can be explained by the influence of environmental, genetic and stochastic factors. This study investigates the genetic and non-genetic determinants of variation in DNA methylation in human infants. Given its central role in provision of methyl groups for DNA methylation, this study focuses on aspects of folate metabolism. Global (LUMA and gene specific (IGF2, ZNT5, IGFBP3 DNA methylation were quantified in 430 infants by Pyrosequencing®. Seven polymorphisms in 6 genes (MTHFR, MTRR, FOLH1, CβS, RFC1, SHMT involved in folate absorption and metabolism were analysed in DNA from both infants and mothers. Red blood cell folate and serum vitamin B(12 concentrations were measured as indices of vitamin status. Relationships between DNA methylation patterns and several covariates viz. sex, gestation length, maternal and infant red cell folate, maternal and infant serum vitamin B(12, maternal age, smoking and genotype were tested. Length of gestation correlated positively with IGF2 methylation (rho = 0.11, p = 0.032 and inversely with ZNT5 methylation (rho = -0.13, p = 0.017. Methylation of the IGFBP3 locus correlated inversely with infant vitamin B(12 concentration (rho = -0.16, p = 0.007, whilst global DNA methylation correlated inversely with maternal vitamin B(12 concentrations (rho = 0.18, p = 0.044. Analysis of common genetic variants in folate pathway genes highlighted several associations including infant MTRR 66G>A genotype with DNA methylation (χ(2 = 8.82, p = 0.003 and maternal MTHFR 677C>T genotype with IGF2 methylation (χ(2 = 2.77, p = 0.006. These data support the hypothesis that both environmental and genetic factors involved in one-carbon metabolism influence DNA methylation in infants. Specifically, the findings highlight the importance of vitamin B(12 status, infant MTRR genotype and maternal MTHFR genotype, all of which may influence the supply of methyl groups for

  11. Incubator Display Software Cost Reduction Toolset Software Requirements Specification

    Science.gov (United States)

    Moran, Susanne; Jeffords, Ralph

    2005-01-01

    The Incubator Display Software Requirements Specification was initially developed by Intrinsyx Technologies Corporation (Intrinsyx) under subcontract to Lockheed Martin, Contract Number NAS2-02090, for the National Aeronautics and Space Administration (NASA) Ames Research Center (ARC) Space Station Biological Research Project (SSBRP). The Incubator Display is a User Payload Application (UPA) used to control an Incubator subrack payload for the SSBRP. The Incubator Display functions on-orbit as part of the subrack payload laptop, on the ground as part of the Communication and Data System (CDS) ground control system, and also as part of the crew training environment.

  12. DNA Methylation Adjusts the Specificity of Memories Depending on the Learning Context and Promotes Relearning in Honeybees.

    Science.gov (United States)

    Biergans, Stephanie D; Claudianos, Charles; Reinhard, Judith; Galizia, C G

    2016-01-01

    The activity of the epigenetic writers DNA methyltransferases (Dnmts) after olfactory reward conditioning is important for both stimulus-specific long-term memory (LTM) formation and extinction. It, however, remains unknown which components of memory formation Dnmts regulate (e.g., associative vs. non-associative) and in what context (e.g., varying training conditions). Here, we address these aspects in order to clarify the role of Dnmt-mediated DNA methylation in memory formation. We used a pharmacological Dnmt inhibitor and classical appetitive conditioning in the honeybee Apis mellifera, a well characterized model for classical conditioning. We quantified the effect of DNA methylation on naïve odor and sugar responses, and on responses following olfactory reward conditioning. We show that (1) Dnmts do not influence naïve odor or sugar responses, (2) Dnmts do not affect the learning of new stimuli, but (3) Dnmts influence odor-coding, i.e., 'correct' (stimulus-specific) LTM formation. Particularly, Dnmts reduce memory specificity when experience is low (one-trial training), and increase memory specificity when experience is high (multiple-trial training), generating an ecologically more useful response to learning. (4) In reversal learning conditions, Dnmts are involved in regulating both excitatory (re-acquisition) and inhibitory (forgetting) processes.

  13. Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

    Science.gov (United States)

    Zeng, Jia; Yi, Soojin V

    2014-10-16

    Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for

  14. Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

    NARCIS (Netherlands)

    Gervin, K. (Kristina); Page, C.M. (Christian Magnus); H.C.D. Aass (Hans Christian Dalsbotten); M.A.E. Jansen (Michelle); Fjeldstad, H.E. (Heidi Elisabeth); B.K. Andreassen (Bettina Kulle); L. Duijts (Liesbeth); J.B.J. van Meurs (Joyce); M.C. van Zelm (Menno); V.W.V. Jaddoe (Vincent); Nordeng, H. (Hedvig); Knudsen, G.P. (Gunn Peggy); P. Magnus (Per); W. Nystad (Wenche); Staff, A.C. (Anne Cathrine); J.F. Felix (Janine); R. Lyle (Robert)

    2016-01-01

    textabstractEpigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2017-04-06

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

  17. Assignment of methyl NMR resonances of a 52 kDa protein with residue-specific 4D correlation maps

    International Nuclear Information System (INIS)

    Mishra, Subrata H.; Frueh, Dominique P.

    2015-01-01

    Methyl groups have become key probes for structural and functional studies by nuclear magnetic resonance. However, their NMR signals cluster in a small spectral region and assigning their resonances can be a tedious process. Here, we present a method that facilitates assignment of methyl resonances from assigned amide groups. Calculating the covariance between sensitive methyl and amide 3D spectra, each providing correlations to C α and C β separately, produces 4D correlation maps directly correlating methyl groups to amide groups. Optimal correlation maps are obtained by extracting residue-specific regions, applying derivative to the dimensions subject to covariance, and multiplying 4D maps stemming from different 3D spectra. The latter procedure rescues weak signals that may be missed in traditional assignment procedures. Using these covariance correlation maps, nearly all assigned isoleucine, leucine, and valine amide resonances of a 52 kDa nonribosomal peptide synthetase cyclization domain were paired with their corresponding methyl groups

  18. Chronic stress induces sex-specific alterations in methylation and expression of corticotropin-releasing factor gene in the rat.

    Directory of Open Access Journals (Sweden)

    Linda Sterrenburg

    Full Text Available BACKGROUND: Although the higher prevalence of depression in women than in men is well known, the neuronal basis of this sex difference is largely elusive. METHODS: Male and female rats were exposed to chronic variable mild stress (CVMS after which immediate early gene products, corticotropin-releasing factor (CRF mRNA and peptide, various epigenetic-associated enzymes and DNA methylation of the Crf gene were determined in the hypothalamic paraventricular nucleus (PVN, oval (BSTov and fusiform (BSTfu parts of the bed nucleus of the stria terminalis, and central amygdala (CeA. RESULTS: CVMS induced site-specific changes in Crf gene methylation in all brain centers studied in female rats and in the male BST and CeA, whereas the histone acetyltransferase, CREB-binding protein was increased in the female BST and the histone-deacetylase-5 decreased in the male CeA. These changes were accompanied by an increased amount of c-Fos in the PVN, BSTfu and CeA in males, and of FosB in the PVN of both sexes and in the male BSTov and BSTfu. In the PVN, CVMS increased CRF mRNA in males and CRF peptide decreased in females. CONCLUSIONS: The data confirm our hypothesis that chronic stress affects gene expression and CRF transcriptional, translational and secretory activities in the PVN, BSTov, BSTfu and CeA, in a brain center-specific and sex-specific manner. Brain region-specific and sex-specific changes in epigenetic activity and neuronal activation may play, too, an important role in the sex specificity of the stress response and the susceptibility to depression.

  19. Alloy 800 specifications in compliance with component requirements

    International Nuclear Information System (INIS)

    Diehl, H.; Bodmann, E.

    1990-01-01

    In view of the importance of the material Alloy 800 in high-temperature reactor plants (HTR), a material data bank was established which is used for statistical evaluation of mechanical and physical material behaviour. Based on investigations on the interconnection between the mechanical properties at high temperatures and the metallurgical parameters, different types of Alloy 800 were specified in compliance with the component requirements. In addition, aspects of corrosion and toughness behaviour were taken into consideration. The specifications and strength characteristics for the different variants of Alloy 800 were incorporated into draft DIN standards after discussion and approval in expert committees. Further important characteristics of the mechanical and physical material behaviour were summarized in HTR material data sheets so as to furnish an improved basis for the design and stress analyses of Alloy 800 components. (orig.)

  20. [Technological innovations in radiation oncology require specific quality controls].

    Science.gov (United States)

    Lenaerts, E; Mathot, M

    2014-01-01

    During the last decade, the field of radiotherapy has benefited from major technological innovations and continuously improving treatment efficacy, comfort and safety of patients. This mainly concerns the imaging techniques that allow 4D CT scan recording the respiratory phases, on-board imaging on linear accelerators that ensure perfect positioning of the patient for treatment and irradiation techniques that reduce very significantly the duration of treatment sessions without compromising quality of the treatment plan, including IMRT (Intensity Modulated Radiation Therapy) and VMAT (Volumetric Modulated Arc therapy). In this context of rapid technological change, it is the responsibility of medical physicists to regularly and precisely monitor the perfect functioning of new techniques to ensure patient safety. This requires the use of specific quality control equipment best suited to these new techniques. We will briefly describe the measurement system Delta4 used to control individualized treatment plan for each patient treated with VMAT technology.

  1. Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure

    Energy Technology Data Exchange (ETDEWEB)

    Ozden, Sibel, E-mail: stopuz@istanbul.edu.tr [Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul (Turkey); Turgut Kara, Neslihan [Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul (Turkey); Sezerman, Osman Ugur [Department of Biostatistics and Medical Informatics, Acibadem University, Istanbul (Turkey); Durasi, İlknur Melis [Biological Sciences and Bioengineering, Faculty of Engineering and Natural Sciences, Sabancı University, Istanbul (Turkey); Chen, Tao [Department of Toxicology, School of Public Health, Soochow University, Suzhou (China); Demirel, Goksun; Alpertunga, Buket [Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Istanbul University, Istanbul (Turkey); Chipman, J. Kevin [School of Biosciences, The University of Birmingham, Birmingham (United Kingdom); Mally, Angela [Department of Toxicology, University of Würzburg, Würzburg (Germany)

    2015-12-01

    Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC–MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity. - Highlights: • Studied non-genotoxic carcinogens caused no change on global DNA hypomethylation. • d-Limonene, DCB and chloroform did not show any genome-wide methylation changes. • Some genes were differentially methylated in response to MPY, TCDD and OTA. • Protein kinase activity

  2. Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure

    International Nuclear Information System (INIS)

    Ozden, Sibel; Turgut Kara, Neslihan; Sezerman, Osman Ugur; Durasi, İlknur Melis; Chen, Tao; Demirel, Goksun; Alpertunga, Buket; Chipman, J. Kevin; Mally, Angela

    2015-01-01

    Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC–MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity. - Highlights: • Studied non-genotoxic carcinogens caused no change on global DNA hypomethylation. • d-Limonene, DCB and chloroform did not show any genome-wide methylation changes. • Some genes were differentially methylated in response to MPY, TCDD and OTA. • Protein kinase activity

  3. Identification of a polyketide synthase required for alternariol (AOH and alternariol-9-methyl ether (AME formation in Alternaria alternata.

    Directory of Open Access Journals (Sweden)

    Debjani Saha

    Full Text Available Alternaria alternata produces more than 60 secondary metabolites, among which alternariol (AOH and alternariol-9-methyl ether (AME are important mycotoxins. Whereas the toxicology of these two polyketide-based compounds has been studied, nothing is known about the genetics of their biosynthesis. One of the postulated core enzymes in the biosynthesis of AOH and AME is polyketide synthase (PKS. In a draft genome sequence of A. alternata we identified 10 putative PKS-encoding genes. The timing of the expression of two PKS genes, pksJ and pksH, correlated with the production of AOH and AME. The PksJ and PksH proteins are predicted to be 2222 and 2821 amino acids in length, respectively. They are both iterative type I reducing polyketide synthases. PksJ harbors a peroxisomal targeting sequence at the C-terminus, suggesting that the biosynthesis occurs at least partly in these organelles. In the vicinity of pksJ we found a transcriptional regulator, altR, involved in pksJ induction and a putative methyl transferase, possibly responsible for AME formation. Downregulation of pksJ and altR caused a large decrease of alternariol formation, suggesting that PksJ is the polyketide synthase required for the postulated Claisen condensations during the biosynthesis. No other enzymes appeared to be required. PksH downregulation affected pksJ expression and thus caused an indirect effect on AOH production.

  4. Partial demethylation of oligogalacturonides by pectin methyl esterase 1 is required for eliciting defence responses in wild strawberry (Fragaria vesca).

    Science.gov (United States)

    Osorio, Sonia; Castillejo, Cristina; Quesada, Miguel A; Medina-Escobar, Nieves; Brownsey, Geoff J; Suau, Rafael; Heredia, Antonio; Botella, Miguel A; Valpuesta, Victoriano

    2008-04-01

    In addition to the role of the cell wall as a physical barrier against pathogens, some of its constituents, such as pectin-derived oligogalacturonides (OGA), are essential components for elicitation of defence responses. To investigate how modifications of pectin alter defence responses, we expressed the fruit-specific Fragaria x ananassa pectin methyl esterase FaPE1 in the wild strawberry Fragaria vesca. Pectin from transgenic ripe fruits differed from the wild-type with regard to the degree and pattern of methyl esterification, as well as the average size of pectin polymers. Purified oligogalacturonides from the transgenic fruits showed a reduced degree of esterification compared to oligogalacturonides from wild-type fruits. This reduced esterification is necessary to elicit defence responses in strawberry. The transgenic F. vesca lines had constitutively activated pathogen defence responses, resulting in higher resistance to the necrotropic fungus Botrytis cinerea. Further studies in F. vesca and Nicotiana benthamiana leaves showed that the elicitation capacity of the oligogalacturonides is more specific than previously envisaged.

  5. Tissue-Specific Methylation of Long Interspersed Nucleotide Element-1 of Homo Sapiens (L1Hs) During Human Embryogenesis and Roles in Neural Tube Defects.

    Science.gov (United States)

    Wang, L; Chang, S; Guan, J; Shangguan, S; Lu, X; Wang, Z; Wu, L; Zou, J; Zhao, H; Bao, Y; Qiu, Z; Niu, B; Zhang, T

    2015-01-01

    Epigenetic regulation of long interspersed nucleotide element-1 (LINE-1) retrotransposition events plays crucial roles during early development. Previously we showed that LINE-1 hypomethylation in neuronal tissues is associated with pathogenesis of neural tube defect (NTD). Herein, we further evaluated LINE-1 Homo sapiens (L1Hs) methylation in tissues derived from three germ layers of stillborn NTD fetuses, to define patterns of tissue specific methylation and site-specific hypomethylation at CpG sites within an L1Hs promoter region. Stable, tissue-specific L1Hs methylation patterns throughout three germ layer lineages of the fetus, placenta, and maternal peripheral blood were observed. Samples from maternal peripheral blood exhibited the highest level of L1Hs methylation (64.95%) and that from placenta showed the lowest (26.82%). Between samples from NTDs and controls, decrease in L1Hs methylation was only significant in NTD-affected brain tissue at 7.35%, especially in females (8.98%). L1Hs hypomethylation in NTDs was also associated with a significant increase in expression level of an L1Hs-encoded transcript in females (r = -0.846, p = 0.004). This could be due to genomic DNA instability and alternation in chromatins accessibility resulted from abnormal L1Hs hypomethylation, as showed in this study with HCT-15 cells treated with methylation inhibitor 5-Aza.

  6. High sensitivity and specificity in proposed clinical diagnostic criteria for anti-N-methyl-D-aspartate receptor encephalitis.

    Science.gov (United States)

    Ho, Alvin C C; Mohammad, Shekeeb S; Pillai, Sekhar C; Tantsis, Esther; Jones, Hannah; Ho, Reena; Lim, Ming; Hacohen, Yael; Vincent, Angela; Dale, Russell C

    2017-12-01

    To determine the validity of the proposed clinical diagnostic criteria for anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis in paediatric patients. The diagnostic criteria for anti-NMDAR encephalitis proposed by Graus et al. (2016) use clinical features and conventional investigations to facilitate early immunotherapy before antibody status is available. The criteria are satisfied if patients develop four out of six symptom groups within 3 months, together with at least one abnormal investigation (electroencephalography/cerebrospinal fluid) and reasonable exclusion of other disorders. We evaluated the validity of the criteria using a retrospective cohort of paediatric patients with encephalitis. Twenty-nine patients with anti-NMDAR encephalitis and 74 comparison children with encephalitis were included. As expected, the percentage of patients with anti-NMDAR encephalitis who fulfilled the clinical criteria increased over time. During the hospital inpatient admission, most patients (26/29, 90%) with anti-NMDAR encephalitis fulfilled the criteria, significantly more than the comparison group (3/74, 4%) (panti-NMDAR encephalitis was 2 weeks from first symptom onset (range 1-6). The sensitivity of the criteria was 90% (95% confidence interval 73-98) and the specificity was 96% (95% confidence interval 89-99). The proposed diagnostic criteria for anti-NMDAR encephalitis have good sensitivity and specificity. Incomplete criteria do not exclude the diagnosis. The proposed clinical diagnostic criteria for anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis by Graus et al. (2016) have high sensitivity and specificity in paediatric patients. The median time of fulfilling the criteria in patients with anti-NMDAR was 2 weeks from first symptom onset. © 2017 Mac Keith Press.

  7. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

    Directory of Open Access Journals (Sweden)

    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  8. Surveillance Analysis Computer System (SACS) software requirements specification (SRS)

    International Nuclear Information System (INIS)

    Glasscock, J.A.; Flanagan, M.J.

    1995-09-01

    This document is the primary document establishing requirements for the Surveillance Analysis Computer System (SACS) Database, an Impact Level 3Q system. The purpose is to provide the customer and the performing organization with the requirements for the SACS Project

  9. Association of methionine requirement with methyl mercury resistant mutants of yeast

    Energy Technology Data Exchange (ETDEWEB)

    Singh, A.; Sherman, F.

    1974-01-25

    It has been known for several years that strains resistant to mercury can be obtained in several bacterial species. Soon after the correlation between resistance to antibiotics and to mercury was recognized, it was established that genetic elements conferring resistance to antibiotics, mercury and other heavy metals in Escherichia coli and Samonella typhimurium and Staphylococcus aureus reside on extrachromosomal resistance transfer factors or plasmids. Among fungi, mercury resistant strains of Botrytis cinerea, Penicillium notatum, Sclerotinia fructicola, Stemphylium sarcinaeforme, and Saccharomyces cerevisiae have been reported. In most cases, this was accomplished by training the normal strains for growth on media supplemented with successively increasing concentrations of mercury compounds, and in some cases the resistance was lost when subcultured on mercury-free media. It is noteworthy that in none of the mercury-adapted strains of fungi has the genetic basis of resistance been determined. In this report we describe a method of isolation and characterization of methyl mercury resistant mutants of S. cerevisiae. This study was undertaken with the view that the examination of physiological changes associated with genetically defined resistant mutants will be useful in studying the mechanisms of cellular detoxification of organic mercurials.

  10. Understanding the technical content of requirements in specification document

    DEFF Research Database (Denmark)

    Sudin, Mohd Nizam Bin; Ahmed-Kristensen, Saeema

    2011-01-01

    development process, is essential to be devised in advance. To achieve this aim, understanding how to formulate a good requirement is necessary and it is only possible if design engineers understand the technical content of a requirement. In aiming to understand the technical content of a requirement...

  11. A functional SNP associated with atopic dermatitis controls cell type-specific methylation of the VSTM1 gene locus

    Directory of Open Access Journals (Sweden)

    Dilip Kumar

    2017-02-01

    Full Text Available Abstract Background Expression quantitative trait loci (eQTL databases represent a valuable resource to link disease-associated SNPs to specific candidate genes whose gene expression is significantly modulated by the SNP under investigation. We previously identified signal inhibitory receptor on leukocytes-1 (SIRL-1 as a powerful regulator of human innate immune cell function. While it is constitutively high expressed on neutrophils, on monocytes the SIRL-1 surface expression varies strongly between individuals. The underlying mechanism of regulation, its genetic control as well as potential clinical implications had not been explored yet. Methods Whole blood eQTL data of a Chinese cohort was used to identify SNPs regulating the expression of VSTM1, the gene encoding SIRL-1. The genotype effect was validated by flow cytometry (cell surface expression, correlated with electrophoretic mobility shift assay (EMSA, chromatin immunoprecipitation (ChIP and bisulfite sequencing (C-methylation and its functional impact studied the inhibition of reactive oxygen species (ROS. Results We found a significant association of a single CpG-SNP, rs612529T/C, located in the promoter of VSTM1. Through flow cytometry analysis we confirmed that primarily in the monocytes the protein level of SIRL-1 is strongly associated with genotype of this SNP. In monocytes, the T allele of this SNP facilitates binding of the transcription factors YY1 and PU.1, of which the latter has been recently shown to act as docking site for modifiers of DNA methylation. In line with this notion rs612529T associates with a complete demethylation of the VSTM1 promoter correlating with the allele-specific upregulation of SIRL-1 expression. In monocytes, this upregulation strongly impacts the IgA-induced production of ROS by these cells. Through targeted association analysis we found a significant Meta P value of 1.14 × 10–6 for rs612529 for association to atopic dermatitis (AD

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  13. 49 CFR 178.35 - General requirements for specification cylinders.

    Science.gov (United States)

    2010-10-01

    ... of part 107 of this chapter; or (2) For DOT Specifications 3B, 3BN, 3E, 4B, 4BA, 4D (water capacity...) The word “spun” or “plug” must be placed near the DOT specification marking when an end closure in the... certifies that the processes of manufacture and heat treatment of cylinders were observed and found...

  14. Disposal of Radioactive Waste. Specific Safety Requirements (Spanish Edition)

    International Nuclear Information System (INIS)

    2012-01-01

    This Safety Requirements publication applies to the disposal of radioactive waste of all types by means of emplacement in designed disposal facilities, subject to the necessary limitations and controls being placed on the disposal of the waste and on the development, operation and closure of facilities. The classification of radioactive waste is discussed. This Safety Requirements publication establishes requirements to provide assurance of the radiation safety of the disposal of radioactive waste, in the operation of a disposal facility and especially after its closure. The fundamental safety objective is to protect people and the environment from harmful effects of ionizing radiation. This is achieved by setting requirements on the site selection and evaluation and design of a disposal facility, and on its construction, operation and closure, including organizational and regulatory requirements.

  15. Safety of Research Reactors. Specific Safety Requirements (French Edition)

    International Nuclear Information System (INIS)

    2017-01-01

    This Safety Requirements publication establishes requirements for all main areas of safety for research reactors, with particular emphasis on requirements for design and operation. It explains the safety objectives and concepts that form the basis for safety and safety assessment for all stages in the lifetime of a research reactor. Technical and administrative requirements for the safety of new research reactors are established in accordance with these objectives and concepts, and they are to be applied to the extent practicable for existing research reactors. The safety requirements established in this publication for the management of safety and regulatory supervision apply to site evaluation, design, manufacturing, construction, commissioning, operation (including utilization and modification), and planning for decommissioning of research reactors (including critical assemblies and subcritical assemblies). The publication is intended for use by regulatory bodies and other organizations with responsibilities in these areas and in safety analysis, verification and review, and the provision of technical support.

  16. Input data required for specific performance assessment codes

    International Nuclear Information System (INIS)

    Seitz, R.R.; Garcia, R.S.; Starmer, R.J.; Dicke, C.A.; Leonard, P.R.; Maheras, S.J.; Rood, A.S.; Smith, R.W.

    1992-02-01

    The Department of Energy's National Low-Level Waste Management Program at the Idaho National Engineering Laboratory generated this report on input data requirements for computer codes to assist States and compacts in their performance assessments. This report gives generators, developers, operators, and users some guidelines on what input data is required to satisfy 22 common performance assessment codes. Each of the codes is summarized and a matrix table is provided to allow comparison of the various input required by the codes. This report does not determine or recommend which codes are preferable

  17. Quantifying Functional Reuse from Object Oriented Requirements Specifications

    NARCIS (Netherlands)

    Condori-Fernandez, Nelly; Condori-Fernández, N.; Pastor, O; Daneva, Maia; Abran, A.; Castro, J.; Quer, C.; Carvallo, J. B.; Fernandes da Silva, L.

    2008-01-01

    Software reuse is essential in improving efficiency and productivity in the software development process. This paper analyses reuse within requirements engineering phase by taking and adapting a standard functional size measurement method, COSMIC FFP. Our proposal attempts to quantify reusability

  18. Specification for Visual Requirements of Work-Centered Software Systems

    National Research Council Canada - National Science Library

    Knapp, James R; Chung, Soon M; Schmidt, Vincent A

    2006-01-01

    .... In order to ensure the coherent development and delivery of work-centered software products, WCSS visual requirements must be specified to capture the cognitive aspects of the user interface design...

  19. Strengthening requirement specification in sustainable procurement - an investigation of challenges

    DEFF Research Database (Denmark)

    Eriksen, Marlene Stenberg Hagen; Bjarløv, Søren Peter; Rode, Carsten

    2017-01-01

    To reap the benefits of sustainability in the construction sector, it is crucial that the stakeholders involved can implement it in practice. Investigations have shown that choices made in the early phases of the building process are of very great importance for the outcome and the initiatives...... and decisions taken by the building owner are crucial.This paper presents research on Danish building practitioners' ability to make requirements for sustainability in procurement. On the basis of an action research strategy, we asked practitioners to help identify the challenges involved in requiring...

  20. 30 CFR 23.7 - Specific requirements for approval.

    Science.gov (United States)

    2010-07-01

    ... adequate instructions for the installation and connection of telephones and signal devices in order that the safety of these devices and other circuits shall not be diminished by improper installation. MSHA reserves the right to require the attachment of wiring diagrams to the cases of telephones and signal...

  1. Risk-Based Confidentiality Requirements Specification for Outsourced IT Systems

    NARCIS (Netherlands)

    Morali, A.; Wieringa, Roelf J.

    Today, companies are required to be in control of their IT assets, and to provide proof of this in the form of independent IT audit reports. However, many companies have outsourced various parts of their IT systems to other companies, which potentially threatens the control they have of their IT

  2. Practical Relevance of Experiments in Comprehensibility of Requirements Specifications

    NARCIS (Netherlands)

    Condori-Fernandez, Nelly; Daneva, Maia; Sikkel, Nicolaas; Herrmann, Andrea

    2011-01-01

    Recently, the Requirements Engineering (RE) community has become increasingly aware of the importance of carrying out industry-relevant research. Researchers and practitioners should be able to evaluate the relevance of their empirical research to increase the likely adoption of RE methods in

  3. System requirements specification for waste information and control system

    International Nuclear Information System (INIS)

    Harris, R.R.

    1994-09-01

    This document defines the requirements for the Waste Information and Control System (WICS). The document defines the functions, constraints, and objectives that pertain to WICS. This shall serve as the baseline document to ensure the needs of the Hazardous Material Control group (HMC) at 222-S Laboratory are met with regard to assurance of accuracy and quality of data taken with WICS

  4. 40 CFR 35.3135 - Specific capitalization grant agreement requirements.

    Science.gov (United States)

    2010-07-01

    ... received a capitalization grant and, if the deposit was expended, it was expended in accordance with title... after it receives the payment, the RA may withhold future quarterly grant payments, and require... procedures regarding the commitment or expenditure of revenues. (h) State accounting and auditing procedures...

  5. Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

    Science.gov (United States)

    Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

    2009-06-01

    It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

  6. Software Requirement Specifications For ASocial Media Threat Assessment Tool

    Science.gov (United States)

    2017-12-01

    media means a much higher volume of threats than before, which taxes law enforcement’s ability to investigate each one. Complicating the investigation...Obtaining the identity of a Twitter account holder requires court paperwork based on probable cause or exigent circumstances. Second, social media...instant connectivity of social media means a much higher number of threats than before, which taxes law enforcement’s ability to investigate each one

  7. Stereoselectivity and substrate specificity in the kinetic resolution of methyl-substituted 1-oxaspiro[2.5]octanes by Rhodotorula glutinis epoxide hydrolase

    NARCIS (Netherlands)

    Weijers, C.A.G.M.; Meeuwse, P.; Herpers, R.L.J.M.; Franssen, M.C.R.; Sudhölter, E.J.R.

    2005-01-01

    [GRAPHICS] The kinetic resolution of a range of methyl-substituted 1-oxaspiro[2.5]octanes by yeast epoxide hydrolase (YEH) from Rhodotorula glutinis has been investigated. The structural determinants of substrate specificity and stereoselectivity of YEH toward these substrates appeared to be the

  8. The fetal programming effect of prenatal smoking on Igf1r and Igf1 methylation is organ- and sex-specific.

    Science.gov (United States)

    Meyer, Karolin F; Verkaik-Schakel, Rikst Nynke; Timens, Wim; Kobzik, Lester; Plösch, Torsten; Hylkema, Machteld N

    2017-01-01

    The impact of prenatal smoke exposure (PSE) on DNA methylation has been demonstrated in blood samples from children of smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. As the identified differentially methylated genes can be associated with developmental processes, and insulin-like growth factors (IGFs) play a critical role in prenatal tissue growth, we hypothesized that PSE induces fetal programming of Igf1r and Igf1. Using a mouse model of smoking during pregnancy, we show that PSE alters promoter methylation of Igf1r and Igf1 and deregulates their gene expression in lung and liver of fetal (E17.5) and neonatal (D3) mouse offspring. By further comparing female versus male, lung versus liver, or fetal versus neonatal time point, our results demonstrate that CpG site-specific aberrant methylation patterns sex-dependently vary per organ and time point. Moreover, PSE reduces gene expression of Igf1r and Igf1, dependent on organ, sex, and offspring's age. Our results indicate that PSE may be a source of organ-specific rather than general systemic fetal programming. This is exemplified here by gene promoter methylation and mRNA levels of Igf1r and Igf1, together with a sex- and organ-specific naturally established correlation of both parameters that is affected by prenatal smoke exposure. Moreover, the comparison of fetuses with neonates suggests a CpG site-dependent reversibility/persistence of PSE-induced differential methylation patterns.

  9. Software requirements specification document for the AREST code development

    International Nuclear Information System (INIS)

    Engel, D.W.; McGrail, B.P.; Whitney, P.D.; Gray, W.J.; Williford, R.E.; White, M.D.; Eslinger, P.W.; Altenhofen, M.K.

    1993-11-01

    The Analysis of the Repository Source Term (AREST) computer code was selected in 1992 by the U.S. Department of Energy. The AREST code will be used to analyze the performance of an underground high level nuclear waste repository. The AREST code is being modified by the Pacific Northwest Laboratory (PNL) in order to evaluate the engineered barrier and waste package designs, model regulatory compliance, analyze sensitivities, and support total systems performance assessment modeling. The current version of the AREST code was developed to be a very useful tool for analyzing model uncertainties and sensitivities to input parameters. The code has also been used successfully in supplying source-terms that were used in a total systems performance assessment. The current version, however, has been found to be inadequate for the comparison and selection of a design for the waste package. This is due to the assumptions and simplifications made in the selection of the process and system models. Thus, the new version of the AREST code will be designed to focus on the details of the individual processes and implementation of more realistic models. This document describes the requirements of the new models that will be implemented. Included in this document is a section describing the near-field environmental conditions for this waste package modeling, description of the new process models that will be implemented, and a description of the computer requirements for the new version of the AREST code

  10. Circuit- and Diagnosis-Specific DNA Methylation Changes at γ-Aminobutyric Acid–Related Genes in Postmortem Human Hippocampus in Schizophrenia and Bipolar Disorder

    Science.gov (United States)

    Ruzicka, W. Brad; Subburaju, Sivan; Benes, Francine M.

    2017-01-01

    IMPORTANCE Dysfunction related to γ-aminobutyric acid (GABA)–ergic neurotransmission in the pathophysiology of major psychosis has been well established by the work of multiple groups across several decades, including the widely replicated downregulation of GAD1. Prior gene expression and network analyses within the human hippocampus implicate a broader network of genes, termed the GAD1 regulatory network, in regulation of GAD1 expression. Several genes within this GAD1 regulatory network show diagnosis- and sector-specific expression changes within the circuitry of the hippocampus, influencing abnormal GAD1 expression in schizophrenia and bipolar disorder. OBJECTIVE To investigate the hypothesis that aberrant DNA methylation contributes to circuit- and diagnosis-specific abnormal expression of GAD1 regulatory network genes in psychotic illness. DESIGN, SETTING, AND PARTICIPANTS This epigenetic association study targeting GAD1 regulatory network genes was conducted between July 1, 2012, and June 30, 2014. Postmortem human hippocampus tissue samples were obtained from 8patients with schizophrenia, 8 patients with bipolar disorder, and 8 healthy control participants matched for age, sex, postmortem interval, and other potential confounds from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont,Massachusetts. We extracted DNA from laser-microdissected stratum oriens tissue of cornu ammonis 2/3 (CA2/3) and CA1 postmortem human hippocampus, bisulfite modified it, and assessed it with the Infinium HumanMethylation450 BeadChip (Illumina, Inc). The subset of CpG loci associated with GAD1 regulatory network genes was analyzed in R version 3.1.0 software (R Foundation) using the minfi package. Findings were validated using bisulfite pyrosequencing. MAIN OUTCOMES AND MEASURES Methylation levels at 1308 GAD1 regulatory network–associated CpG loci were assessed both as individual sites to identify differentially methylated positions and by sharing

  11. Circuit- and Diagnosis-Specific DNA Methylation Changes at γ-Aminobutyric Acid-Related Genes in Postmortem Human Hippocampus in Schizophrenia and Bipolar Disorder.

    Science.gov (United States)

    Ruzicka, W Brad; Subburaju, Sivan; Benes, Francine M

    2015-06-01

    Dysfunction related to γ-aminobutyric acid (GABA)-ergic neurotransmission in the pathophysiology of major psychosis has been well established by the work of multiple groups across several decades, including the widely replicated downregulation of GAD1. Prior gene expression and network analyses within the human hippocampus implicate a broader network of genes, termed the GAD1 regulatory network, in regulation of GAD1 expression. Several genes within this GAD1 regulatory network show diagnosis- and sector-specific expression changes within the circuitry of the hippocampus, influencing abnormal GAD1 expression in schizophrenia and bipolar disorder. To investigate the hypothesis that aberrant DNA methylation contributes to circuit- and diagnosis-specific abnormal expression of GAD1 regulatory network genes in psychotic illness. This epigenetic association study targeting GAD1 regulatory network genes was conducted between July 1, 2012, and June 30, 2014. Postmortem human hippocampus tissue samples were obtained from 8 patients with schizophrenia, 8 patients with bipolar disorder, and 8 healthy control participants matched for age, sex, postmortem interval, and other potential confounds from the Harvard Brain Tissue Resource Center, McLean Hospital, Belmont, Massachusetts. We extracted DNA from laser-microdissected stratum oriens tissue of cornu ammonis 2/3 (CA2/3) and CA1 postmortem human hippocampus, bisulfite modified it, and assessed it with the Infinium HumanMethylation450 BeadChip (Illumina, Inc). The subset of CpG loci associated with GAD1 regulatory network genes was analyzed in R version 3.1.0 software (R Foundation) using the minfi package. Findings were validated using bisulfite pyrosequencing. Methylation levels at 1308 GAD1 regulatory network-associated CpG loci were assessed both as individual sites to identify differentially methylated positions and by sharing information among colocalized probes to identify differentially methylated regions. A total of

  12. Cultivar-Specific Changes in Primary and Secondary Metabolites in Pak Choi (Brassica Rapa, Chinensis Group by Methyl Jasmonate

    Directory of Open Access Journals (Sweden)

    Moo Jung Kim

    2017-05-01

    Full Text Available Glucosinolates, their hydrolysis products and primary metabolites were analyzed in five pak choi cultivars to determine the effect of methyl jasmonate (MeJA on metabolite flux from primary metabolites to glucosinolates and their hydrolysis products. Among detected glucosinolates (total 14 glucosinolates; 9 aliphatic, 4 indole and 1 aromatic glucosinolates, indole glucosinolate concentrations (153–229% and their hydrolysis products increased with MeJA treatment. Changes in the total isothiocyanates by MeJA were associated with epithiospecifier protein activity estimated as nitrile formation. Goitrin, a goitrogenic compound, significantly decreased by MeJA treatment in all cultivars. Changes in glucosinolates, especially aliphatic, significantly differed among cultivars. Primary metabolites including amino acids, organic acids and sugars also changed with MeJA treatment in a cultivar-specific manner. A decreased sugar level suggests that they might be a carbon source for secondary metabolite biosynthesis in MeJA-treated pak choi. The result of the present study suggests that MeJA can be an effective agent to elevate indole glucosinolates and their hydrolysis products and to reduce a goitrogenic compound in pak choi. The total glucosinolate concentration was the highest in “Chinese cabbage” in the control group (32.5 µmol/g DW, but indole glucosinolates increased the greatest in “Asian” when treated with MeJA.

  13. Are patient specific meshes required for EIT head imaging?

    Science.gov (United States)

    Jehl, Markus; Aristovich, Kirill; Faulkner, Mayo; Holder, David

    2016-06-01

    Head imaging with electrical impedance tomography (EIT) is usually done with time-differential measurements, to reduce time-invariant modelling errors. Previous research suggested that more accurate head models improved image quality, but no thorough analysis has been done on the required accuracy. We propose a novel pipeline for creation of precise head meshes from magnetic resonance imaging and computed tomography scans, which was applied to four different heads. Voltages were simulated on all four heads for perturbations of different magnitude, haemorrhage and ischaemia, in five different positions and for three levels of instrumentation noise. Statistical analysis showed that reconstructions on the correct mesh were on average 25% better than on the other meshes. However, the stroke detection rates were not improved. We conclude that a generic head mesh is sufficient for monitoring patients for secondary strokes following head trauma.

  14. Specification of advanced safety modeling requirements (Rev. 0)

    International Nuclear Information System (INIS)

    Fanning, T. H.; Tautges, T. J.

    2008-01-01

    The U.S. Department of Energy's Global Nuclear Energy Partnership has lead to renewed interest in liquid-metal-cooled fast reactors for the purpose of closing the nuclear fuel cycle and making more efficient use of future repository capacity. However, the U.S. has not designed or constructed a fast reactor in nearly 30 years. Accurate, high-fidelity, whole-plant dynamics safety simulations will play a crucial role by providing confidence that component and system designs will satisfy established design limits and safety margins under a wide variety of operational, design basis, and beyond design basis transient conditions. Current modeling capabilities for fast reactor safety analyses have resulted from several hundred person-years of code development effort supported by experimental validation. The broad spectrum of mechanistic and phenomenological models that have been developed represent an enormous amount of institutional knowledge that needs to be maintained. Complicating this, the existing code architectures for safety modeling evolved from programming practices of the 1970s. This has lead to monolithic applications with interdependent data models which require significant knowledge of the complexities of the entire code in order for each component to be maintained. In order to develop an advanced fast reactor safety modeling capability, the limitations of the existing code architecture must be overcome while preserving the capabilities that already exist. To accomplish this, a set of advanced safety modeling requirements is defined, based on modern programming practices, that focuses on modular development within a flexible coupling framework. An approach for integrating the existing capabilities of the SAS4A/SASSYS-1 fast reactor safety analysis code into the SHARP framework is provided in order to preserve existing capabilities while providing a smooth transition to advanced modeling capabilities. In doing this, the advanced fast reactor safety models will

  15. 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...

  16. Synthesis of high specific activity tritium labelled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)benzene acetamide, a specific irreversible ligand for kappa opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Costa, B.R. de; Thurkauf, A.; Rothman, R.R. (National Inst. of Mental Health, Bethesda, MD (USA)); Jacobson, A.E.; Rice, K.C. (National Inst. of Digestive Diabetes, and Kidney Diseases, Bethesda, MD (USA))

    1990-11-01

    Optically pure tritium labeled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl )benzeneacetamide, an affinity ligand specific for the kappa opioid receptor was synthesized from optically pure 1S,2S-(-)-trans-2-amino-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide via the sequence of dibromination (57%) followed by catalytic tritiation of the dibromide. The resulting tritium labelled aniline (14% yield, specific activity 31.2 Ci/mmol) was transformed to the title compound in 13.3% yield and 99+% radiochemical purity by treatment with thiophosgene. (author).

  17. Specification of advanced safety modeling requirements (Rev. 0).

    Energy Technology Data Exchange (ETDEWEB)

    Fanning, T. H.; Tautges, T. J.

    2008-06-30

    The U.S. Department of Energy's Global Nuclear Energy Partnership has lead to renewed interest in liquid-metal-cooled fast reactors for the purpose of closing the nuclear fuel cycle and making more efficient use of future repository capacity. However, the U.S. has not designed or constructed a fast reactor in nearly 30 years. Accurate, high-fidelity, whole-plant dynamics safety simulations will play a crucial role by providing confidence that component and system designs will satisfy established design limits and safety margins under a wide variety of operational, design basis, and beyond design basis transient conditions. Current modeling capabilities for fast reactor safety analyses have resulted from several hundred person-years of code development effort supported by experimental validation. The broad spectrum of mechanistic and phenomenological models that have been developed represent an enormous amount of institutional knowledge that needs to be maintained. Complicating this, the existing code architectures for safety modeling evolved from programming practices of the 1970s. This has lead to monolithic applications with interdependent data models which require significant knowledge of the complexities of the entire code in order for each component to be maintained. In order to develop an advanced fast reactor safety modeling capability, the limitations of the existing code architecture must be overcome while preserving the capabilities that already exist. To accomplish this, a set of advanced safety modeling requirements is defined, based on modern programming practices, that focuses on modular development within a flexible coupling framework. An approach for integrating the existing capabilities of the SAS4A/SASSYS-1 fast reactor safety analysis code into the SHARP framework is provided in order to preserve existing capabilities while providing a smooth transition to advanced modeling capabilities. In doing this, the advanced fast reactor safety models

  18. Asymmetric cell division requires specific mechanisms for adjusting global transcription.

    Science.gov (United States)

    Mena, Adriana; Medina, Daniel A; García-Martínez, José; Begley, Victoria; Singh, Abhyudai; Chávez, Sebastián; Muñoz-Centeno, Mari C; Pérez-Ortín, José E

    2017-12-01

    Most cells divide symmetrically into two approximately identical cells. There are many examples, however, of asymmetric cell division that can generate sibling cell size differences. Whereas physical asymmetric division mechanisms and cell fate consequences have been investigated, the specific problem caused by asymmetric division at the transcription level has not yet been addressed. In symmetrically dividing cells the nascent transcription rate increases in parallel to cell volume to compensate it by keeping the actual mRNA synthesis rate constant. This cannot apply to the yeast Saccharomyces cerevisiae, where this mechanism would provoke a never-ending increasing mRNA synthesis rate in smaller daughter cells. We show here that, contrarily to other eukaryotes with symmetric division, budding yeast keeps the nascent transcription rates of its RNA polymerases constant and increases mRNA stability. This control on RNA pol II-dependent transcription rate is obtained by controlling the cellular concentration of this enzyme. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Gene-specific DNA methylation association with serum levels of C-reactive protein in African Americans.

    Directory of Open Access Journals (Sweden)

    Yan V Sun

    Full Text Available A more thorough understanding of the differences in DNA methylation (DNAm profiles in populations may hold promise for identifying molecular mechanisms through which genetic and environmental factors jointly contribute to human diseases. Inflammation is a key molecular mechanism underlying several chronic diseases including cardiovascular disease, and it affects DNAm profile on both global and locus-specific levels. To understand the impact of inflammation on the DNAm of the human genome, we investigated DNAm profiles of peripheral blood leukocytes from 966 African American participants in the Genetic Epidemiology Network of Arteriopathy (GENOA study. By testing the association of DNAm sites on CpG islands of over 14,000 genes with C-reactive protein (CRP, an inflammatory biomarker of cardiovascular disease, we identified 257 DNAm sites in 240 genes significantly associated with serum levels of CRP adjusted for age, sex, body mass index and smoking status, and corrected for multiple testing. Of the significantly associated DNAm sites, 80.5% were hypomethylated with higher CRP levels. The most significant Gene Ontology terms enriched in the genes associated with the CRP levels were immune system process, immune response, defense response, response to stimulus, and response to stress, which are all linked to the functions of leukocytes. While the CRP-associated DNAm may be cell-type specific, understanding the DNAm association with CRP in peripheral blood leukocytes of multi-ethnic populations can assist in unveiling the molecular mechanism of how the process of inflammation affects the risks of developing common disease through epigenetic modifications.

  20. Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl β-cyclodextrin or cholesterol-loaded cyclodextrin.

    Science.gov (United States)

    Blanch, Eva; Tomás, Cristina; Hernández, Marta; Roca, Jordi; Martínez, Emilio A; Vázquez, Juan M; Mocé, Eva

    2014-04-24

    Egg yolk (EY) and glycerol are common constituents of extenders used for sperm cryopreservation. It has been demonstrated that using cholesterol-loaded cyclodextrins (CLC) improves sperm cryosurvival in several species. However, standard freezing extenders might not be the most appropriate for CLC-treated sperm. This study evaluated the EY and glycerol requirements for freezing CLC-treated boar spermatozoa. Semen samples from 34 ejaculates coming from 4 boars were used. Each ejaculate was split into three aliquots: one was used untreated (control), and the other two were treated with 1 mg of CLC or methyl-β-cyclodextrin/120 × 10(6) sperm for 15 min at 22 C prior to cryopreservation. Our results indicated that reducing the concentration of EY was detrimental for sperm viability after thawing (31.57 ± 2 vs. 19.89% ± 2 for 20 and 10% EY, respectively; P semen treated with CLC. On the other hand, it was observed that the traditional concentration of glycerol (3%) was not the appropriate for freezing CLC-treated sperm (61.10 ± 3 vs. 47.87% ± 3 viable sperm for control and CLC-treated sperm, respectively; P extenders for CLC-treated sperm. Nevertheless, additional studies will be needed to evaluate alternative cryoprotectants and to determine the effect of high glycerol concentrations on sperm functionality.

  1. Nucleolin is required for DNA methylation state and the expression of rRNA gene variants in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Frédéric Pontvianne

    2010-11-01

    Full Text Available In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1. Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre-rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes. Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis.

  2. Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Miralem Mrkonjic

    Full Text Available We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734 and microsatellite unstable (MSI-H colorectal cancers (CRCs in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls and from Seattle (591 cases, 629 controls. Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4 when the SNP was examined alone.The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

  3. Identification of Methyl Halide-Utilizing Genes in the Methyl Bromide-Utilizing Bacterial Strain IMB-1 Suggests a High Degree of Conservation of Methyl Halide-Specific Genes in Gram-Negative Bacteria

    Science.gov (United States)

    Woodall, C.A.; Warner, K.L.; Oremland, R.S.; Murrell, J.C.; McDonald, I.R.

    2001-01-01

    Strain IMB-1, an aerobic methylotrophic member of the alpha subgroup of the Proteobacteria, can grow with methyl bromide as a sole carbon and energy source. A single cmu gene cluster was identified in IMB-1 that contained six open reading frames: cmuC, cmuA, orf146, paaE, hutI, and partial metF. CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyl-transferase motif. However, cmuB, identified in M. chloromethanicum and H. chloromethanicum, was not detected in IMB-1.

  4. Robust design requirements specification: a quantitative method for requirements development using quality loss functions

    DEFF Research Database (Denmark)

    Pedersen, Søren Nygaard; Christensen, Martin Ebro; Howard, Thomas J.

    2016-01-01

    Product requirements serve many purposes in the product development process. Most importantly, they are meant to capture and facilitate product goals and acceptance criteria, as defined by stakeholders. Accurately communicating stakeholder goals and acceptance criteria can be challenging and more...

  5. Inter- and intra-individual variation in allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology.

    Directory of Open Access Journals (Sweden)

    Nahid Turan

    2010-07-01

    Full Text Available Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART, suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth.

  6. 40 CFR 166.32 - Reporting and recordkeeping requirements for specific, quarantine, and public health exemptions.

    Science.gov (United States)

    2010-07-01

    ... requirements for specific, quarantine, and public health exemptions. 166.32 Section 166.32 Protection of... AGENCIES FOR USE OF PESTICIDES UNDER EMERGENCY CONDITIONS Specific, Quarantine, and Public Health Exemptions § 166.32 Reporting and recordkeeping requirements for specific, quarantine, and public health...

  7. Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

    Directory of Open Access Journals (Sweden)

    Chung Jae

    2009-06-01

    Full Text Available Abstract Background Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition. Methods To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis. Results Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99 between the total number of hypermethylated CGIs and GI50 values (i.e., increased drug resistance following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells. Conclusion Selective epigenetic disruption of distinct biological

  8. Structure-guided mutational analysis reveals the functional requirements for product specificity of DOT1 enzymes.

    Science.gov (United States)

    Dindar, Gülcin; Anger, Andreas M; Mehlhorn, Christine; Hake, Sandra B; Janzen, Christian J

    2014-11-12

    DOT1 enzymes are conserved methyltransferases that catalyse the methylation of lysine 79 on histone H3 (H3K79). Most eukaryotes contain one DOT1 enzyme, whereas African trypanosomes have two homologues, DOT1A and DOT1B, with different enzymatic activities. DOT1A mediates mono- and dimethylation of H3K76, the homologue of H3K79 in other organisms, whereas DOT1B additionally catalyses H3K76 trimethylation. However, it is unclear how these different enzymatic activities are achieved. Here we employ a trypanosomal nucleosome reconstitution system and structure-guided homology modelling to identify critical residues within and outside the catalytic centre that modulate product specificity. Exchange of these residues transfers the product specificity from one enzyme to the other, and reveals the existence of distinct regulatory domains adjacent to the catalytic centre. Our study provides the first evidence that a few crucial residues in DOT1 enzymes are sufficient to catalyse methyl-state-specific reactions. These results might also have far-reaching consequences for the functional understanding of homologous enzymes in higher eukaryotes.

  9. Computer-Aided Discovery of Formal Specification Behavioral Requirements and Requirement to Implementation Mappings

    Science.gov (United States)

    2014-01-01

    the executable SRM is developed according to the specification and marketing documents. Hence, for example, the Vehicles, Car, and Truck classes in...transitions ternary relation: transitions ⊆ states x transitionIDs x states, such as <"Init", "Tr1", " stP "> • A conditions unary relation bound to

  10. CH2M Hill Hanford Group, Inc., Standards and Requirements Identification Document (SRID) Requirements Management System and Requirements Specification

    International Nuclear Information System (INIS)

    JOHNSON, A.L.

    2000-01-01

    The current Tank Farm Contractor (TFC) for the U. S. Department of Energy, Office of River Protection (ORP), River Protection Project (RPP), CH2M Hill Hanford Group, Inc. (CHG), will use a computer based requirements management system. The system will serve as a tool to assist in identifying, capturing, and maintaining the Standards/Requirements Identification Document (S/RID) requirements and links to implementing procedures and other documents. By managing requirements as one integrated set, CHG will be able to carry out its mission more efficiently and effectively. CHG has chosen the Dynamic Object Oriented Requirements System (DOORS(trademark)) as the preferred computer based requirements management system. Accordingly, the S/RID program will use DOORS(trademark). DOORS(trademark) will replace the Environmental Requirements Management Interface (ERMI) system as the tool for S/RID data management. The DOORS(trademark) S/RID test project currently resides on the DOORSTM test server. The S/RID project will be migrated to the DOORS(trademark) production server. After the migration the S/RID project will be considered a production project and will no longer reside on the test server

  11. [Definition and specification requirements for PAC-systems (picture archiving and communication system). A performance index with reference to the standard "IEEE Recommended Practice for Software Requirement Specifications"].

    Science.gov (United States)

    König, H; Klose, K J

    1999-04-01

    The formulation of requirements is necessary to control the goals of a PACS project. Furthermore, in this way, the scope of functionality necessary to support radiological working processes becomes clear. Definitions of requirements and specification are formulated independently of systems according to the IEEE standard "Recommended Practice for Software Requirements Specifications". Definitions are given in the Request for Information, specifications in the Request for Proposal. Functional and non-functional requirements are distinguished. The solutions are rated with respect to scope, appropriateness and quality of implementation. A PACS checklist was created according to the methods described above. It is published on the homepage of the "Arbeitsgemeinschaft Informationstechnologie" (AGIT) within the "Deutsche Röntgengesellschaft" (DRG) (http://www.uni-marburg.de/mzr/agit). The checklist provides a discussion forum which should contribute to an agreement on accepted basic PACS functionalities.

  12. Mutations in TET2 and DNMT3A genes are associated with changes in global and gene-specific methylation in acute myeloid leukemia.

    Science.gov (United States)

    Ponciano-Gómez, Alberto; Martínez-Tovar, Adolfo; Vela-Ojeda, Jorge; Olarte-Carrillo, Irma; Centeno-Cruz, Federico; Garrido, Efraín

    2017-10-01

    Acute myeloid leukemia is characterized by its high biological and clinical heterogeneity, which represents an important barrier for a precise disease classification and accurate therapy. While epigenetic aberrations play a pivotal role in acute myeloid leukemia pathophysiology, molecular signatures such as change in the DNA methylation patterns and genetic mutations in enzymes needed to the methylation process can also be helpful for classifying acute myeloid leukemia. Our study aims to unveil the relevance of DNMT3A and TET2 genes in global and specific methylation patterns in acute myeloid leukemia. Peripheral blood samples from 110 untreated patients with acute myeloid leukemia and 15 healthy control individuals were collected. Global 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from peripheral blood leukocytes were measured by using the MethylFlashTM Quantification kits. DNMT3A and TET2 expression levels were evaluated by real-time quantitative polymerase chain reaction. The R882A hotspot of DNMT3A and exons 6-10 of TET2 were amplified by polymerase chain reaction and sequenced using the Sanger method. Methylation patterns of 16 gene promoters were evaluated by pyrosequencing after treating DNA with sodium bisulfite, and their transcriptional products were measured by real-time quantitative polymerase chain reaction.Here, we demonstrate altered levels of 5-methylcytosine and 5-hydroxymethylcytosine and highly variable transcript levels of DNMT3A and TET2 in peripheral blood leukocytes from acute myeloid leukemia patients. We found a mutation prevalence of 2.7% for DNMT3A and 11.8% for TET2 in the Mexican population with this disease. The average overall survival of acute myeloid leukemia patients with DNMT3A mutations was only 4 months. In addition, we showed that mutations in DNMT3A and TET2 may cause irregular DNA methylation patterns and transcriptional expression levels in 16 genes known to be involved in acute myeloid leukemia pathogenesis

  13. Definition and specification for PACS. A checklist based on the standard ''IEEE Recommended Practice for Software Requirements Specifications''

    International Nuclear Information System (INIS)

    Koenig, H.; Klose, K.J.

    1999-01-01

    Problem: The formulation of requirements is necessary to control the goals of a PACS project. Furthermore, in this way, the scope of functionality necessary to support radiological working processes becomes clear. Method: Definitions of requirements and specification are formulated independently of systems according to the IEEE standard 'Recommended Practice for Software Requirements Specifications'. Definitions are given in the Request for Information, specifications in the Request for Proposal. Functional and non-functional requirements are distinguished. The solutions are rated with respect to scope, appropriateness and quality of implementation. Results: A PACS checklist was created according to the methods described above. It is published on the homepage of the 'Arbeitsgemeinschaft Informationstechnologie' (AGIT) within the 'Deutsche Roentgengesellschaft' (DRG) (http://www.uni-marburg.de/mzr.agit). Conclusion: The checklist provides a discussion forum which should contribute to an agreement on accepted basic PACS functionalities. (orig.) [de

  14. 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 B

    2014-01-01

    (DMRs) was validated using Methylation Sensitive High Resolution Melting (MS-HRM) in a case control study on a panel of breast carcinomas (N = 275) and non-malignant controls (N = 74). RESULTS: Based on microarray results we selected 19 DMRs for large-scale screening of cases and controls. Analysis...

  15. Functions and Requirements and Specifications for Replacement of the Computer Automated Surveillance System (CASS)

    International Nuclear Information System (INIS)

    SCAIEF, C.C.

    1999-01-01

    This functions, requirements and specifications document defines the baseline requirements and criteria for the design, purchase, fabrication, construction, installation, and operation of the system to replace the Computer Automated Surveillance System (CASS) alarm monitoring

  16. Structure, function and carcinogenicity of metabolites of methylated and non-methylated polycyclic aromatic hydrocarbons: a comprehensive review.

    Science.gov (United States)

    Flesher, James W; Lehner, Andreas F

    2016-01-01

    The Unified Theory of PAH Carcinogenicity accommodates the activities of methylated and non-methylated polycyclic aromatic hydrocarbons (PAHs) and states that substitution of methyl groups on meso-methyl substituted PAHs with hydroxy, acetoxy, chloride, bromide or sulfuric acid ester groups imparts potent cancer producing properties. It incorporates specific predictions from past researchers on the mechanism of carcinogenesis by methyl-substituted hydrocarbons, including (1) requirement for metabolism to an ArCH2X type structure where X is a good leaving group and (2) biological substitution of a meso-methyl group at the most reactive center in non-methylated hydrocarbons. The Theory incorporates strong inferences of Fieser: (1) The mechanism of carcinogenesis involves a specific metabolic substitution of a hydrocarbon at its most reactive center and (2) Metabolic elimination of a carcinogen is a detoxifying process competitive with that of carcinogenesis and occurring by a different mechanism. According to this outlook, chemical or biochemical substitution of a methyl group at the reactive meso-position of non-methylated hydrocarbons is the first step in the mechanism of carcinogenesis for most, if not all, PAHs and the most potent metabolites of PAHs are to be found among the meso methyl-substituted hydrocarbons. Some PAHs and their known or potential metabolites and closely related compounds have been tested in rats for production of sarcomas at the site of subcutaneous injection and the results strongly support the specific predictions of the Unified Theory.

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

    Directory of Open Access Journals (Sweden)

    Schwarzenbach Heidi

    2010-06-01

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

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  19. 78 FR 47015 - Software Requirement Specifications for Digital Computer Software Used in Safety Systems of...

    Science.gov (United States)

    2013-08-02

    ... NUCLEAR REGULATORY COMMISSION [NRC-2012-0195] Software Requirement Specifications for Digital Computer Software Used in Safety Systems of Nuclear Power Plants AGENCY: Nuclear Regulatory Commission... issuing a revised regulatory guide (RG), revision 1 of RG 1.172, ``Software Requirement Specifications for...

  20. Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

    International Nuclear Information System (INIS)

    Pan, Yun-Zu; Quade, Bradley; Brewer, Kyle D.; Szabo, Monika; Swarbrick, James D.; Graham, Bim; Rizo, Josep

    2016-01-01

    Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca 2+ -dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.

  1. Sequence-specific assignment of methyl groups from the neuronal SNARE complex using lanthanide-induced pseudocontact shifts

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yun-Zu; Quade, Bradley; Brewer, Kyle D. [University of Texas Southwestern Medical Center, Department of Biophysics (United States); Szabo, Monika; Swarbrick, James D.; Graham, Bim [Monash Institute of Pharmaceutical Sciences, Monash University (Australia); Rizo, Josep, E-mail: Jose.Rizo-Rey@UTSouthwestern.edu [University of Texas Southwestern Medical Center, Department of Biophysics (United States)

    2016-12-15

    Neurotransmitter release depends critically on the neuronal SNARE complex formed by syntaxin-1, SNAP-25 and synaptobrevin, as well as on other proteins such as Munc18-1, Munc13-1 and synaptotagmin-1. Although three-dimensional structures are available for these components, it is still unclear how they are assembled between the synaptic vesicle and plasma membranes to trigger fast, Ca{sup 2+}-dependent membrane fusion. Methyl TROSY NMR experiments provide a powerful tool to study complexes between these proteins, but assignment of the methyl groups of the SNARE complex is hindered by its limited solubility. Here we report the assignment of the isoleucine, leucine, methionine and valine methyl groups of the four SNARE motifs of syntaxin-1, SNAP-25 and synaptobrevin within the SNARE complex based solely on measurements of lanthanide-induced pseudocontact shifts. Our results illustrate the power of this approach to assign protein resonances without the need of triple resonance experiments and provide an invaluable tool for future structural studies of how the SNARE complex binds to other components of the release machinery.

  2. Brown adipose tissue (BAT specific vaspin expression is increased after obesogenic diets and cold exposure and linked to acute changes in DNA-methylation

    Directory of Open Access Journals (Sweden)

    Juliane Weiner

    2017-06-01

    Full Text Available Objective: Several studies have demonstrated anti-diabetic and anti-obesogenic properties of visceral adipose tissue-derived serine protease inhibitor (vaspin and so evoked its potential use for treatment of obesity-related diseases. The aim of the study was to unravel physiological regulators of vaspin expression and secretion with a particular focus on its role in brown adipose tissue (BAT biology. Methods: We analyzed the effects of obesogenic diets and cold exposure on vaspin expression in liver and white and brown adipose tissue (AT and plasma levels. Vaspin expression was analyzed in isolated white and brown adipocytes during adipogenesis and in response to adrenergic stimuli. DNA-methylation within the vaspin promoter was analyzed to investigate acute epigenetic changes after cold-exposure in BAT. Results: Our results demonstrate a strong induction of vaspin mRNA and protein expression specifically in BAT of both cold-exposed and high-fat (HF or high-sugar (HS fed mice. While obesogenic diets also upregulated hepatic vaspin mRNA levels, cold exposure tended to increase vaspin gene expression of inguinal white adipose tissue (iWAT depots. Concomitantly, vaspin plasma levels were decreased upon obesogenic or thermogenic triggers. Vaspin expression was increased during adipogenesis but unaffected by sympathetic activation in brown adipocytes. Analysis of vaspin promoter methylation in AT revealed lowest methylation levels in BAT, which were acutely reduced after cold exposure. Conclusions: Our data demonstrate a novel BAT-specific regulation of vaspin gene expression upon physiological stimuli in vivo with acute epigenetic changes that may contribute to cold-induced expression in BAT. We conclude that these findings indicate functional relevance and potentially beneficial effects of vaspin in BAT function. Keywords: Brown adipose tissue, Browning, Cold exposure, DNA methylation, High-fat diet, High-sucrose diet, SerpinA12, Thermogenesis

  3. Small Interfering RNA Specific for N-Methyl-D-Aspartate Receptor 2B Offers Neuroprotection to Dopamine Neurons through Activation of MAP Kinase

    Directory of Open Access Journals (Sweden)

    Olivia T.W. Ng

    2012-02-01

    Full Text Available In the present study, N-methyl-D-aspartate receptor 2B (NR2B-specific siRNA was applied in parkinsonian models. Our previous results showed that reduction in expression of N-methyl-D-aspartate receptor 1 (NR1, the key subunit of N-methyl-D-aspartate receptors, by antisense oligos amelio-rated the motor symptoms in the 6-hydroxydopamine (6-OHDA-lesioned rat, an animal model of Parkinson's disease (PD [Lai et al.: Neurochem Int 2004;45:11-22]. To further the investigation on the efficacy of gene silencing, small interference RNA (siRNA specific for the NR2B subunit was designed and administered in the striatum of 6-OHDA-lesioned rats. The present results show that administration of NR2B-specific siRNA decreased the number of apomorphine-induced rotations in the lesioned rats and that there was a significant reduction in NR2B proteins levels after NR2B-specific siRNA administration. Furthermore, attenuation of the loss of dopaminergic neurons was found in both the striatal and substantia nigra regions of the 6-OHDA-lesioned rats that had been continuously infused with siRNA for 7 days. In addition, a significant upregulation of p-p44/42 MAPK (ERK1/2; Thr202/Tyr204 and p-CREB (Ser133 in striatal neurons was found. These results suggest that application of the gene silencing targeting NR2B could be a potential treatment of PD, and they also revealed the possibility of NR2B-specific siRNA being involved in the prosurvival pathway.

  4. Electronic Health Record in Occupational Medicine: Specific Aspects and Requirements of Data Structuring and Standardization

    Directory of Open Access Journals (Sweden)

    Dorin TRIFF

    2009-07-01

    Full Text Available The service of occupational medicine of a specific economic agent, as integrated part of the System of Labor Health and Safety, requires efficient, well-organized information management through standardized and computerized data processing and exploitation. Legal requirements and practical aspects of information management in occupational medicine trigger necessary operational modifications in the Electronic Health File. The goal of the paper is to present basic requirements of structuring the electronic health file and the necessary standards in recording specific data.

  5. PRC2 is required to maintain expression of the maternal Gtl2-Rian-Mirg locus by preventing de novo DNA methylation in mouse embryonic stem cells

    Science.gov (United States)

    Das, Partha Pratim; Hendrix, David A.; Apostolou, Effie; Buchner, Alice H.; Canver, Matthew C.; Beyaz, Semir; Ljuboja, Damir; Kuintzle, Rachael; Kim, Woojin; Karnik, Rahul; Shao, Zhen; Xie, Huafeng; Xu, Jian; De Los Angeles, Alejandro; Zhang, Yingying; Choe, Junho; Jun, Don Leong Jia; Shen, Xiaohua; Gregory, Richard I.; Daley, George Q.; Meissner, Alexander; Kellis, Manolis; Hochedlinger, Konrad; Kim, Jonghwan; Orkin, Stuart H.

    2017-01-01

    SUMMARY Polycomb Repressive Complex 2 (PRC2) function and DNA methylation (DNAme) are typically correlated with the gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2 the entire locus becomes transcriptionally repressed due to gain of DNA methylation at the intergenic differentially methylated regions (IG-DMR). Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Mechanistic study reveals that PRC2 interacts physically with Dnmt3 methyltransferases and prevents their recruitment and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations provide a novel mechanism by which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency. PMID:26299972

  6. A Hybrid Parallel Execution Model for Logic Based Requirement Specifications (Invited Paper

    Directory of Open Access Journals (Sweden)

    Jeffrey J. P. Tsai

    1999-05-01

    Full Text Available It is well known that undiscovered errors in a requirements specification is extremely expensive to be fixed when discovered in the software maintenance phase. Errors in the requirement phase can be reduced through the validation and verification of the requirements specification. Many logic-based requirements specification languages have been developed to achieve these goals. However, the execution and reasoning of a logic-based requirements specification can be very slow. An effective way to improve their performance is to execute and reason the logic-based requirements specification in parallel. In this paper, we present a hybrid model to facilitate the parallel execution of a logic-based requirements specification language. A logic-based specification is first applied by a data dependency analysis technique which can find all the mode combinations that exist within a specification clause. This mode information is used to support a novel hybrid parallel execution model, which combines both top-down and bottom-up evaluation strategies. This new execution model can find the failure in the deepest node of the search tree at the early stage of the evaluation, thus this new execution model can reduce the total number of nodes searched in the tree, the total processes needed to be generated, and the total communication channels needed in the search process. A simulator has been implemented to analyze the execution behavior of the new model. Experiments show significant improvement based on several criteria.

  7. 49 CFR 179.301 - Individual specification requirements for multi-unit tank car tanks.

    Science.gov (United States)

    2010-10-01

    ...-unit tank car tanks. 179.301 Section 179.301 Transportation Other Regulations Relating to... MATERIALS REGULATIONS SPECIFICATIONS FOR TANK CARS Specifications for Multi-Unit Tank Car Tanks (Classes DOT-106A and 110AW) § 179.301 Individual specification requirements for multi-unit tank car tanks. (a) In...

  8. Effects of Using Requirements Catalogs on Effectiveness and Productivity of Requirements Specification in a Software Project Management Course

    Science.gov (United States)

    Fernández-Alemán, José Luis; Carrillo-de-Gea, Juan Manuel; Meca, Joaquín Vidal; Ros, Joaquín Nicolás; Toval, Ambrosio; Idri, Ali

    2016-01-01

    This paper presents the results of two educational experiments carried out to determine whether the process of specifying requirements (catalog-based reuse as opposed to conventional specification) has an impact on effectiveness and productivity in co-located and distributed software development environments. The participants in the experiments…

  9. Functional promoter upstream p53 regulatory sequence of IGFBP3 that is silenced by tumor specific methylation

    International Nuclear Information System (INIS)

    Hanafusa, Tadashi; Shinji, Toshiyuki; Shiraha, Hidenori; Nouso, Kazuhiro; Iwasaki, Yoshiaki; Yumoto, Eichiro; Ono, Toshiro; Koide, Norio

    2005-01-01

    Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells

  10. Comparison of CpG island methylator phenotype (CIMP frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

    Directory of Open Access Journals (Sweden)

    Marianne Berg

    Full Text Available BACKGROUND: In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP. However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. METHODS: Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. RESULTS: For 47 samples (71%, the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20% consistently scored as CIMP positive. Only four of 31 probes (13% investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; p<0.005 was demonstrated. CONCLUSIONS: A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition

  11. Comparison of CpG island methylator phenotype (CIMP) frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

    Science.gov (United States)

    Berg, Marianne; Hagland, Hanne R; Søreide, Kjetil

    2014-01-01

    In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; pCIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.

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

    Science.gov (United States)

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

    2017-03-09

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

  13. Certification of packagings: compliance with DOT specification 7A packaging requirements

    International Nuclear Information System (INIS)

    Edling, D.A.

    1976-01-01

    A study was conducted to determine which of the packagings currently listed in CFR 49 Section 173.395 a.1-5, meet the Specification 7A requirements (CFR 49 Section 173.350). According to DOT HM-111 the present listing of various authorized DOT specifications in Section 173.394 and Section 173.395 (Type A containers) of ICC Tariff No. 27 would be deleted with complete reliance being placed on the use of DOT 7A, Type A general packaging specification. Each user of a Specification 7A package would be required to document and maintain on file for one year a written record of his determination of compliance with the DOT Specification 7A performance requirements. All the specification packagings listed in CFR 49 Section 173.395a.1-5 were tested and shown to meet the Specification 7A criteria; however, in many cases qualifications were placed on their use. Forty-nine specification packagings were tested and shown to meet the DOT Specification 7A performance requirements and since there were several styles of some specific packagings, this amounts to greater than 80 packagings. The extensive testing generally indicated a high degree of containment integrity in the packagings tested and the documentation discussed is a valuable tool for shippers of Type A quantities of radioactive materials

  14. The Mitochondrial Lon Protease Is Required for Age-Specific and Sex-Specific Adaptation to Oxidative Stress.

    Science.gov (United States)

    Pomatto, Laura C D; Carney, Caroline; Shen, Brenda; Wong, Sarah; Halaszynski, Kelly; Salomon, Matthew P; Davies, Kelvin J A; Tower, John

    2017-01-09

    Multiple human diseases involving chronic oxidative stress show a significant sex bias, including neurodegenerative diseases, cancer, immune dysfunction, diabetes, and cardiovascular disease. However, a possible molecular mechanism for the sex bias in physiological adaptation to oxidative stress remains unclear. Here, we report that Drosophila melanogaster females but not males adapt to hydrogen peroxide stress, whereas males but not females adapt to paraquat (superoxide) stress. Stress adaptation in each sex requires the conserved mitochondrial Lon protease and is associated with sex-specific expression of Lon protein isoforms and proteolytic activity. Adaptation to oxidative stress is lost with age in both sexes. Transgenic expression of transformer gene during development transforms chromosomal males into pseudo-females and confers the female-specific pattern of Lon isoform expression, Lon proteolytic activity induction, and H 2 O 2 stress adaptation; these effects were also observed using adult-specific transformation. Conversely, knockdown of transformer in chromosomal females eliminates the female-specific Lon isoform expression, Lon proteolytic activity induction, and H 2 O 2 stress adaptation and produces the male-specific paraquat (superoxide) stress adaptation. Sex-specific expression of alternative Lon isoforms was also observed in mouse tissues. The results develop Drosophila melanogaster as a model for sex-specific stress adaptation regulated by the Lon protease, with potential implications for understanding sexual dimorphism in human disease. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. DNA methylation analysis of paediatric low-grade astrocytomas identifies a tumour-specific hypomethylation signature in pilocytic astrocytomas.

    Science.gov (United States)

    Jeyapalan, Jennie N; Doctor, Gabriel T; Jones, Tania A; Alberman, Samuel N; Tep, Alexander; Haria, Chirag M; Schwalbe, Edward C; Morley, Isabel C F; Hill, Alfred A; LeCain, Magdalena; Ottaviani, Diego; Clifford, Steven C; Qaddoumi, Ibrahim; Tatevossian, Ruth G; Ellison, David W; Sheer, Denise

    2016-05-27

    Low-grade gliomas (LGGs) account for about a third of all brain tumours in children. We conducted a detailed study of DNA methylation and gene expression to improve our understanding of the biology of pilocytic and diffuse astrocytomas. Pilocytic astrocytomas were found to have a distinctive signature at 315 CpG sites, of which 312 were hypomethylated and 3 were hypermethylated. Genomic analysis revealed that 182 of these sites are within annotated enhancers. The signature was not present in diffuse astrocytomas, or in published profiles of other brain tumours and normal brain tissue. The AP-1 transcription factor was predicted to bind within 200 bp of a subset of the 315 differentially methylated CpG sites; the AP-1 factors, FOS and FOSL1 were found to be up-regulated in pilocytic astrocytomas. We also analysed splice variants of the AP-1 target gene, CCND1, which encodes cell cycle regulator cyclin D1. CCND1a was found to be highly expressed in both pilocytic and diffuse astrocytomas, but diffuse astrocytomas have far higher expression of the oncogenic variant, CCND1b. These findings highlight novel genetic and epigenetic differences between pilocytic and diffuse astrocytoma, in addition to well-described alterations involving BRAF, MYB and FGFR1.

  16. [COMPARATIVE SENSITIVITY OF CHOLINESTERASES IN VERTEBRATES AND INVERTEBRATES TO HIGHLY SPECIFIC ORGANOPHOSPHORUS INHIBITORS DIISOPROPYL FLUOROPHOSPHATE (DFP) AND (2-ETHOXYMETHYL PHOSPHORYL THIOETHYL) ETHYL (METHYL) SULPHONIUM SULPHOMETHYLAT (GD-42)].

    Science.gov (United States)

    Basova, N E; Kormilitsyn, B N; Perchenok, A Yu; Rozengart, E V; Saakov, V S; Suvorov, A A

    2015-01-01

    The review presents data on comparative reactivity of 68 cholinesterase preparation from various organs and tissues in a number of vertebrates and invertebrates based on sensitivity to two highly specific and most studied organophosphorus inhibitors--diisopropyl fluorophosphates (DFP) and (2-ethoxymethyl phosphoryl thioethyl) ethyl (methyl) sulphonium sulphomethylat (GD-42). Analysis of these data suggests a great diversity in enzymologic characteristics of cholinesterase preparation in representatives of vertebrates and invertebrates, this variety observed even for closely related enzymes in animals of almost the same level of development.

  17. Specific requirements of GS-R3 related to process implementation

    International Nuclear Information System (INIS)

    Florescu, N.

    2009-01-01

    The PowerPoint presentation gives: an overview of IAEA management system requirements or topics; - the requirements specific to processes and process implementation; - the key practical challenge of using the process approach specified in IAEA SG GS-G3.1 and GS-G3.5. The following items are thoroughly discussed: - Requirements related to specific process implementation and developing processes; - Process management; Generic management; - System processes: - Control of documents; Control of products; Control of records; - Purchasing; - Communication; - Managing organizational change; - Other requirements concerning the process management system; - General management system; - Grading; - Documentation; - Fulfilling the requirements of interested parties; - Management responsibility; - Planning responsibility and authority for the management system monitoring and measurement; - Independent assessment; - Management system review; - Non-conformances, corrective and preventive actions; - Improvement key practical challenge of using the process approach specified in IAEA SG GS-G3.1 and GS-G3.5; - Key challenge: - Process common to all stages; - Phases of process development proposed by IAEA. The following conclusions complete the presentation: GS-R-3 sets basic requirements for process-based integrated management system; - Some key generic processes required, no specific process model favoured namely, no reference to management, core and support processes; - Up to organization to determine appropriate process model; - Easily applicable to a wide range of facilities and activities, including those of a regulatory body; - Specific requirements are found in specific Safety Guide. (author)

  18. Software requirements specification for the program analysis and control system risk management module

    International Nuclear Information System (INIS)

    SCHAEFER, J.C.

    1999-01-01

    TWR Program Analysis and Control System Risk Module is used to facilitate specific data processes surrounding the Risk Management program of the Tank Waste Retrieval environment. This document contains the Risk Management system requirements of the database system

  19. Specification of Behavioural Requirements within Compositional Multi-Agent System Design

    OpenAIRE

    Herlea, D.E.; Jonker, C.M.; Treur, J.; Wijngaards, N.J.E.

    1999-01-01

    In this paper it is shown how informal and formal specification of behavioural requirements and scenarios for agents and multi-agent systems can be integrated within multi-agent system design. In particular, it is addressed how a compositional

  20. System requirement specifications for the Z-plant materials information tracking system (ZMITS)

    International Nuclear Information System (INIS)

    NEGIN, C.A.

    1999-01-01

    This is a system requirement specification for a database which will be developed to track classified information related to nuclear materials stored at PFP. The system will supplement existing databases to support both processing and disposition information needs

  1. Appraisal of work ability in relation to job-specific health requirements in ambulance workers

    OpenAIRE

    van Schaaijk, A.; Boschman, J. S.; Frings-Dresen, M. H. W.; Sluiter, J. K.

    2016-01-01

    Purpose To gain insight into which job-specific health requirements relate to work ability, the following two research questions were formulated: Which job-specific health requirements are associated with the appraisal of work ability in ambulance drivers and paramedics? How are appraisals of physical and mental work ability associated with the appraisal of overall work ability in ambulance drivers and paramedics? Method Workers Health Surveillance cross-sectional data of 506 ambulance worker...

  2. Synthesis and characterization of a radiolabeled derivative of the phencyclidine/N-methyl-D-aspartate receptor ligand (+)MK-801 with high specific radioactivity

    International Nuclear Information System (INIS)

    Keana, J.F.W.; Scherz, M.W.; Quarum, M.; Sonders, M.S.; Weber, E.

    1988-01-01

    A [ 3 H]-labelled derivative of the drug (+)MK-801 with a high specific radioactivity was synthesized by first preparing a tribromo derivative of (+)MK-801 followed by catalytic reduction in the presence of [ 3 H]-gas and subsequent purification of the radioactive product by reversed-phase high performance liquid chromatography (RP-HPLC). This resulted in pure (+) [ 3 H]MK-801 with a specific radioactivity of 97 Ci/mmol. The (+) [ 3 H]MK-801 was shown to interact with high affinity and selectivity with the phencyclidine (PCP) receptor in guinea pig brain membrane suspensions. The PCP receptor is associated with a cation channel that is chemically gated by glutamate and N-methyl-D-aspartate (NMDA). Drugs that interact with the PCP receptor block this channel. The (+) [ 3 H]MK-801 described here will be useful to investigate the biochemistry of PCP/NMDA receptors in experiments where a high specific radioactivity is essential

  3. Dynamic instability of genomic methylation patterns in pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Ooi Steen KT

    2010-09-01

    Full Text Available Abstract Background Genomic methylation patterns are established during gametogenesis, and perpetuated in somatic cells by faithful maintenance methylation. There have been previous indications that genomic methylation patterns may be less stable in embryonic stem (ES cells than in differentiated somatic cells, but it is not known whether different mechanisms of de novo and maintenance methylation operate in pluripotent stem cells compared with differentiating somatic cells. Results In this paper, we show that ablation of the DNA methyltransferase regulator DNMT3L (DNA methyltransferase 3-like in mouse ES cells renders them essentially incapable of de novo methylation of newly integrated retroviral DNA. We also show that ES cells lacking DNMT3L lose DNA methylation over time in culture, suggesting that DNA methylation in ES cells is the result of dynamic loss and gain of DNA methylation. We found that wild-type female ES cells lose DNA methylation at a much faster rate than do male ES cells; this defect could not be attributed to sex-specific differences in expression of DNMT3L or of any DNA methyltransferase. We also found that human ES and induced pluripotent stem cell lines showed marked but variable loss of methylation that could not be attributed to sex chromosome constitution or time in culture. Conclusions These data indicate that DNA methylation in pluripotent stem cells is much more dynamic and error-prone than is maintenance methylation in differentiated cells. DNA methylation requires DNMT3L in stem cells, but DNMT3L is not expressed in differentiating somatic cells. Error-prone maintenance methylation will introduce unpredictable phenotypic variation into clonal populations of pluripotent stem cells, and this variation is likely to be much more pronounced in cultured female cells. This epigenetic variability has obvious negative implications for the clinical applications of stem cells.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  5. Brown adipose tissue (BAT) specific vaspin expression is increased after obesogenic diets and cold exposure and linked to acute changes in DNA-methylation.

    Science.gov (United States)

    Weiner, Juliane; Rohde, Kerstin; Krause, Kerstin; Zieger, Konstanze; Klöting, Nora; Kralisch, Susan; Kovacs, Peter; Stumvoll, Michael; Blüher, Matthias; Böttcher, Yvonne; Heiker, John T

    2017-06-01

    Several studies have demonstrated anti-diabetic and anti-obesogenic properties of visceral adipose tissue-derived serine protease inhibitor (vaspin) and so evoked its potential use for treatment of obesity-related diseases. The aim of the study was to unravel physiological regulators of vaspin expression and secretion with a particular focus on its role in brown adipose tissue (BAT) biology. We analyzed the effects of obesogenic diets and cold exposure on vaspin expression in liver and white and brown adipose tissue (AT) and plasma levels. Vaspin expression was analyzed in isolated white and brown adipocytes during adipogenesis and in response to adrenergic stimuli. DNA-methylation within the vaspin promoter was analyzed to investigate acute epigenetic changes after cold-exposure in BAT. Our results demonstrate a strong induction of vaspin mRNA and protein expression specifically in BAT of both cold-exposed and high-fat (HF) or high-sugar (HS) fed mice. While obesogenic diets also upregulated hepatic vaspin mRNA levels, cold exposure tended to increase vaspin gene expression of inguinal white adipose tissue (iWAT) depots. Concomitantly, vaspin plasma levels were decreased upon obesogenic or thermogenic triggers. Vaspin expression was increased during adipogenesis but unaffected by sympathetic activation in brown adipocytes. Analysis of vaspin promoter methylation in AT revealed lowest methylation levels in BAT, which were acutely reduced after cold exposure. Our data demonstrate a novel BAT-specific regulation of vaspin gene expression upon physiological stimuli in vivo with acute epigenetic changes that may contribute to cold-induced expression in BAT. We conclude that these findings indicate functional relevance and potentially beneficial effects of vaspin in BAT function.

  6. Pan-cancer stratification of solid human epithelial tumors and cancer cell lines reveals commonalities and tissue-specific features of the CpG island methylator phenotype.

    Science.gov (United States)

    Sánchez-Vega, Francisco; Gotea, Valer; Margolin, Gennady; Elnitski, Laura

    2015-01-01

    The term CpG island methylator phenotype (CIMP) has been used to describe widespread DNA hypermethylation at CpG-rich genomic regions affecting clinically distinct subsets of cancer patients. Even though there have been numerous studies of CIMP in individual cancer types, a uniform analysis across tissues is still lacking. We analyze genome-wide patterns of CpG island hypermethylation in 5,253 solid epithelial tumors from 15 cancer types from TCGA and 23 cancer cell lines from ENCODE. We identify differentially methylated loci that define CIMP+ and CIMP- samples, and we use unsupervised clustering to provide a robust molecular stratification of tumor methylomes for 12 cancer types and all cancer cell lines. With a minimal set of 89 discriminative loci, we demonstrate accurate pan-cancer separation of the 12 CIMP+/- subpopulations, based on their average levels of methylation. Tumor samples in different CIMP subclasses show distinctive correlations with gene expression profiles and recurrence of somatic mutations, copy number variations, and epigenetic silencing. Enrichment analyses indicate shared canonical pathways and upstream regulators for CIMP-targeted regions across cancer types. Furthermore, genomic alterations showing consistent associations with CIMP+/- status include genes involved in DNA repair, chromatin remodeling genes, and several histone methyltransferases. Associations of CIMP status with specific clinical features, including overall survival in several cancer types, highlight the importance of the CIMP+/- designation for individual tumor evaluation and personalized medicine. We present a comprehensive computational study of CIMP that reveals pan-cancer commonalities and tissue-specific differences underlying concurrent hypermethylation of CpG islands across tumors. Our stratification of solid tumors and cancer cell lines based on CIMP status is data-driven and agnostic to tumor type by design, which protects against known biases that have hindered

  7. 24 CFR 200.936 - Supplementary specific procedural requirements under HUD building products certification program...

    Science.gov (United States)

    2010-04-01

    ... requirements under HUD building products certification program for solid fuel type room heaters and fireplace... Supplementary specific procedural requirements under HUD building products certification program for solid fuel... fireplace stoves certified under the HUD Building Products Certification Program shall be designed...

  8. 77 FR 50726 - Software Requirement Specifications for Digital Computer Software and Complex Electronics Used in...

    Science.gov (United States)

    2012-08-22

    ... Computer Software and Complex Electronics Used in Safety Systems of Nuclear Power Plants AGENCY: Nuclear...-1209, ``Software Requirement Specifications for Digital Computer Software and Complex Electronics used... Electronics Engineers (ANSI/IEEE) Standard 830-1998, ``IEEE Recommended Practice for Software Requirements...

  9. Treated effluent disposal system process control computer software requirements and specification

    International Nuclear Information System (INIS)

    Graf, F.A. Jr.

    1994-01-01

    The software requirements for the monitor and control system that will be associated with the effluent collection pipeline system known as the 200 Area Treated Effluent Disposal System is covered. The control logic for the two pump stations and specific requirements for the graphic displays are detailed

  10. 24 CFR 200.940 - Supplementary specific requirements under the HUD building product standards and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standards and certification program for sealed insulating glass units... Supplementary specific requirements under the HUD building product standards and certification program for... specific building product certification program. In the case of sealed insulating glass units, testing and...

  11. 24 CFR 200.955 - Supplementary specific requirements under the HUD building product standard and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standard and certification program for fenestration products (windows and... Supplementary specific requirements under the HUD building product standard and certification program for... testing for a product shall be described in the specific building product certification program. In the...

  12. 40 CFR 53.51 - Demonstration of compliance with design specifications and manufacturing and test requirements.

    Science.gov (United States)

    2010-07-01

    ... Methods and Class I and Class II Equivalent Methods for PM2.5 or PM10â2.5 § 53.51 Demonstration of... standard specification 8625F, Type II, Class I (reference 4 in appendix A of this subpart) in the same way... specifications and manufacturing and test requirements. 53.51 Section 53.51 Protection of Environment...

  13. Getting the balance right between functional and non-functional requirements: the case of requirement specification in IT procurement

    Directory of Open Access Journals (Sweden)

    Björn Johansson

    2013-01-01

    Full Text Available IT procurement represents a business process of high importance, including the ability to articulate requirements that the procurement deals with. Furthermore, specifying requirements is of importance for both procurer and potential supplier, as it functions as central contractual element between the two. The purpose of this article is two-fold: (i to show how established terminology for requirement specification is represented in current call for bids for the procurement of IT; and (ii to introduce an organizing framework that may assist procurers in actively addressing functional requirements and business requirements. Ten “call for bids” were examined from a Swedish national procurement database. From the analysis of the bids, it can be concluded that: (i the call for bids displays a high degree of precision regarding hardware aspects, but less precision regarding software; (ii supplier experience and competence is stressed, but rarely elaborated on in detail; and (iii call for bids vagueness may be used as a lock-in opportunity for suppliers. From the discussion on this, a tentative procurement framework is suggested, aiming on increasing the logical transparency for the procurement of IT.

  14. Methylated genes as new cancer biomarkers

    DEFF Research Database (Denmark)

    Brunner, Nils; Duffy, M.J; Napieralski, R.

    2009-01-01

    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 meas......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...

  15. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Chinese Ed.)

    International Nuclear Information System (INIS)

    2012-01-01

    On the basis of the principles included in the Fundamental Safety Principles, IAEA Safety Standards Series No. SF-1, this Safety Requirements publication establishes requirements applicable to the design of nuclear power plants. It covers the design phase and provides input for the safe operation of the power plant. It elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  16. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (French Ed.)

    International Nuclear Information System (INIS)

    2012-01-01

    On the basis of the principles included in the Fundamental Safety Principles, IAEA Safety Standards Series No. SF-1, this Safety Requirements publication establishes requirements applicable to the design of nuclear power plants. It covers the design phase and provides input for the safe operation of the power plant. It elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  17. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Arabic Ed.)

    International Nuclear Information System (INIS)

    2012-01-01

    On the basis of the principles included in the Fundamental Safety Principles, IAEA Safety Standards Series No. SF-1, this Safety Requirements publication establishes requirements applicable to the design of nuclear power plants. It covers the design phase and provides input for the safe operation of the power plant. It elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  18. PRC2 Is Required to Maintain Expression of the Maternal Gtl2-Rian-Mirg Locus by Preventing De Novo DNA Methylation in Mouse Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Partha Pratim Das

    2015-09-01

    Full Text Available Polycomb Repressive Complex 2 (PRC2 function and DNA methylation (DNAme are typically correlated with gene repression. Here, we show that PRC2 is required to maintain expression of maternal microRNAs (miRNAs and long non-coding RNAs (lncRNAs from the Gtl2-Rian-Mirg locus, which is essential for full pluripotency of iPSCs. In the absence of PRC2, the entire locus becomes transcriptionally repressed due to gain of DNAme at the intergenic differentially methylated regions (IG-DMRs. Furthermore, we demonstrate that the IG-DMR serves as an enhancer of the maternal Gtl2-Rian-Mirg locus. Further analysis reveals that PRC2 interacts physically with Dnmt3 methyltransferases and reduces recruitment to and subsequent DNAme at the IG-DMR, thereby allowing for proper expression of the maternal Gtl2-Rian-Mirg locus. Our observations are consistent with a mechanism through which PRC2 counteracts the action of Dnmt3 methyltransferases at an imprinted locus required for full pluripotency.

  19. Certification of ERDA contractors' packaging with respect to compliance with DOT specification 7A performance requirements

    International Nuclear Information System (INIS)

    Edling, D.A.; Griffin, J.F.

    1975-01-01

    The purpose of this study was to have one ERDA contractor: (1) compile a list of specification packagings, proposed by ERDA contractors, for shipping Type A quantities of radioactive material, and (2) analyze these packages for conformance to Specification 7A requirements. This study was divided into two phases. Phase I provides a report on those packages which could be shown, based on existing test data and engineering analyses, to conform to DOT Specification 7A packaging requirements. The results of Phase I are discussed in detail in the publication, ''Certification of AEC Contractor's Packagings With Respect to Compliance with DOT Specification 7A Performance Requirements -- Phase I Summary Report,'' D. A. Edling, H. E. Meyer and G. L. Phillabaum (Schedule 189C, May 26, 1974). The objectives of Phase II were: (1) identification of packages from Phase I for which available information was not adequate for certification. (Those specification containers used by ERDA contractors and those containers for which adequate information was available for certification are listed in the Phase I summary report.); (2) identification of specific test/engineering analysis data required; (3) generation/procurement of these data; and (4) documentation of study results for use by all ERDA contractors and private industry. The results of Phase II of the study are presented. (U.S.)

  20. Handbook of methods for risk-based analysis of technical specification requirements

    International Nuclear Information System (INIS)

    Samanta, P.K.; Vesely, W.E.

    1994-01-01

    Technical Specifications (TS) requirements for nuclear power plants define the Limiting Conditions for Operation (LCOs) and Surveillance Requirements (SRs) to assure safety during operation. In general, these requirements were based on deterministic analysis and engineering judgments. Experiences with plant operation indicate that some elements of the requirements are unnecessarily restrictive, while others may not be conducive to safety. Improvements in these requirements are facilitated by the availability of plant specific Probabilistic Safety Assessments (PSAs). The use of risk and reliability-based methods to improve TS requirements has gained wide interest because these methods can: Quantitatively evaluate the risk and justify changes based on objective risk arguments; Provide a defensible basis for these requirements for regulatory applications. The US NRC Office of Research is sponsoring research to develop systematic risk-based methods to improve various aspects of TS requirements. The handbook of methods, which is being prepared, summarizes such risk-based methods. The scope of the handbook includes reliability and risk-based methods for evaluating allowed outage times (AOTs), action statements requiring shutdown where shutdown risk may be substantial, surveillance test intervals (STIs), defenses against common-cause failures, managing plant configurations, and scheduling maintenances. For each topic, the handbook summarizes methods of analysis and data needs, outlines the insights to be gained, lists additional references, and presents examples of evaluations

  1. Handbook of methods for risk-based analysis of Technical Specification requirements

    International Nuclear Information System (INIS)

    Samanta, P.K.; Vesely, W.E.

    1993-01-01

    Technical Specifications (TS) requirements for nuclear power plants define the Limiting Conditions for Operation (LCOs) and Surveillance Requirements (SRs) to assure safety during operation. In general, these requirements were based on deterministic analysis and engineering judgments. Experiences with plant operation indicate that some elements of the requirements are unnecessarily restrictive, while others may not be conducive to safety. Improvements in these requirements are facilitated by the availability of plant specific Probabilistic Safety Assessments (PSAs). The use of risk and reliability-based methods to improve TS requirements has gained wide interest because these methods can: quantitatively evaluate the risk impact and justify changes based on objective risk arguments. Provide a defensible basis for these requirements for regulatory applications. The United States Nuclear Regulatory Commission (USNRC) Office of Research is sponsoring research to develop systematic risk-based methods to improve various aspects of TS requirements. The handbook of methods, which is being prepared, summarizes such risk-based methods. The scope of the handbook includes reliability and risk-based methods for evaluating allowed outage times (AOTs), action statements requiring shutdown where shutdown risk may be substantial, surveillance test intervals (STIs), defenses against common-cause failures, managing plant configurations, and scheduling maintenances. For each topic, the handbook summarizes methods of analysis and data needs, outlines the insights to be gained, lists additional references, and presents examples of evaluations

  2. Oligodeoxynucleotides Can Transiently Up- and Downregulate CHS Gene Expression in Flax by Changing DNA Methylation in a Sequence-Specific Manner

    Directory of Open Access Journals (Sweden)

    Magdalena Dzialo

    2017-05-01

    G island. The methylation motifs also led to rearrangement of the nucleosome location. The obtained results suggest high specificity of ODN action and establish a potential valuable alternative for improvement of crops.

  3. The impact of technical specification surveillance requirements and allowable outage times on plant availability

    International Nuclear Information System (INIS)

    Webster, S.A.; Finnicum, D.J.

    1985-01-01

    Surveillances required to be conducted by a plant's Technical Specifications have resulted in plant shutdowns and lost availability. This paper looks at shutdowns which have occurred due to required surveillance testing and insufficient repair time allowed by Technical Specifications. A loss of plant availability of almost 3% per plant year was found for U.S. pressurized water reactors during the five year period, 1979 to 1984. This figure excludes major problems which required plant shutdown whether or not mandated by the Technical Specifications. In addition to their affect on availability, such shutdowns can add to the challenges to plant safety systems and can affect plant aging by increasing the thermal cycles on plant components

  4. Genome-Wide Maps of m6A circRNAs Identify Widespread and Cell-Type-Specific Methylation Patterns that Are Distinct from mRNAs

    Directory of Open Access Journals (Sweden)

    Chan Zhou

    2017-08-01

    Full Text Available N6-methyladenosine (m6A is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ RNAs. We developed a computational pipeline (AutoCirc that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.

  5. A cross-study analysis of prenatal exposures to environmental contaminants and the epigenome: support for stress-responsive transcription factor occupancy as a mediator of gene-specific CpG methylation patterning

    Science.gov (United States)

    Martin, Elizabeth M.; Fry, Rebecca C.

    2016-01-01

    Abstract A biological mechanism by which exposure to environmental contaminants results in gene-specific CpG methylation patterning is currently unknown. We hypothesize that gene-specific CpG methylation is related to environmentally perturbed transcription factor occupancy. To test this hypothesis, a database of 396 genes with altered CpG methylation either in cord blood leukocytes or placental tissue was compiled from 14 studies representing assessments of six environmental contaminants. Subsequently, an in silico approach was used to identify transcription factor binding sites enriched among the genes with altered CpG methylation in relationship to the suite of environmental contaminants. For each study, the sequences of the promoter regions (representing −1000 to +500 bp from the transcription start site) of all genes with altered CpG methylation were analyzed for enrichment of transcription factor binding sites. Binding sites for a total of 56 unique transcription factors were identified to be enriched within the promoter regions of the genes. Binding sites for the Kidney-Enriched Krupple-like Factor 15, a known responder to endogenous stress, were enriched ( P  contaminants. These data support the transcription factor occupancy theory as a potential mechanism underlying environmentally-induced gene-specific CpG methylation. PMID:27066266

  6. Evaluation procedure of software requirements specification for digital I and C of KNGR

    International Nuclear Information System (INIS)

    Lee, Jang Soo; Park, Jong Kyun; Lee, Ki Young; Kim, Jang Yeol; Cheon, Se Woo

    2001-06-01

    The accuracy of the specification of requirements of a digital system is of prime importance to the acceptance and success of the system. The development, use, and regulation of computer systems in nuclear reactor Instrumentation and Control (I and C) systems to enhance reliability and safety is a complex issue. This report is one of a series of reports from the Korean Next Generation Reactor (KNGR) Software Safety Verification and Validation (SSVV) Task, Korea Atomic Energy Research Institute, which investigates different aspects of computer software in reactor I and C systems, and describes the engineering procedures for developing such a software. The purpose of this guideline is to give the software safety evaluator the trail map between the code and standards layer and the design methodology and documents layer for the software important to safety in nuclear power plants. Recently, the requirements specification of safety-critical software systems and safety analysis of them are being recognized as one of the important issues in the software life cycle, and being developed new regulatory positions and standards by the regulatory and the standardization organizations such as IAEA, IEC, and IEEE. We presented the procedure for evaluating the software requirements specifications of the KNGR protection systems. We believe it can be useful for both licenser and licensee to conduct an evaluation of the safety in the requirements phase of developing the software. The guideline consists of the requirements engineering for software of KNGR protection systems in chapter 1, the evaluation checklist of software requirements specification in chapter2.3, and the safety evaluation procedure of KNGR software requirements specification in chapter 2.4

  7. Specification of safety requirements for waste packages with respect to practicable quality control measures

    International Nuclear Information System (INIS)

    Gruendler, D.; Wurtinger, W.

    1987-01-01

    Waste packages for disposal in a repository in the Federal Republic of Germany have to meet safety requirements derived from site specific safety analyses. The examination of the waste packages with regard to compliance with these requirements is the main objective of quality control measures. With respect to quality control the requirements have to be specified in a way that practicable control measures can be applied. This is dealt with for the quality control of the activity inventory and the quality control of the waste form. The paper discusses the determination of the activity of hard-to-measure radionuclides and the specification of safety related requirements for the waste form and the packaging using typical examples

  8. Guidance and methods for satisfying low specific activity material and surface contaminated object regulatory requirements

    International Nuclear Information System (INIS)

    Pope, R.B.; Shappert, L.B.; Michelhaugh, R.D.; Boyle, R.W.; Easton, E.P.; Coodk, J.R.

    1998-01-01

    The U.S. Department of Transportation (DOT) and the U.S. Nuclear Regulatory Commission (NRC) have prepared a comprehensive set of draft guidance for shippers and inspectors to use when applying the newly imposed regulatory requirements for low specific activity (LSA) material and surface contaminated objects (SCOs). These requirements represent significant departures in some areas from the manner in which these materials and objects were regulated by the earlier versions of the regulations. The proper interpretation and application of the regulatory criteria can require a fairly complex set of decisions be made. To assist those trying these regulatory requirements, a detailed set of logic-flow diagrams representing decisions related to multiple factors were prepared and included in the draft report for comment on Categorizing and Transporting Low Specific Activity Materials and Surface Contaminated Objects, (DOT/NRC, 1997). These logic-flow diagrams, as developed, are specific to the U.S. regulations, but were readily adaptable to the IAEA regulations. The diagrams have been modified accordingly and tied directly to specific paragraphs in IAEA Safety Series No. 6. This paper provides the logic-flow diagrams adapted in the IAEA regulations, and demonstrated how these diagrams can be used to assist consignors and inspectors in assessing compliance of shipments with the LSA material and SCO regulatory requirements. (authors)

  9. Satellite-enabled educational services specification and requirements analysis based on user feedback

    OpenAIRE

    Tsekeridou, Sofia; Tiropanis, Thanassis; Rorris, Dimitris; Constantinos, Makropoulos; Serif, Tacha; Stergioulas, Lampros

    2008-01-01

    Advanced tele-education services provision in remote geographically dispersed user communities (such as agriculture and maritime), based on the specific needs and requirements of such communities, implies significant infrastructural and broadband connectivity requirements for rich media, timely and quality-assured content delivery and interactivity. The solution to broadband access anywhere is provided by satellite-enabled communication infrastructures. This paper aims to present such satelli...

  10. Surveillance Analysis Computer System (SACS): Software requirements specification (SRS). Revision 2

    International Nuclear Information System (INIS)

    Glasscock, J.A.

    1995-01-01

    This document is the primary document establishing requirements for the Surveillance Analysis Computer System (SACS) database, an Impact Level 3Q system. SACS stores information on tank temperatures, surface levels, and interstitial liquid levels. This information is retrieved by the customer through a PC-based interface and is then available to a number of other software tools. The software requirements specification (SRS) describes the system requirements for the SACS Project, and follows the Standard Engineering Practices (WHC-CM-6-1), Software Practices (WHC-CM-3-10) and Quality Assurance (WHC-CM-4-2, QR 19.0) policies

  11. Identification of Differentially Methylated Sites with Weak Methylation Effects

    Directory of Open Access Journals (Sweden)

    Hong Tran

    2018-02-01

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

  12. The applies specific technical requirements to the services of radiological protection according to the 17025

    Energy Technology Data Exchange (ETDEWEB)

    Marrero Garcia, M.; Fernandez Gomez, I. M.; Prendes Alonso, M.; Molina Perez, D.

    2004-07-01

    The employment in the Center of Radiation Protection and Hygiene (CRPH) of Cuba, of the standard 17025 for the administration of the quality of the calibration laboratories and test that offer services of radiological protection requires of a technical complement to really determine the technical competence. In the standard 17025, the specific requirements, are mentioned in the annex B (with informative character), where it points out that: {sup T}he requirements specified in the standard 17025 are established in general terms and, although they can be applied to all the test laboratories and applications, they could be necessary applications{sup .} In the CRPH these applications are translated in indispensable technical requirements to evaluate the performance of the services of radiological protection that offers, specifically those of personal monitoring, the calibration of instruments and the metrology of radionuclides. Although the standard 17025 is quite rigorous in the evaluation of technical requirements, to have specific materials not serves alone of tool to the laboratories, but also for the body of accreditation . In the CRPH the permanent employment of this tool has not constituted a practice, but had been good before our clients certain nonconformities to evaluate the performance of the laboratory and to achieve the recovery of the trust in its results. With this work we seek to expose some of these experiences. The experiences of the application of some of the technical requirements, in our case, they have been applied to the service of personal monitoring that offers the Laboratory of Personal Dosimetry. This laboratory is accredited by the National Accreditation Board of the Republic of Cuba (NABC) and besides fulfilling that settled down in 17025, the Approaches of Accreditation for test laboratories and calibration and it should also implant that settled down as specific technical requirements for the recognition of technical competence. (Author

  13. Challenges in balancing the amount of solution information in requirement specifications for embedded products

    DEFF Research Database (Denmark)

    Savolainen, Juha; Hauksdóttir, Dagný; Mannion, Mike

    2013-01-01

    customers have such intimate knowledge of their products that their requirements tend to be very specific, and if the customer knows the exact solution needed that naturally will reduce the cost of the requirements elicitation as well as design activities. Practitioners are challenged to understand when......Requirements are traditionally viewed as being free of the details of an envisioned solution and specified using purely problem domain entities. Preventing premature design in the requirements permits the available design space not to be restricted too early which might inhibit innovative designs....... In practice, on many industrial projects, separating the problem and solution domain entities can be difficult, and arguably there are benefits for not doing so. Many customers feel more confident describing their requirements, often as the difference between the existing products and their needs, some...

  14. Identification of factors required for m6 A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.

    Science.gov (United States)

    Růžička, Kamil; Zhang, Mi; Campilho, Ana; Bodi, Zsuzsanna; Kashif, Muhammad; Saleh, Mária; Eeckhout, Dominique; El-Showk, Sedeer; Li, Hongying; Zhong, Silin; De Jaeger, Geert; Mongan, Nigel P; Hejátko, Jan; Helariutta, Ykä; Fray, Rupert G

    2017-07-01

    N6-adenosine methylation (m 6 A) of mRNA is an essential process in most eukaryotes, but its role and the status of factors accompanying this modification are still poorly understood. Using combined methods of genetics, proteomics and RNA biochemistry, we identified a core set of mRNA m 6 A writer proteins in Arabidopsis thaliana. The components required for m 6 A in Arabidopsis included MTA, MTB, FIP37, VIRILIZER and the E3 ubiquitin ligase HAKAI. Downregulation of these proteins led to reduced relative m 6 A levels and shared pleiotropic phenotypes, which included aberrant vascular formation in the root, indicating that correct m 6 A methylation plays a role in developmental decisions during pattern formation. The conservation of these proteins amongst eukaryotes and the demonstration of a role in writing m 6 A for the E3 ubiquitin ligase HAKAI is likely to be of considerable relevance beyond the plant sciences. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  15. Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.

    Science.gov (United States)

    Wirebrand, Lisa; Madhushani, Anjana W K; Irie, Yasuhiko; Shingler, Victoria

    2018-01-01

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here, we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. 24 CFR 200.943 - Supplementary specific requirements under the HUD building product standards and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standards and certification program for the grademarking of lumber. 200... Supplementary specific requirements under the HUD building product standards and certification program for the... compliance with the U.S. Department of Commerce Voluntary Product Standard PS 20-94 American Softwood Lumber...

  17. 24 CFR 200.952 - Supplementary specific requirements under the HUD building product standards and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standards and certification program for particleboard interior stair... Supplementary specific requirements under the HUD building product standards and certification program for... forth in § 200.935(d)(6) concerning labeling of a product, the administrator's validation mark and the...

  18. 24 CFR 200.954 - Supplementary specific requirements under the HUD building product standard and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standard and certification program for construction adhesives for wood... § 200.954 Supplementary specific requirements under the HUD building product standard and certification...) concerning labeling of a product, the administrator's validation mark and the manufacturer's certification of...

  19. Development Modules for Specification of Requirements for a System of Verification of Parallel Algorithms

    Directory of Open Access Journals (Sweden)

    Vasiliy Yu. Meltsov

    2012-05-01

    Full Text Available This paper presents the results of the development of one of the modules of the system verification of parallel algorithms that are used to verify the inference engine. This module is designed to build the specification requirements, the feasibility of which on the algorithm is necessary to prove (test.

  20. The impact of customer-specific requirements on supply chain management

    Directory of Open Access Journals (Sweden)

    Hubert I.P. Conceivious

    2010-11-01

    Full Text Available The complexities of being a supplier to motorcar manufacturers, also known as original equipment manufacturers (OEMs, provide an array of challenges to component manufacturers. Customer-specific requirements (CSRs add to the convolutions of a supplier’s quality management systems when producing components for the various motor manufacturers. The catalytic converter industry (CCI forms part of the component supply chain in the motor industry. The CCI consists of a plethora of suppliers to produce the catalytic converter. This paper focuses on three of the five main suppliers, namely the ‘monolith substrate manufacturers’, the ‘coaters’, and the ‘canners’. Most OEMs required that critical and strategic suppliers should be ISO/TS 16949:2009 certified. ISO/TS 16949:2009 refers to an internationally recognised specification, specifically adapted for the motor industry. The specification indicates the minimum requirements and also makes provision for additional requirements known as CSRs that can be specified by the OEM.

  1. 40 CFR 270.24 - Specific part B information requirements for process vents.

    Science.gov (United States)

    2010-07-01

    ... incinerator, flare, boiler, process heater, condenser, or carbon adsorption system to comply with the... each compliance test required by § 264.1033(k). (3) A design analysis, specifications, drawings... texts acceptable to the Regional Administrator that present basic control device information. The design...

  2. Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α,24(R), 25-trihydroxy-[26,27-methyl-3H]-vitamin D3

    International Nuclear Information System (INIS)

    Chandler, J.S.; Pike, J.W.; Haussler, M.R.

    1982-01-01

    A procedure for the biosynthesis and purification of 1α,24(R),25-trihydroxy[26,27-methyl- 3 H]-vitamin D 3 (1,24,25-(OH) 3 [ 3 H]D 3 ) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1α,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) was used for C-24-hydroxylation of 1α,25-dihydroxy[26,27-methyl- 3 H]-vitamin D 3 (1,25-(OH) 2 [ 3 H]D 3 ), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24,25-(OH) 3 [ 3 H]D 3 with a specific radioactivity equivalent to the initial substrate (166 Ci/mmol). The authenticity of the generated metabolite was assessed by co-migration with synthetic 1,24,25-(OH) 3 D 3 on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH) 3 D 3 for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH) 2 D 3 for hormone binding sites. Further analysis of 1,24,25-(OH) 3 D 3 -receptor interaction reveals a high-affinity, saturable binding with an apparent Ksub(d) of 2.2 x 10 -9 M. These studies demonstrate that although slightly less active than 1,25-(OH) 2 D 3 , 1,24,25-(OH) 3 D 3 is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance. (author)

  3. Sharing data between mobile devices, connected vehicles, and infrastructure task 3: system requirements specifications (SyRS) final.

    Science.gov (United States)

    2016-07-14

    This report describes the system requirements specifications (SyRS) for the use of mobile devices in a connected vehicle environment. Specifically, it defines the different types of requirements (functional, interface, performance, security, data, an...

  4. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Spanish Edition)

    International Nuclear Information System (INIS)

    2012-01-01

    This publication is a revision of Safety Requirements No. NS-R-1, Safety of Nuclear Power Plants: Design. It establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in the design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  5. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Russian Edition)

    International Nuclear Information System (INIS)

    2012-01-01

    This publication is a revision of Safety Requirements No. NS-R-1, Safety of Nuclear Power Plants: Design. It establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in the design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  6. A PLC generic requirements and specification for safety-related applications in nuclear power plants

    International Nuclear Information System (INIS)

    Han, Jea Bok; Lee, C. K.; Lee, D. Y.

    2001-12-01

    This report presents the requirements and specification to be applied to the generic qualification of programmable Logic Controller(PLC), which is being developed as part of the KNICS project, 'Development of the Digital Reactor Safety Systems' of which purpose is the application to safety-related instrumentation and control systems in nuclear power plants. This report defines the essential and critical characteristics that shall be included as part of a PLC design for safety-related application. The characteristics include performance, reliability, accuracy, the overall response time from an input to the PLC exceeding it trip condition to the resulting outputs, and the specification of processors and memories in digital controller. It also specifies the quality assurance process for software development, dealing with executive software, firmware, application software tools for developing the application software, and human machine interface(HMI). In addition, this report reviews the published standards and guidelines that are required for the PLC development and the quality assurance processes such as environment requirements, seismic withstand requirements, EMI/RFI withstand requirements, and isolation test

  7. Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

    Science.gov (United States)

    Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

    2016-09-01

    Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

  8. A discussion of higher order software concepts as they apply to functional requirements and specifications. [space shuttles and guidance

    Science.gov (United States)

    Hamilton, M.

    1973-01-01

    The entry guidance software functional requirements (requirements design phase), its architectural requirements (specifications design phase), and the entry guidance software verified code are discussed. It was found that the proper integration of designs at both the requirements and specifications levels are of high priority consideration.

  9. Requirement for specific gravity and creatinine adjustments for urinary steroids and luteinizing hormone concentrations in adolescents.

    Science.gov (United States)

    Singh, Gurmeet K S; Balzer, Ben W R; Desai, Reena; Jimenez, Mark; Steinbeck, Katharine S; Handelsman, David J

    2015-11-01

    Urinary hormone concentrations are often adjusted to correct for hydration status. We aimed to determine whether first morning void urine hormones in growing adolescents require adjustments and, if so, whether urinary creatinine or specific gravity are better adjustments. The study population was adolescents aged 10.1 to 14.3 years initially who provided fasting morning blood samples at 0 and 12 months (n = 343) and first morning urine every three months (n = 644). Unadjusted, creatinine and specific gravity-adjusted hormonal concentrations were compared by Deming regression and Bland-Altman analysis and grouped according to self-rated Tanner stage or chronological age. F-ratios for self-rated Tanner stages and age groups were used to compare unadjusted and adjusted hormonal changes in growing young adolescents. Correlations of paired serum and urinary hormonal concentration of unadjusted and creatinine and specific gravity-adjusted were also compared. Fasting first morning void hormone concentrations correlated well and were unbiased between unadjusted or adjusted by either creatinine or specific gravity. Urine creatinine concentration increases with Tanner stages, age and male gender whereas urine specific gravity was not influenced by Tanner stage, age or gender. Adjustment by creatinine or specific gravity of urinary luteinizing hormone, estradiol, testosterone, dihydrotestosterone and dehydroepiandrosterone concentrations did not improve correlation with paired serum concentrations. Urine steroid and luteinizing hormone concentrations in first morning void samples of adolescents are not significantly influenced by hydration status and may not require adjustments; however, if desired, both creatinine and specific gravity adjustments are equally suitable. © The Author(s) 2015.

  10. Testing Requirements to Manage Data Exchange Specifications in Enterprise Integration - A Schema Design Quality Focus.

    Energy Technology Data Exchange (ETDEWEB)

    Kulvatunyou, Boonserm [ORNL; Ivezic, Nenad [ORNL; Buhwan, Jeong [POSTECH University, South Korea

    2004-07-01

    In this paper, we describe the requirements to test W3C XML Schema usage when defining message schemas for data exchange in any large and evolving enterprise integration project. We then decompose the XML Schema testing into four (4) aspects including the message schema conformance to the XML Schema specification grammar, the message schema conformance to the XML Schema specification semantics, the message schema conformance to design quality testing, and canonical semantics testing of the message schema. We describe these four testing aspects in some detail and point to other related efforts. We further focus to provide some technical details for the message schema design quality testing. As a future work, we describe the requirements for canonical semantics testing and potential solution approaches. Finally, we describe an implementation architecture for the message schema design quality testing.

  11. Statistical Study to Check the Conformity of Aggregate in Kirkuk City to Requirement of Iraqi Specification

    OpenAIRE

    Ammar Saleem Khazaal; Nizar N Ismeel; Abdel fattah K. Hussein

    2018-01-01

    This research reviews a statistical study to check the conformity of aggregates (Coarse and Fine) was used in Kirkuk city to the requirements of the Iraqi specifications. The data of sieve analysis (215 samples) of aggregates being obtained from of National Central Construction Laboratory and Technical College Construction Laboratory in Kirkuk city have analyzed using the statistical program SAS. The results showed that 5%, 17%, and 18% of fine aggregate samples are passing sieve sizes 10 mm,...

  12. Appraisal of work ability in relation to job-specific health requirements in ambulance workers.

    Science.gov (United States)

    van Schaaijk, A; Boschman, J S; Frings-Dresen, M H W; Sluiter, J K

    2017-01-01

    To gain insight into which job-specific health requirements relate to work ability, the following two research questions were formulated: Which job-specific health requirements are associated with the appraisal of work ability in ambulance drivers and paramedics? How are appraisals of physical and mental work ability associated with the appraisal of overall work ability in ambulance drivers and paramedics? Workers Health Surveillance cross-sectional data of 506 ambulance workers (236 drivers and 270 paramedics) were used. The tests for specific job requirements were divided into six categories. Work ability was appraised as overall, physical and mental/emotional. Multiple linear stepwise regression analyses were used to model the associations. Outcomes in 'raised alertness and judgment ability' (R 2  = 0.09), 'job-specific physical abilities' (R 2  = 0.10) and 'emotional peak load' (R 2  = 0.07) significantly explained appraised overall, physical and mental/emotional work ability. Physical and mental/emotional work ability together explained 48.3% of the variance of overall work ability. The explained variance by physical and mental/emotional work ability was almost 4% higher in drivers than in paramedics. Overall work ability was significantly explained by outcomes in 'raised alertness and judgment ability' and 'emotional peak load.' Physical work ability was significantly explained by 'job-specific physical abilities' and 'raised alertness and judgment ability' outcomes, while 'emotional peak load' and 'raised alertness and judgment ability' outcomes significantly explained mental/emotional work ability. Physical and mental/emotional work ability explains the same proportion of variance in overall work ability.

  13. Effect of maternal and post weaning folate supply on gene-specific DNA methylation in the small intestine of weaning and adult Apc+/Min and wild type mice.

    Directory of Open Access Journals (Sweden)

    Jill Ann Mckay

    2011-05-01

    Full Text Available Increasing evidence supports the developmental origins of adult health and disease hypothesis which argues for a causal relationship between adverse early life nutrition and increased disease risk in adulthood. Modulation of epigenetic marks, e.g. DNA methylation and consequential altered gene expression, has been proposed as a mechanism mediating these effects. Via its role as a methyl donor, dietary folate supply may influence DNA methylation. As aberrant methylation is an early event in colorectal cancer (CRC pathogenesis, we hypothesised low maternal and/or post-weaning folate intake may influence methylation of genes involved in CRC development. We investigated the effects of maternal folate depletion during pregnancy and lactation on selected gene methylation in the small intestine (SI of wild type (WT and Apc+/Min mice at weaning and as adults. We also investigated the effects of folate depletion post-weaning on gene methylation in adult mice. Female C57Bl6/J mice were fed low or normal folate diets from mating with Apc+/Min males to the end of lactation. A sub set of offspring were killed at weaning. Remaining offspring were weaned on to low or normal folate diets, resulting in 4 treatment groups of Apc+/Min and WT mice. p53 was more methylated in weaning and adult WT compared with Apc+/Min mice (p>0.001. Igf2 and Apc were hypermethylated in adult Apc+/Mi n compared with WT mice (p=0.004 & p=0.012 respectively. Low maternal folate reduced p53 methylation in adults (p=0.04. Low post-weaning folate increased Apc methylation in Apc+/Min mice only (p=0.008 for interaction. These observations demonstrate that folate depletion in early life can alter epigenetic marks in a gene specific manner. Also, the differential effects of altered folate supply on DNA methylation in WT and Apc+/Min mice suggest that genotype may modulate epigenetic responses to environmental cues and may have implications for the development of personalised nutrition.

  14. AMTD: update of engineering specifications derived from science requirements for future UVOIR space telescopes

    Science.gov (United States)

    Stahl, H. Philip; Postman, Marc; Mosier, Gary; Smith, W. Scott; Blaurock, Carl; Ha, Kong; Stark, Christopher C.

    2014-08-01

    The Advance Mirror Technology Development (AMTD) project is in Phase 2 of a multiyear effort, initiated in FY12, to mature by at least a half TRL step six critical technologies required to enable 4 meter or larger UVOIR space telescope primary mirror assemblies for both general astrophysics and ultra-high contrast observations of exoplanets. AMTD uses a science-driven systems engineering approach. We mature technologies required to enable the highest priority science AND provide a high-performance low-cost low-risk system. To give the science community options, we are pursuing multiple technology paths. A key task is deriving engineering specifications for advanced normal-incidence monolithic and segmented mirror systems needed to enable both general astrophysics and ultra-high contrast observations of exoplanets missions as a function of potential launch vehicles and their mass and volume constraints. A key finding of this effort is that the science requires an 8 meter or larger aperture telescope.

  15. DNA-methylation dependent regulation of embryo-specific 5S ribosomal DNA cluster transcription in adult tissues of sea urchin Paracentrotus lividus.

    Science.gov (United States)

    Bellavia, Daniele; Dimarco, Eufrosina; Naselli, Flores; Caradonna, Fabio

    2013-10-01

    We have previously reported a molecular and cytogenetic characterization of three different 5S rDNA clusters in the sea urchin Paracentrotus lividus and recently, demonstrated the presence of high heterogeneity in functional 5S rRNA. In this paper, we show some important distinctive data on 5S rRNA transcription for this organism. Using single strand conformation polymorphism (SSCP) analysis, we demonstrate the existence of two classes of 5S rRNA, one which is embryo-specific and encoded by the smallest (700 bp) cluster and the other which is expressed at every stage and encoded by longer clusters (900 and 950 bp). We also demonstrate that the embryo-specific class of 5S rRNA is expressed in oocytes and embryonic stages and is silenced in adult tissue and that this phenomenon appears to be due exclusively to DNA methylation, as indicated by sensitivity to 5-azacytidine, unlike Xenopus where this mechanism is necessary but not sufficient to maintain the silenced status. © 2013 Elsevier Inc. All rights reserved.

  16. Identifying context-specific competencies required by community Australian Football sports trainers.

    Science.gov (United States)

    Donaldson, Alex; Finch, Caroline F

    2012-08-01

    First-aid is a recommended injury prevention and risk management strategy in community sport; however, little is known about the sport-specific competencies required by first-aid providers. To achieve expert consensus on the competencies required by community Australian Football (community-AF) sports trainers. A three-round online Delphi process. Community-AF. 16 Australian sports first-aid and community-AF experts. Rating of competencies as either 'essential', 'expected', 'ideal' or 'not required'. Results After Round 3, 47 of the 77 (61%) competencies were endorsed as 'essential' or 'expected' for a sports trainer to effectively perform the activities required to the standards expected at a community-AF club by ≥75% of experts. These competencies covered: the role of the sports trainer; the responsibilities of the sports trainer; emergency management; injury and illness assessment and immediate management; taping; and injury prevention and risk management. Four competencies (5%) were endorsed as 'ideal' or 'not required' by ≥85% of experts and were excluded from further consideration. The 26 competencies where consensus was not reached were retained as second-tier, optional competencies. Sports trainers are important members of on-field first-aid teams, providing support to both injured players and other sports medicine professionals. The competencies identified in this study provide the basis of a proposed two-tiered community-AF-specific sports trainer education structure that can be implemented by the peak sports body. This includes six mandatory modules, relating to the 'required' competencies, and a further six optional modules covering competencies on which consensus was not reached.

  17. Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing.

    Science.gov (United States)

    Kenyon, Jonathan; Nickel-Meester, Gabrielle; Qing, Yulan; Santos-Guasch, Gabriela; Drake, Ellen; PingfuFu; Sun, Shuying; Bai, Xiaodong; Wald, David; Arts, Eric; Gerson, Stanton L

    Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1 , an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylation upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from -938 to -337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1 . We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34 + selected hematopoietic stem and progenitor cells.

  18. DNA Methylation in Peripheral Blood Cells of Pigs Cloned by Somatic Cell Nuclear Transfer

    DEFF Research Database (Denmark)

    Gao, Fei; Li, Shengting; Lin, Lin

    2011-01-01

    To date, the genome-wide DNA methylation status of cloned pigs has not been investigated. Due to the relatively low success rate of pig cloning by somatic cell nuclear transfer, a better understanding of the epigenetic reprogramming and the global methylation patterns associated with development...... in cloned pigs is required. In this study we applied methylation-specific digital karyotyping tag sequencing by Solexa technology and investigated the genome-wide DNA methylation profiles of peripheral blood cells in cloned pigs with normal phenotypes in comparison with their naturally bred controls....... In the result, we found that globally there was no significant difference of DNA methylation patterns between the two groups. Locus-specifically, some genes involved in embryonic development presented a generally increased level of methylation. Our findings suggest that in cloned pigs with normal phenotypes...

  19. No difference in the frequency of locus-specific methylation in the peripheral blood DNA of women diagnosed with breast cancer and age-matched controls

    DEFF Research Database (Denmark)

    Wojdacz, Tomasz K; Thestrup, Britta Boserup; Cold, Søren

    2011-01-01

    with no signs of breast cancer. No significant differences in the frequency of methylation of the above genes were found between cases and controls in our study. Hence, testing for the presence of methylation of cancer-related genes in PBL DNA from women diagnosed with sporadic breast cancer and classified...... might predispose for cancer development. Here, we have used the methlyation-sensitive high-resolution melting approach to examine the methylation status of the BRCA1, BRCA2, APC, RASSF1A and RARβ2 genes in PBLs of a group of women diagnosed with breast cancer, and an age-matched control group......, to the pathology of different diseases, remains open. Recently, a number of studies addressed the question of the prevalence of aberrant methylation of cancer-related genes in peripheral blood leukocyte (PBL) DNA and indicated a strong possibility that the presence of constitutional methylation of different genes...

  20. An effective technique for the software requirements analysis of NPP safety-critical systems, based on software inspection, requirements traceability, and formal specification

    International Nuclear Information System (INIS)

    Koo, Seo Ryong; Seong, Poong Hyun; Yoo, Junbeom; Cha, Sung Deok; Yoo, Yeong Jae

    2005-01-01

    A thorough requirements analysis is indispensable for developing and implementing safety-critical software systems such as nuclear power plant (NPP) software systems because a single error in the requirements can generate serious software faults. However, it is very difficult to completely analyze system requirements. In this paper, an effective technique for the software requirements analysis is suggested. For requirements verification and validation (V and V) tasks, our technique uses software inspection, requirement traceability, and formal specification with structural decomposition. Software inspection and requirements traceability analysis are widely considered the most effective software V and V methods. Although formal methods are also considered an effective V and V activity, they are difficult to use properly in the nuclear fields as well as in other fields because of their mathematical nature. In this work, we propose an integrated environment (IE) approach for requirements, which is an integrated approach that enables easy inspection by combining requirement traceability and effective use of a formal method. The paper also introduces computer-aided tools for supporting IE approach for requirements. Called the nuclear software inspection support and requirements traceability (NuSISRT), the tool incorporates software inspection, requirement traceability, and formal specification capabilities. We designed the NuSISRT to partially automate software inspection and analysis of requirement traceability. In addition, for the formal specification and analysis, we used the formal requirements specification and analysis tool for nuclear engineering (NuSRS)

  1. The NIMA Kinase Is Required To Execute Stage-Specific Mitotic Functions after Initiation of Mitosis

    Science.gov (United States)

    Govindaraghavan, Meera; Lad, Alisha A.

    2014-01-01

    The G2-M transition in Aspergillus nidulans requires the NIMA kinase, the founding member of the Nek kinase family. Inactivation of NIMA results in a late G2 arrest, while overexpression of NIMA is sufficient to promote mitotic events independently of cell cycle phase. Endogenously tagged NIMA-GFP has dynamic mitotic localizations appearing first at the spindle pole body and then at nuclear pore complexes before transitioning to within nuclei and the mitotic spindle and back at the spindle pole bodies at mitotic exit, suggesting that it functions sequentially at these locations. Since NIMA is indispensable for mitotic entry, it has been difficult to determine the requirement of NIMA for subaspects of mitosis. We show here that when NIMA is partially inactivated, although mitosis can be initiated, a proportion of cells fail to successfully generate two daughter nuclei. We further define the mitotic defects to show that normal NIMA function is required for the formation of a bipolar spindle, nuclear pore complex disassembly, completion of chromatin segregation, and the normal structural rearrangements of the nuclear envelope required to generate two nuclei from one. In the remaining population of cells that enter mitosis with inadequate NIMA, two daughter nuclei are generated in a manner dependent on the spindle assembly checkpoint, indicating highly penetrant defects in mitotic progression without sufficient NIMA activity. This study shows that NIMA is required not only for mitotic entry but also sequentially for successful completion of stage-specific mitotic events. PMID:24186954

  2. Disease specific stress of tumor patients at the beginning of radiotherapy. Effect on psychosocial support requirement

    International Nuclear Information System (INIS)

    Sehlen, S.; Hollenhorst, H.; Schymura, B.; Firsching, M.; Duehmke, E.; Herschbach, P.

    2001-01-01

    Purpose: Radiotherapy brings a tumor patient into a special life situation in which different variables play a role of often unknown importance. The goal of this study was to investigate disease specific stress of tumor patients at the beginning of radiotherapy with established psychodiagnostic questionnaires and to evaluate the effect on psychosocial support requirement in order to reduce stress and to improve quality of life and compliance during radiotherapeutical treatment. Patients and Methods: 732 patients were screened, of whom 446 (60.9%) fulfilled the criteria for inclusion (refusals 21.0%, low Karnofsky performance status 6.6%, management problems 3.4%, language barriers 3.0%, cognitive restrictions 2.6%, death 2.5%). Disease specific aspects of stress in the questionnaire (Fragebogen zur Belastung von Krebspatienten, FBK), life situation (LS) and self-defined care requirements (BB) were self-rated by patients with different tumor types before radiotherapy. Medical and sociodemographic data were also documented. We investigated 446 patients (262 male, 184 female; median age 60.0 years) with different diagnoses. Results: Stress was observed mainly due to reduction of efficiency, anxiety and pain on the subscales. Women had a significant higher stress on subscales of pain (p=0.016) and anxiety (p=0.009), patients younger than 45 years in the subscale information (p=0.002) and patients older than 45 and younger than 60 years in the subscale anxiety (p=0.002) and the total score (p=0.003). Patients with mamma carcinoma had the highest stress. The maximum percentages of patients under high stress were found for the subscales of efficiency (43%) and anxiety (40%). The support requirement was characterized by the need of more medical information and dialogue with the doctor. We saw a significant correlation of high stress and high care requirement. Conclusions: Psychosocial support should be founded on psychosocial stress diagnostic and self-defined care

  3. HPV specific testing: a requirement for oropharyngeal squamous cell carcinoma patients.

    Science.gov (United States)

    Robinson, Max; Schache, Andrew; Sloan, Philip; Thavaraj, Selvam

    2012-07-01

    Human papillomavirus (HPV) testing is now recommended as part of the work up for patients with oropharyngeal squamous cell carcinoma (OPSCC) and those patients with cervical lymph node metastasis of unknown origin. The laboratory testing strategy should accurately assess the presence or absence of oncogenic HPV infection in routinely collected tumour samples that are subject to standard fixation protocols, alcohol-fixed cytological preparations and formalin-fixed tissue samples. The HPV status should correlate with biologically relevant outcome measures such as overall, disease-specific and disease-free survival. Whilst increased expression of p16 by immunohistochemistry is considered to be a surrogate marker of oncogenic HPV infection and is a validated independent prognostic biomarker, only HPV specific tests provide definitive evidence of the aetiological agent. We provide an overview of HPV testing in OPSCC, justifying the use of HPV specific tests. We examine the analytical accuracy of HPV specific tests against the 'reference' test--high risk HPV mRNA in fresh tissue--and contrast this with the performance of p16 immunohistochemistry as a stand alone test. We highlight the added value of HPV specific tests in prognostication, clinical trial design, and population-based disease surveillance. We consider that HPV specific testing is the starting point for developing increasingly informative biomarker panels in the context of 'stratified medicine'. We briefly frame test information in the context of disclosure of HPV status to patients. We conclude that only a testing strategy that includes HPV specific tests can deliver more effective care for patients with OPSCC. The international head and neck oncology community should work together to clearly define the minimum requirements for assigning a diagnosis of HPV-related OPSCC in order to ensure consistent reporting of this emerging and increasingly prevalent disease.

  4. Poliovirus intrahost evolution is required to overcome tissue-specific innate immune responses.

    Science.gov (United States)

    Xiao, Yinghong; Dolan, Patrick Timothy; Goldstein, Elizabeth Faul; Li, Min; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

    2017-08-29

    RNA viruses, such as poliovirus, have a great evolutionary capacity, allowing them to quickly adapt and overcome challenges encountered during infection. Here we show that poliovirus infection in immune-competent mice requires adaptation to tissue-specific innate immune microenvironments. The ability of the virus to establish robust infection and virulence correlates with its evolutionary capacity. We further identify a region in the multi-functional poliovirus protein 2B as a hotspot for the accumulation of minor alleles that facilitate a more effective suppression of the interferon response. We propose that population genetic dynamics enables poliovirus spread between tissues through optimization of the genetic composition of low frequency variants, which together cooperate to circumvent tissue-specific challenges. Thus, intrahost virus evolution determines pathogenesis, allowing a dynamic regulation of viral functions required to overcome barriers to infection.RNA viruses, such as polioviruses, have a great evolutionary capacity and can adapt quickly during infection. Here, the authors show that poliovirus infection in mice requires adaptation to innate immune microenvironments encountered in different tissues.

  5. Hazard Analysis of Software Requirements Specification for Process Module of FPGA-based Controllers in NPP

    Energy Technology Data Exchange (ETDEWEB)

    Jung; Sejin; Kim, Eui-Sub; Yoo, Junbeom [Konkuk University, Seoul (Korea, Republic of); Keum, Jong Yong; Lee, Jang-Soo [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2016-10-15

    Software in PLC, FPGA which are used to develop I and C system also should be analyzed to hazards and risks before used. NUREG/CR-6430 proposes the method for performing software hazard analysis. It suggests analysis technique for software affected hazards and it reveals that software hazard analysis should be performed with the aspects of software life cycle such as requirements analysis, design, detailed design, implements. It also provides the guide phrases for applying software hazard analysis. HAZOP (Hazard and operability analysis) is one of the analysis technique which is introduced in NUREG/CR-6430 and it is useful technique to use guide phrases. HAZOP is sometimes used to analyze the safety of software. Analysis method of NUREG/CR-6430 had been used in Korea nuclear power plant software for PLC development. Appropriate guide phrases and analysis process are selected to apply efficiently and NUREG/CR-6430 provides applicable methods for software hazard analysis is identified in these researches. We perform software hazard analysis of FPGA software requirements specification with two approaches which are NUREG/CR-6430 and HAZOP with using general GW. We also perform the comparative analysis with them. NUREG/CR-6430 approach has several pros and cons comparing with the HAZOP with general guide words and approach. It is enough applicable to analyze the software requirements specification of FPGA.

  6. RET Functions as a Dual-Specificity Kinase that Requires Allosteric Inputs from Juxtamembrane Elements

    Directory of Open Access Journals (Sweden)

    Iván Plaza-Menacho

    2016-12-01

    Full Text Available Receptor tyrosine kinases exhibit a variety of activation mechanisms despite highly homologous catalytic domains. Such diversity arises through coupling of extracellular ligand-binding portions with highly variable intracellular sequences flanking the tyrosine kinase domain and specific patterns of autophosphorylation sites. Here, we show that the juxtamembrane (JM segment enhances RET catalytic domain activity through Y687. This phospho-site is also required by the JM region to rescue an otherwise catalytically deficient RET activation-loop mutant lacking tyrosines. Structure-function analyses identified interactions between the JM hinge, αC helix, and an unconventional activation-loop serine phosphorylation site that engages the HRD motif and promotes phospho-tyrosine conformational accessibility and regulatory spine assembly. We demonstrate that this phospho-S909 arises from an intrinsic RET dual-specificity kinase activity and show that an equivalent serine is required for RET signaling in Drosophila. Our findings reveal dual-specificity and allosteric components for the mechanism of RET activation and signaling with direct implications for drug discovery.

  7. The Extent to Which Methyl Salicylate Is Required for Signaling Systemic Acquired Resistance Is Dependent on Exposure to Light after Infection1[OA

    Science.gov (United States)

    Liu, Po-Pu; von Dahl, Caroline C.; Klessig, Daniel F.

    2011-01-01

    Systemic acquired resistance (SAR) is a state of heightened defense to a broad spectrum of pathogens that is activated throughout a plant following local infection. Development of SAR requires the translocation of one or more mobile signals from the site of infection through the vascular system to distal (systemic) tissues. The first such signal identified was methyl salicylate (MeSA) in tobacco (Nicotiana tabacum). Subsequent studies demonstrated that MeSA also serves as a SAR signal in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum). By contrast, another study suggested that MeSA is not required for SAR in Arabidopsis and raised questions regarding its signaling role in tobacco. Differences in experimental design, including the developmental age of the plants, the light intensity, and/or the strain of bacterial pathogen, were proposed to explain these conflicting results. Here, we demonstrate that the length of light exposure that plants receive after the primary infection determines the extent to which MeSA is required for SAR signaling. When the primary infection occurred late in the day and as a result infected plants received very little light exposure before entering the night/dark period, MeSA and its metabolizing enzymes were essential for SAR development. In contrast, when infection was done in the morning followed by 3.5 h or more of exposure to light, SAR developed in the absence of MeSA. However, MeSA was generally required for optimal SAR development. In addition to resolving the conflicting results concerning MeSA and SAR, this study underscores the importance of environmental factors on the plant’s response to infection. PMID:22021417

  8. The extent to which methyl salicylate is required for signaling systemic acquired resistance is dependent on exposure to light after infection.

    Science.gov (United States)

    Liu, Po-Pu; von Dahl, Caroline C; Klessig, Daniel F

    2011-12-01

    Systemic acquired resistance (SAR) is a state of heightened defense to a broad spectrum of pathogens that is activated throughout a plant following local infection. Development of SAR requires the translocation of one or more mobile signals from the site of infection through the vascular system to distal (systemic) tissues. The first such signal identified was methyl salicylate (MeSA) in tobacco (Nicotiana tabacum). Subsequent studies demonstrated that MeSA also serves as a SAR signal in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum). By contrast, another study suggested that MeSA is not required for SAR in Arabidopsis and raised questions regarding its signaling role in tobacco. Differences in experimental design, including the developmental age of the plants, the light intensity, and/or the strain of bacterial pathogen, were proposed to explain these conflicting results. Here, we demonstrate that the length of light exposure that plants receive after the primary infection determines the extent to which MeSA is required for SAR signaling. When the primary infection occurred late in the day and as a result infected plants received very little light exposure before entering the night/dark period, MeSA and its metabolizing enzymes were essential for SAR development. In contrast, when infection was done in the morning followed by 3.5 h or more of exposure to light, SAR developed in the absence of MeSA. However, MeSA was generally required for optimal SAR development. In addition to resolving the conflicting results concerning MeSA and SAR, this study underscores the importance of environmental factors on the plant's response to infection.

  9. Variation in the DNA methylation pattern of expressed and nonexpressed genes in chicken.

    Science.gov (United States)

    Cooper, D N; Errington, L H; Clayton, R M

    1983-01-01

    Using methyl-sensitive and -insensitive restriction enzymes, Hpa II and Msp I, the methylation status of various chicken genes was examined in different tissues and developmental stages. Tissue-specific differences in methylation were found for the delta-crystallin, beta-tubulin, G3PDH, rDNA, and actin genes but not for the histone genes. Developmental decreases in methylation were noted for the delta-crystallin and actin genes in chicken kidney between embryo and adult. Since most of the sequences examined were housekeeping genes, transcriptional differences are apparently not a necessary accompaniment to changes in DNA methylation at the CpG sites examined. The only exception is sperm DNA where the delta-crystallin, beta-tubulin, and actin genes are highly methylated and almost certainly not transcribed. However the G3PDH genes are no more highly methylated in sperm than in other somatic tissues. Many sequences homologous to the rDNA and histone probes used are unmethylated in all tissues examined including sperm, but a methylated rDNA subfraction is more heavily methylated in sperm than in other tissues. We speculate as to the significance of these differences in sperm DNA methylation in the light of possible requirements for early gene activation and the probable deleterious mutagenic effects of heavy methylation within coding sequences.

  10. High-Level software requirements specification for the TWRS controlled baseline database system

    International Nuclear Information System (INIS)

    Spencer, S.G.

    1998-01-01

    This Software Requirements Specification (SRS) is an as-built document that presents the Tank Waste Remediation System (TWRS) Controlled Baseline Database (TCBD) in its current state. It was originally known as the Performance Measurement Control System (PMCS). Conversion to the new system name has not occurred within the current production system. Therefore, for simplicity, all references to TCBD are equivalent to PMCS references. This SRS will reference the PMCS designator from this point forward to capture the as-built SRS. This SRS is written at a high-level and is intended to provide the design basis for the PMCS. The PMCS was first released as the electronic data repository for cost, schedule, and technical administrative baseline information for the TAAS Program. During its initial development, the PMCS was accepted by the customer, TARS Business Management, with no formal documentation to capture the initial requirements

  11. Economics of the specification 6M safety re-evaluation and regulatory requirements

    International Nuclear Information System (INIS)

    Hopper, C.M.

    1985-01-01

    The objective of this work was to examine the potential economic impact of the DOT Specification 6M criticality safety re-evaluation and regulatory requirements. The examination was based upon comparative analyses of current authorized fissile material load limits for the 6M, current Federal regulations (and interpretations) limiting the contents of Type B fissile material packages, limiting aggregates of fissile material packages, and recent proposed fissile material mass limits derived from specialized criticality safety analyses of the 6M package. The work examines influences on cost in transportation, handling, and storage of fissile materials. Depending upon facility throughput requirements (and assumed incremental costs of fissile material packaging, storage, and transport), operating, facility storage capacity, and transportation costs can be reduced significantly. As an example of the pricing algorithm application based upon reasonable cost influences, the magnitude of the first year cost reductions could extend beyond four times the cost of the packaging nuclear criticality safety re-evaluation. 1 tab

  12. A study to develop the domestic functional requirements of the specific safety systems of CANDU

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Man Woong [Korea Institute of Nuclear Safety, Taejon (Korea, Republic of); Lee, Jae Young; Park, Kun Chul [Handong Global Univ., Pohang (Korea, Republic of)] (and others)

    2003-03-15

    The present research has been made to develop and review critically the functional requirements of the specific safety systems of CANDU such as SDS-1, SDS2, ECCS, and containment. Based on R documents for this, a systematic study was made to develop the domestic regulation statements. Also, the conventional laws are carefully reviewed to see the compatibility to CANDU. Also, the safety assessment method for CANDU was studied by reviewing C documents and recommendation of IAEA. Through the present works, the vague policy in the CANDU safety regulation is cleaning up in a systematic form and a new frame to measure the objective risk of nuclear power plants was developed.

  13. Requirements, design and specifications of a multipurpose 7 Tesla NMR-system

    International Nuclear Information System (INIS)

    Mehlkopf, A.F.

    1978-01-01

    The requirements as well as the design and the specifications of a multi-purpose high resolution nuclear magnetic resonance spectrometer with superconducting magnet are described. The spectrometer operates at a magnetic fluxdensity of 7 Tesla and has 5 transmitter channels with 4 different frequencies, which can have values between 20 and 300 MHz. With this spectrometer almost all the current high resolution magnetic resonance techniques can be performed under computer control. It can also operate in the non-computer controlled slow passage mode. (Auth.)

  14. A study to develop the domestic functional requirements of the specific safety systems of CANDU

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Man Woong; Lee, Jae Young; Bang, Kwang Hyun [Handong Global Univ., Pohang (Korea, Republic of)] (and others)

    2001-03-15

    The present research has been made to develop and review critically the functional requirements of the specific safety systems of CANDU such as SOS-1, SOS-2, ECCS and containment. Based on R documents for this, a systematic study was made to develop the domestic regulation statements. Also, the conventional laws are carefully reviewed to see the compatibility to CANDU. Also, the safety assessment method for CANDU was studied by reviewing C documents and recommendation of IAEA. Through the present works, the vague policy in the CANDU safety regulation is cleaning up in a systematic form and a new frame to measure the objective risk of nuclear power plants was developed.

  15. Statistical Study to Check the Conformity of Aggregate in Kirkuk City to Requirement of Iraqi Specification

    Directory of Open Access Journals (Sweden)

    Ammar Saleem Khazaal

    2018-01-01

    Full Text Available This research reviews a statistical study to check the conformity of aggregates (Coarse and Fine was used in Kirkuk city to the requirements of the Iraqi specifications. The data of sieve analysis (215 samples of aggregates being obtained from of National Central Construction Laboratory and Technical College Construction Laboratory in Kirkuk city have analyzed using the statistical program SAS. The results showed that 5%, 17%, and 18% of fine aggregate samples are passing sieve sizes 10 mm, 4.75 mm, and 2.36 mm, respectively, which were less than the minimum limit allowed by the Iraqi specifications for each sieve. The percentages passing sieve sizes 1.18mm, 600micrometers, and 300micrometers were more than the upper limit of specification by 5%, 20%, and 30% respectively. The samples were passing sieve sizes 1.18mm, and 600micrometers less than the minimum limit of specification by 17%, and 4%, respectively. The results showed that the deviation in a sieve size of 150 micrometers for the upper limit of the specification performs 2% of the total number of samples. For Coarse aggregate, the samples passing sieves size 37.5mm and 20mm were comforting the Iraqi specifications by 100% and 83% respectively, it has found that the samples were passing sieve sizes 10 mm was 5% was more than the higher limit of Iraqi specifications, and 27% of these samples were less than the minimum limit, whereas sample passing sieve size 5mm was 1% which is more than the upper limit of the Iraqi specification. As a result of statistical analysis of data for fine aggregate, it has found that the samples were passing sieve sizes 10 mm, 2.36 mm, 1.18 mm and 150micrometers conforming from statistical point of view the Iraqi specifications, whereas the samples were passing sieve sizes 4.75 mm, 600micrometers and 300 micrometers didn’t conform. Statistical analysis of the results of the coarse aggregates also showed that conforming to sieve sizes of 37.5 mm and 20 mm and

  16. Functional characterization of O-methyltransferases used to catalyse site-specific methylation in the post-tailoring steps of pradimicin biosynthesis.

    Science.gov (United States)

    Han, J W; Ng, B G; Sohng, J K; Yoon, Y J; Choi, G J; Kim, B S

    2018-01-01

    To identify the roles of the two O-methyltransferase homologous genes pdmF and pdmT in the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2. Pradimicins are pentangular polyphenol antibiotics synthesized by bacterial type II polyketide synthases (PKSs) and tailoring enzymes. Pradimicins are naturally derivatized by combinatorial O-methylation at two positions (i.e., 7-OH and 11-OH) of the benzo[α]naphthacenequinone structure. PdmF and PdmT null mutants (PFKO and PTKO) were generated. PFKO produced the 11-O-demethyl shunt metabolites 11-O-demethylpradimicinone II (1), 11-O-demethyl-7-methoxypradimicinone II (2), 11-O-demethylpradimicinone I (3) and 11-O-demethylpradimicin A (4), while PTKO generated the 7-O-demethyl derivatives pradimicinone II (5) and 7-hydroxypradimicin A (6). Pradimicinones 1, 2, 3, and 5 were fed to a heterologous host Escherichia coli harbouring expression plasmid pET-22b::pdmF or pET-28a::pdmT. PdmF catalysed 11-O-methylation of pradimicinones 1, 2, and 3 regardless of O-methylation at the C-7 position, while PdmT was unable to catalyse 7-O-methylation when the C-11 hydroxyl group was methylated (5). PdmF and PdmT were involved in 11-O- and 7-O-methylations of the benzo[α]naphthacenequinone moiety of pradimicin, respectively. Methylation of the C-7 hydroxyl group precedes methylation of the C-11 hydroxyl group in pradimicin biosynthesis. This is the first reported demonstration of the functions of PdmF and PdmT for regiospecific O-methylation, which contributes to better understanding of the post-PKS modifications in pradimicin biosynthesis as well as to rational engineering of the pradimicin biosynthetic machinery. © 2017 The Society for Applied Microbiology.

  17. Neurotransmitter Transporter-Like: a male germline-specific SLC6 transporter required for Drosophila spermiogenesis.

    Directory of Open Access Journals (Sweden)

    Nabanita Chatterjee

    2011-01-01

    Full Text Available The SLC6 class of membrane transporters, known primarily as neurotransmitter transporters, is increasingly appreciated for its roles in nutritional uptake of amino acids and other developmentally specific functions. A Drosophila SLC6 gene, Neurotransmitter transporter-like (Ntl, is expressed only in the male germline. Mobilization of a transposon inserted near the 3' end of the Ntl coding region yields male-sterile mutants defining a single complementation group. Germline transformation with Ntl cDNAs under control of male germline-specific control elements restores Ntl/Ntl homozygotes to normal fertility, indicating that Ntl is required only in the germ cells. In mutant males, sperm morphogenesis appears normal, with elongated, individualized and coiled spermiogenic cysts accumulating at the base of the testes. However, no sperm are transferred to the seminal vesicle. The level of polyglycylation of Ntl mutant sperm tubulin appears to be significantly lower than that of wild type controls. Glycine transporters are the most closely related SLC6 transporters to Ntl, suggesting that Ntl functions as a glycine transporter in developing sperm, where augmentation of the cytosolic pool of glycine may be required for the polyglycylation of the massive amounts of tubulin in the fly's giant sperm. The male-sterile phenotype of Ntl mutants may provide a powerful genetic system for studying the function of an SLC6 transporter family in a model organism.

  18. Differential requirements for Gli2 and Gli3 in the regional specification of the mouse hypothalamus

    Directory of Open Access Journals (Sweden)

    Roberta eHaddad-Tóvolli

    2015-03-01

    Full Text Available Secreted protein Sonic hedgehog (Shh ventralizes the neural tube by modulating the crucial balance between activating and repressing functions (GliA, GliR of transcription factors Gli2 and Gli3. This balance—the Shh-Gli code—is species- and context-dependent and has been elucidated for the mouse spinal cord. The hypothalamus, a forebrain region regulating vital functions like homeostasis and hormone secretion, shows dynamic and intricate Shh expression as well as complex regional differentiation. Here we asked if particular combinations of Gli2 and Gli3 and of GliA and GliR functions contribute to the variety of hypothalamic regions, i.e. we wanted to clarify the hypothalamic version of the Shh-Gli code. Based on mouse mutant analysis, we show that: 1 hypothalamic regional heterogeneity is based in part on differentially stringent requirements for Gli2 or Gli3; 2 another source of diversity are differential requirements for Shh of neural vs non-neural origin; 3 Gli2 is indispensable for the specification of a medial progenitor domain generating several essential hypothalamic nuclei plus the pituitary and median eminence; 4 the suppression of Gli3R by neural and non-neural Shh is essential for hypothalamic specification. Finally, we have mapped our results on a recent model which considers the hypothalamus as a transverse region with alar and basal portions. Our data confirm the model and are explained by it.

  19. Nodal-dependent mesendoderm specification requires the combinatorial activities of FoxH1 and Eomesodermin.

    Directory of Open Access Journals (Sweden)

    Christopher E Slagle

    2011-05-01

    Full Text Available Vertebrate mesendoderm specification requires the Nodal signaling pathway and its transcriptional effector FoxH1. However, loss of FoxH1 in several species does not reliably cause the full range of loss-of-Nodal phenotypes, indicating that Nodal signals through additional transcription factors during early development. We investigated the FoxH1-dependent and -independent roles of Nodal signaling during mesendoderm patterning using a novel recessive zebrafish FoxH1 mutation called midway, which produces a C-terminally truncated FoxH1 protein lacking the Smad-interaction domain but retaining DNA-binding capability. Using a combination of gel shift assays, Nodal overexpression experiments, and genetic epistasis analyses, we demonstrate that midway more accurately represents a complete loss of FoxH1-dependent Nodal signaling than the existing zebrafish FoxH1 mutant schmalspur. Maternal-zygotic midway mutants lack notochords, in agreement with FoxH1 loss in other organisms, but retain near wild-type expression of markers of endoderm and various nonaxial mesoderm fates, including paraxial and intermediate mesoderm and blood precursors. We found that the activity of the T-box transcription factor Eomesodermin accounts for specification of these tissues in midway embryos. Inhibition of Eomesodermin in midway mutants severely reduces the specification of these tissues and effectively phenocopies the defects seen upon complete loss of Nodal signaling. Our results indicate that the specific combinations of transcription factors available for signal transduction play critical and separable roles in determining Nodal pathway output during mesendoderm patterning. Our findings also offer novel insights into the co-evolution of the Nodal signaling pathway, the notochord specification program, and the chordate branch of the deuterostome family of animals.

  20. A small asparagine-rich protein required for S-allele-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    McClure, B; Mou, B; Canevascini, S; Bernatzky, R

    1999-11-09

    Although S-locus RNases (S-RNases) determine the specificity of pollen rejection in self-incompatible (SI) solanaceous plants, they alone are not sufficient to cause S-allele-specific pollen rejection. To identify non-S-RNase sequences that are required for pollen rejection, a Nicotiana alata cDNA library was screened by differential hybridization. One clone, designated HT, hybridized strongly to RNA from N. alata styles but not to RNA from Nicotiana plumbaginifolia, a species known to lack one or more factors necessary for S-allele-specific pollen rejection. Sequence analysis revealed a 101-residue ORF including a putative secretion signal and an asparagine-rich domain near the C terminus. RNA blot analysis showed that the HT-transcript accumulates in the stigma and style before anthesis. The timing of HT-expression lags slightly behind S(C10)-RNase in SI N. alata S(C10)S(C10) and is well correlated with the onset of S-allele-specific pollen rejection in the style. An antisense-HT construct was prepared to test for a role in pollen rejection. Transformed (N. plumbaginifolia x SI N. alata S(C10)S(C10)) hybrids with reduced levels of HT-protein continued to express S(C10)-RNase but failed to reject S(C10)-pollen. Control hybrids expressing both S(C10)-RNase and HT-protein showed a normal S-allele-specific pollen rejection response. We conclude that HT-protein is directly implicated in pollen rejection.

  1. Exogenous Methyl Jasmonate and Salicylic Acid Induce Subspecies-Specific Patterns of Glucosinolate Accumulation and Gene Expression in Brassica oleracea L.

    Science.gov (United States)

    Yi, Go-Eun; Robin, Arif Hasan Khan; Yang, Kiwoung; Park, Jong-In; Hwang, Byung Ho; Nou, Ill-Sup

    2016-10-24

    Glucosinolates have anti-carcinogenic properties. In the recent decades, the genetics of glucosinolate biosynthesis has been widely studied, however, the expression of specific genes involved in glucosinolate biosynthesis under exogenous phytohormone treatment has not been explored at the subspecies level in Brassica oleracea . Such data are vital for strategies aimed at selective exploitation of glucosinolate profiles. This study quantified the expression of 38 glucosinolate biosynthesis-related genes in three B. oleracea subspecies, namely cabbage, broccoli and kale, and catalogued associations between gene expression and increased contents of individual glucosinolates under methyl jasmonate (MeJA) and salicylic acid (SA) treatments. Glucosinolate accumulation and gene expression in response to phytohormone elicitation was subspecies specific. For instance, cabbage leaves showed enhanced accumulation of the aliphatic glucoiberin, progoitrin, sinigrin and indolic neoglucobrassicin under both MeJA and SA treatment. MeJA treatment induced strikingly higher accumulation of glucobrassicin (GBS) in cabbage and kale and of neoglucobrassicin (NGBS) in broccoli compared to controls. Notably higher expression of ST5a (Bol026200), CYP81F1 (Bol028913, Bol028914) and CYP81F4 genes was associated with significantly higher GBS accumulation under MeJA treatment compared to controls in all three subspecies. CYP81F4 genes, trans-activated by MYB34 genes, were expressed at remarkably high levels in all three subspecies under MeJA treatment, which also induced in higher indolic NGBS accumulation in all three subspecies. Remarkably higher expression of MYB28 (Bol036286), ST5b , ST5c , AOP2 , FMOGS-OX5 (Bol031350) and GSL-OH (Bol033373) was associated with much higher contents of aliphatic glucosinolates in kale leaves compared to the other two subspecies. The genes expressed highly could be utilized in strategies to selectively increase glucosinolate compounds in B. oleracea

  2. Context-specific requirements of functional domains of the Spectraplakin Short stop in vivo.

    Science.gov (United States)

    Bottenberg, Wolfgang; Sanchez-Soriano, Natalia; Alves-Silva, Juliana; Hahn, Ines; Mende, Michael; Prokop, Andreas

    2009-07-01

    Spectraplakins are large multifunctional cytoskeletal interacting molecules implicated in various processes, including gastrulation, wound healing, skin blistering and neuronal degeneration. It has been speculated that the various functional domains and regions found in Spectraplakins are used in context-specific manners, a model which would provide a crucial explanation for the multifunctional nature of Spectraplakins. Here we tested this possibility by studying domain requirements of the Drosophila Spectraplakin Short stop (Shot) in three different cellular contexts in vivo: (1) neuronal growth, which requires dynamic actin-microtubule interaction; (2) formation and maintenance of tendon cells, which depends on highly stabilised arrays of actin filaments and microtubules, and (3) compartmentalisation in neurons, which is likely to involve cortical F-actin networks. Using these cellular contexts for rescue experiments with Shot deletion constructs in shot mutant background, a number of differential domain requirements were uncovered. First, binding of Shot to F-actin through the first Calponin domain is essential in neuronal contexts but dispensable in tendon cells. This finding is supported by our analyses of shot(kakP2) mutant embryos, which produce only endogenous isoforms lacking the first Calponin domain. Thus, our data demonstrate a functional relevance for these isoforms in vivo. Second, we provide the first functional role for the Plakin domain of Shot, which has a strong requirement for compartmentalisation in neurons and axonal growth, demonstrating that Plakin domains of long Spectraplakin isoforms are of functional relevance. Like the Calponin domain, also the Plakin domain is dispensable in tendon cells, and the currently assumed role of Shot as a linker of microtubules to the tendon cell surface may have to be reconsidered. Third, we demonstrate a function of Shot as an actin-microtubule linker in dendritic growth, thus shedding new light into

  3. Exploring QSAR with E-state index: selectivity requirements for COX-2 versus COX-1 binding of terphenyl methyl sulfones and sulfonamides.

    Science.gov (United States)

    Chakraborty, Santanu; Sengupta, Chandana; Roy, Kunal

    2004-09-20

    An attempt has been made to explore selectivity requirements for cyclooxygenase-2 (COX-2) versus cyclooxygenase-1 (COX-1) binding of terphenyl methyl sulfones and sulfonamides using electrotopological state (E-state) index and suitable indicator parameters. Multiple linear regression analyses produced statistically acceptable equations: the best relation based on 'all-possible-subsets regression' for COX-1 binding (n=18) showed predicted variance and explained variance of 0.675 and 0.777, respectively, while in case of the best equation for COX-2 binding (n=38), these values rose to 0.842 and 0.874, respectively. For the selectivity relation (n=17), predicted variance and explained variance values were 0.601 and 0.687, respectively. Based on the results of the analyses, three important sites have been suggested: sites A (methylsulfonyl or aminosulfonyl moiety), B (central phenyl ring), and C (terminal phenyl ring containing different substituents). All three sites are important for COX-2 binding while sites B and C are important for COX-1 binding. For COX-2 selectivity, only site C plays an important role. The study shows the utility of E-state index in developing statistically acceptable model having direct physicochemical significance.

  4. Requirement for Pectin Methyl Esterase and Preference for Fragmented over Native Pectins for Wall-associated Kinase-activated, EDS1/PAD4-dependent Stress Response in Arabidopsis*

    Science.gov (United States)

    Kohorn, Bruce D.; Kohorn, Susan L.; Saba, Nicholas J.; Martinez, Victoriano Meco

    2014-01-01

    The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. PMID:24855660

  5. Requirement for pectin methyl esterase and preference for fragmented over native pectins for wall-associated kinase-activated, EDS1/PAD4-dependent stress response in Arabidopsis.

    Science.gov (United States)

    Kohorn, Bruce D; Kohorn, Susan L; Saba, Nicholas J; Martinez, Victoriano Meco

    2014-07-04

    The wall-associated kinases (WAKs) have a cytoplasmic protein kinase domain that spans the plasma membrane and binds pectin in the extracellular matrix of plants. WAKs are required for cell expansion during Arabidopsis seedling development but are also an integral part of the response to pathogens and stress that present oligogalacturonides (OGs), which subsequently bind to WAKs and activate a MPK6 (mitogen-activated protein kinase)-dependent pathway. It was unclear how WAKs distinguish native pectin polymers and OGs to activate one or the other of these two pathways. A dominant allele of WAK2 constitutively activates the stress response, and we show here that the effect is dependent upon EDS1 and PAD4, transcriptional activators involved in the pathogen response. Moreover, the WAK2 dominant allele is suppressed by a null allele of a pectin methyl esterase (PME3) whose activity normally leads to cross-linking of pectins in the cell wall. Although OGs activate a transcriptional response in wild type, the response is enhanced in a pme3/pme3 null, consistent with a competition by OG and native polymers for activation of WAKs. This provides a plausible mechanism for WAKs to distinguish an expansion from a stress pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Methylation by a unique α-class N4-cytosine methyltransferase is required for DNA transformation of Caldicellulosiruptor bescii DSM6725.

    Directory of Open Access Journals (Sweden)

    Daehwan Chung

    Full Text Available Thermophilic microorganisms capable of using complex substrates offer special advantages for the conversion of lignocellulosic biomass to biofuels and bioproducts. Members of the gram-positive bacterial genus Caldicellulosiruptor are anaerobic thermophiles with optimum growth temperatures between 65°C and 78°C and are the most thermophilic cellulolytic organisms known. In fact, they efficiently use biomass non-pretreated as their sole carbon source and in successive rounds of application digest 70% of total switchgrass substrate. The ability to genetically manipulate these organisms is a prerequisite to engineering them for use in conversion of these complex substrates to products of interest as well as identifying gene products critical for their ability to utilize non-pretreated biomass. Here, we report the first example of DNA transformation of a member of this genus, C. bescii. We show that restriction of DNA is a major barrier to transformation (in this case apparently absolute and that methylation with an endogenous unique α-class N4-Cytosine methyltransferase is required for transformation of DNA isolated from E. coli. The use of modified DNA leads to the development of an efficient and reproducible method for DNA transformation and the combined frequencies of transformation and recombination allow marker replacement between non-replicating plasmids and chromosomal genes providing the basis for rapid and efficient methods of genetic manipulation.

  7. Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis.

    Directory of Open Access Journals (Sweden)

    Michalis Barkoulas

    2016-09-01

    Full Text Available Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.

  8. DNA methylation and memory formation.

    Science.gov (United States)

    Day, Jeremy J; Sweatt, J David

    2010-11-01

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

  9. Environmental Molecular Sciences Laboratory Operations System: Version 4.0 - system requirements specification

    Energy Technology Data Exchange (ETDEWEB)

    Kashporenko, D.

    1996-07-01

    This document is intended to provide an operations standard for the Environmental Molecular Sciences Laboratory OPerations System (EMSL OPS). It is directed toward three primary audiences: (1) Environmental Molecular Sciences Laboratory (EMSL) facility and operations personnel; (2) laboratory line managers and staff; and (3) researchers, equipment operators, and laboratory users. It is also a statement of system requirements for software developers of EMSL OPS. The need for a finely tuned, superior research environment as provided by the US Department of Energy`s (DOE) Environmental Molecular Sciences Laboratory has never been greater. The abrupt end of the Cold War and the realignment of national priorities caused major US and competing overseas laboratories to reposition themselves in a highly competitive research marketplace. For a new laboratory such as the EMSL, this means coming into existence in a rapidly changing external environment. For any major laboratory, these changes create funding uncertainties and increasing global competition along with concomitant demands for higher standards of research product quality and innovation. While more laboratories are chasing fewer funding dollars, research ideas and proposals, especially for molecular-level research in the materials and biological sciences, are burgeoning. In such an economically constrained atmosphere, reduced costs, improved productivity, and strategic research project portfolio building become essential to establish and maintain any distinct competitive advantage. For EMSL, this environment and these demands require clear operational objectives, specific goals, and a well-crafted strategy. Specific goals will evolve and change with the evolution of the nature and definition of DOE`s environmental research needs. Hence, EMSL OPS is designed to facilitate migration of these changes with ease into every pertinent job function, creating a facile {open_quotes}learning organization.{close_quotes}

  10. 49 CFR 173.427 - Transport requirements for low specific activity (LSA) Class 7 (radioactive) materials and...

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Transport requirements for low specific activity... SHIPMENTS AND PACKAGINGS Class 7 (Radioactive) Materials § 173.427 Transport requirements for low specific... must be transported in accordance with the following conditions: (1) The external dose rate may not...

  11. Direct effects of ionizing radiation on integral membrane proteins. Noncovalent energy transfer requires specific interpeptide interactions

    International Nuclear Information System (INIS)

    Jhun, E.; Jhun, B.H.; Jones, L.R.; Jung, C.Y.

    1991-01-01

    The 12 transmembrane alpha helices (TMHs) of human erythrocyte glucose transporter were individually cut by pepsin digestion as membrane-bound 2.5-3.5-kDa peptide fragments. Radiation-induced chemical degradation of these fragments showed an average target size of 34 kDa. This is 10-12 x larger than the average size of an individual TMH, demonstrating that a significant energy transfer occurs among these TMHs in the absence of covalent linkage. Heating this TMH preparation at 100 degree C for 15 min reduced the target size to 5 kDa or less, suggesting that the noncovalent energy transfer requires specific helix-helix interactions. Purified phospholamban, a small (6-kDa) integral membrane protein containing a single TMH, formed a pentameric assembly in sodium dodecyl sulfate. The chemical degradation target size of this phospholamban pentamer was 5-6 kDa, illustrating that not all integral membrane protein assemblies permit intersubunit energy transfer. These findings together with other published observations suggest strongly that significant noncovalent energy transfer can occur within the tertiary and quaternary structure of membrane proteins and that as yet undefined proper molecular interactions are required for such covalent energy transfer. Our results with pepsin-digested glucose transporter also illustrate the importance of the interhelical interaction as a predominating force in maintaining the tertiary structure of a transmembrane protein

  12. Recommendations for equipment requirements and specifications for digital and interventional radiology: Dosimetric aspects

    International Nuclear Information System (INIS)

    Suliman, I.I.; Zoetelief, J.

    2002-01-01

    The recognition of radiation induced injuries from fluoroscopically guided interventional procedures has resulted in the current demand for development of recommendations and standards to limit dose to both patients and staff. This paper outlines the recommendations drafted within the framework of European Project DIMOND III. The actual work involves survey and review of national and international documents as well as scientific publications in areas relevant to the digital and/or interventional radiology with an aim of developing recommendations for equipment requirements and specifications for digital and interventional radiology. A pilot study of experimental investigations in at least three hospitals will be conducted to test the requirements and the specifications, the result of which will be presented. The recommendations are expected to provide an effective means of dose reduction to both patients and staff while maintaining image quality adequate for the specific diagnosis or interventional procedure. Different components of x-ray systems that have direct impact on patient and staff doses have been considered. Where necessary a compromise between patient dose and image quality has been made. The dosimetric aspects of the recommendations propose detailed descriptions and limits to dosimetric information relevant to patient and staff doses. International recommendations on maximum patient entrance surface dose rate vary in the range from 25 to 65 mGy.min -1 for normal mode fluoroscopy. Maximum image intensifier or image receptor input dose rate around 0.1 Gy min -1 at a distance 30 cm from the image intensifier input surface has been generally recommended. Maximum fluoroscopic dose rate in air must not exceed 50 mGy.min -1 at a location depending on the configuration e.g. for undertable x-ray tube at 10 mm from the patient support on the patient side of the support. The use of pulsed fluoroscopy or low dose fluoroscopy is proposed as good options to minimize

  13. 40 CFR 152.95 - Citation of all studies in the Agency's files pertinent to a specific data requirement.

    Science.gov (United States)

    2010-07-01

    ... PROCEDURES Procedures To Ensure Protection of Data Submitters' Rights § 152.95 Citation of all studies in the... requirement. The applicant who selects this cite-all option must submit to the Agency: (a) A general offer to... may be limited to apply only to data pertinent to the specific data requirement(s) for which the cite...

  14. Niche-Specific Requirement for Hyphal Wall protein 1 in Virulence of Candida albicans

    Science.gov (United States)

    Staab, Janet F.; Datta, Kausik; Rhee, Peter

    2013-01-01

    Specialized Candida albicans cell surface proteins called adhesins mediate binding of the fungus to host cells. The mammalian transglutaminase (TG) substrate and adhesin, Hyphal wall protein 1 (Hwp1), is expressed on the hyphal form of C. albicans where it mediates fungal adhesion to epithelial cells. Hwp1 is also required for biofilm formation and mating thus the protein functions in both fungal-host and self-interactions. Hwp1 is required for full virulence of C. albicans in murine models of disseminated candidiasis and of esophageal candidiasis. Previous studies correlated TG activity on the surface of oral epithelial cells, produced by epithelial TG (TG1), with tight binding of C. albicans via Hwp1 to the host cell surfaces. However, the contribution of other Tgs, specifically tissue TG (TG2), to disseminated candidiasis mediated by Hwp1 was not known. A newly created hwp1 null strain in the wild type SC5314 background was as virulent as the parental strain in C57BL/6 mice, and virulence was retained in C57BL/6 mice deleted for Tgm2 (TG2). Further, the hwp1 null strains displayed modestly reduced virulence in BALB/c mice as did strain DD27-U1, an independently created hwp1Δ/Δ in CAI4 corrected for its ura3Δ defect at the URA3 locus. Hwp1 was still needed to produce wild type biofilms, and persist on murine tongues in an oral model of oropharyngeal candidiasis consistent with previous studies by us and others. Finally, lack of Hwp1 affected the translocation of C. albicans from the mouse intestine into the bloodstream of mice. Together, Hwp1 appears to have a minor role in disseminated candidiasis, independent of tissue TG, but a key function in host- and self-association to the surface of oral mucosa. PMID:24260489

  15. The rapidly evolving centromere-specific histone has stringent functional requirements in Arabidopsis thaliana.

    Science.gov (United States)

    Ravi, Maruthachalam; Kwong, Pak N; Menorca, Ron M G; Valencia, Joel T; Ramahi, Joseph S; Stewart, Jodi L; Tran, Robert K; Sundaresan, Venkatesan; Comai, Luca; Chan, Simon W-L

    2010-10-01

    Centromeres control chromosome inheritance in eukaryotes, yet their DNA structure and primary sequence are hypervariable. Most animals and plants have megabases of tandem repeats at their centromeres, unlike yeast with unique centromere sequences. Centromere function requires the centromere-specific histone CENH3 (CENP-A in human), which replaces histone H3 in centromeric nucleosomes. CENH3 evolves rapidly, particularly in its N-terminal tail domain. A portion of the CENH3 histone-fold domain, the CENP-A targeting domain (CATD), has been previously shown to confer kinetochore localization and centromere function when swapped into human H3. Furthermore, CENP-A in human cells can be functionally replaced by CENH3 from distantly related organisms including Saccharomyces cerevisiae. We have used cenh3-1 (a null mutant in Arabidopsis thaliana) to replace endogenous CENH3 with GFP-tagged variants. A H3.3 tail domain-CENH3 histone-fold domain chimera rescued viability of cenh3-1, but CENH3's lacking a tail domain were nonfunctional. In contrast to human results, H3 containing the A. thaliana CATD cannot complement cenh3-1. GFP-CENH3 from the sister species A. arenosa functionally replaces A. thaliana CENH3. GFP-CENH3 from the close relative Brassica rapa was targeted to centromeres, but did not complement cenh3-1, indicating that kinetochore localization and centromere function can be uncoupled. We conclude that CENH3 function in A. thaliana, an organism with large tandem repeat centromeres, has stringent requirements for functional complementation in mitosis.

  16. 78 FR 30245 - Electric Reliability Organization Interpretation of Specific Requirements of the Disturbance...

    Science.gov (United States)

    2013-05-22

    ... contingency reserve policy; and (3) refers to the ERO rather than the NERC Operating Committee in Requirements... the proposed interpretation, Requirements R3 and R4 provide: R3. Each Balancing Authority or Reserve... Requirement R4 requires that: ``[A] Balancing Authority or Reserve Sharing Group shall meet the Disturbance...

  17. Solid Waste Information Tracking System (SWITS), Backlog Waste Modifications, Software Requirements Specification (SRS)

    International Nuclear Information System (INIS)

    Clark, R.E.

    1995-01-01

    Purpose of this document is to define the system requirements necessary to improve computer support for the WHC backlog waste business process through enhancements to the backlog waste function of the SWITS system. This SRS document covers enhancements to the SWITS system to support changes to the existing Backlog Waste screens including new data elements, label changes, and new pop-up screens. The pop-ups will allow the user to flag the processes that a waste container must have performed on it, and will provide history tracking of changes to data. A new screen will also be provided allowing Acceptable Services to perform mass updates to specific data in Backlog Waste table. The SWITS Backlog Waste enhancements in this document will support the project goals in WHC-SD-WM-003 and its Revision 1 (Radioactive Solid Waste Tracking System Conceptual Definition) for the control, tracing, and inventory management of waste as the packages are generated and moved through final disposal (cradle-to-grave)

  18. Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures.

    Science.gov (United States)

    Bohlen, Christopher J; Bennett, F Chris; Tucker, Andrew F; Collins, Hannah Y; Mulinyawe, Sara B; Barres, Ben A

    2017-05-17

    Microglia, the resident macrophages of the CNS, engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    Directory of Open Access Journals (Sweden)

    Aymerick Eudes

    2016-07-01

    Full Text Available Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet. In this study, we demonstrate in Arabidopsis stems that targeted expression of S-adenosylmethionine hydrolase (AdoMetase, E.C. 3.3.1.2 in secondary cell-wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H units and a reduction of dimethylated syringyl (S units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock.

  20. O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside

    Directory of Open Access Journals (Sweden)

    Roman Sommer

    2016-12-01

    Full Text Available Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

  1. O-Alkylated heavy atom carbohydrate probes for protein X-ray crystallography: Studies towards the synthesis of methyl 2-O-methyl-L-selenofucopyranoside.

    Science.gov (United States)

    Sommer, Roman; Hauck, Dirk; Varrot, Annabelle; Imberty, Anne; Künzler, Markus; Titz, Alexander

    2016-01-01

    Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O -methylated selenoglycoside, specifically methyl 2- O -methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor . The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2- O -methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.

  2. Requirement for Pdx1 in specification of latent endocrine progenitors in zebrafish

    Directory of Open Access Journals (Sweden)

    Ellertsdottir Elin

    2011-10-01

    Full Text Available Abstract Background Insulin-producing beta cells emerge during pancreas development in two sequential waves. Recently described later-forming beta cells in zebrafish show high similarity to second wave mammalian beta cells in developmental capacity. Loss-of-function studies in mouse and zebrafish demonstrated that the homeobox transcription factors Pdx1 and Hb9 are both critical for pancreas and beta cell development and discrete stage-specific requirements for these genes have been uncovered. Previously, exocrine and endocrine cell recovery was shown to follow loss of pdx1 in zebrafish, but the progenitor cells and molecular mechanisms responsible have not been clearly defined. In addition, interactions of pdx1 and hb9 in beta cell formation have not been addressed. Results To learn more about endocrine progenitor specification, we examined beta cell formation following morpholino-mediated depletion of pdx1 and hb9. We find that after early beta cell reduction, recovery occurs following loss of either pdx1 or hb9 function. Unexpectedly, simultaneous knockdown of both hb9 and pdx1 leads to virtually complete and persistent beta cell deficiency. We used a NeuroD:EGFP transgenic line to examine endocrine cell behavior in vivo and developed a novel live-imaging technique to document emergence and migration of late-forming endocrine precursors in real time. Our data show that Notch-responsive progenitors for late-arising endocrine cells are predominantly post mitotic and depend on pdx1. By contrast, early-arising endocrine cells are specified and differentiate independent of pdx1. Conclusions The nearly complete beta cell deficiency after combined loss of hb9 and pdx1 suggests functional cooperation, which we clarify as distinct roles in early and late endocrine cell formation. A novel imaging approach permitted visualization of the emergence of late endocrine cells within developing embryos for the first time. We demonstrate a pdx1-dependent

  3. SB certification handout material requirements, test methods, responsibilities, and minimum classification levels for mixture-based specification for flexible base.

    Science.gov (United States)

    2012-10-01

    A handout with tables representing the material requirements, test methods, responsibilities, and minimum classification levels mixture-based specification for flexible base and details on aggregate and test methods employed, along with agency and co...

  4. Transcription factor Tbx3 is required for the specification of the atrioventricular conduction system

    NARCIS (Netherlands)

    Bakker, Martijn L.; Boukens, Bastiaan J.; Mommersteeg, Mathilda T. M.; Brons, Janynke F.; Wakker, Vincent; Moorman, Antoon F. M.; Christoffels, Vincent M.

    2008-01-01

    The cardiac conduction system consists of distinctive heart muscle cells that initiate and propagate the electric impulse required for coordinated contraction. The conduction system expresses the transcriptional repressor Tbx3, which is required for vertebrate development and controls the formation

  5. Development of requirements and functional specifications for crash event data recorders : final report

    Science.gov (United States)

    2004-12-01

    The U.S. DOT has conducted research on the requirements for a Crash Event Data Recorder to facilitate the reconstruction of commercial motor vehicle crashes. This report documents the work performed on the Development of Requirements and Functiona...

  6. 49 CFR 179.201 - Individual specification requirements applicable to non-pressure tank car tanks.

    Science.gov (United States)

    2010-10-01

    ... to non-pressure tank car tanks. 179.201 Section 179.201 Transportation Other Regulations Relating to... MATERIALS REGULATIONS SPECIFICATIONS FOR TANK CARS Specifications for Non-Pressure Tank Car Tanks (Classes... car tanks. ...

  7. Requirements Analysis Study for Master Pump Shutdown System Project Development Specification [SEC 1 and 2

    International Nuclear Information System (INIS)

    BEVINS, R.R.

    2000-01-01

    This document has been updated during the definitive design portion of the first phase of the W-314 Project to capture additional software requirements and is planned to be updated during the second phase of the W-314 Project to cover the second phase of the Project's scope. The objective is to provide requirement traceability by recording the analysis/basis for the functional descriptions of the master pump shutdown system. This document identifies the sources of the requirements and/or how these were derived. Each requirement is validated either by quoting the source or an analysis process involving the required functionality, performance characteristics, operations input or engineering judgment

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

    Science.gov (United States)

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

    2018-04-14

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

  9. Requirements Analysis Study for Master Pump Shutdown System Project Development Specification

    International Nuclear Information System (INIS)

    BEVINS, R.R.

    2000-01-01

    This study is a requirements document that presents analysis for the functional description for the master pump shutdown system. This document identifies the sources of the requirements and/or how these were derived. Each requirement is validated either by quoting the source or an analysis process involving the required functionality, performance characteristics, operations input or engineering judgment. The requirements in this study apply to the first phase of the W314 Project. This document has been updated during the definitive design portion of the first phase of the W314 Project to capture additional software requirements and is planned to be updated during the second phase of the W314 Project to cover the second phase of the project's scope

  10. Circuit architecture derivation starting from a formal requirements specification considering a DDS as example

    Science.gov (United States)

    Garbe, H.; Richter, R.; Jentschel, H.-J.

    2004-05-01

    Based on a formal specification of a direct digital synthesis (DDS) and assuming the availability of a set of possible circuit architectures we derive a customised system configuration. e calculate the design parameters that can be used for the specification to synthesise the circuit components. We show how the derived parameters and the selected IC technology influence the complexity of the circuit implementation.

  11. 46 CFR 164.009-3 - Noncombustible materials not requiring specific approval.

    Science.gov (United States)

    2010-10-01

    ...) EQUIPMENT, CONSTRUCTION, AND MATERIALS: SPECIFICATIONS AND APPROVAL MATERIALS Noncombustible Materials for... noncombustible materials may be used in merchant vessel construction though not specifically approved under this subpart: (a) Sheet glass, block glass, clay, ceramics, and uncoated fibers. (b) All metals, except...

  12. 49 CFR 179.101 - Individual specification requirements applicable to pressure tank car tanks.

    Science.gov (United States)

    2010-10-01

    ... to pressure tank car tanks. 179.101 Section 179.101 Transportation Other Regulations Relating to... MATERIALS REGULATIONS SPECIFICATIONS FOR TANK CARS Specifications for Pressure Tank Car Tanks (Classes DOT... tank car tanks. Editorial Note: At 66 FR 45186, Aug. 28, 2001, an amendment published amending a table...

  13. Generic requirements specification for qualifying a commercially available PLC for safety-related applications in nuclear power plants. Final report

    International Nuclear Information System (INIS)

    Ostenso, A.; May, R.

    1996-12-01

    This is a specification for qualifying a commercially available PLC for application to safety systems in nuclear power plants. The specifications are suitable for evaluating a particular PLC product line as a platform for safety-related applications, establishing a suitable qualification test program, and confirming that the manufacturer has a quality assurance program that is adequate for safety-related applications or is sufficiently complete that, with a reasonable set of compensatory actions, it can be brought into conformance. The specification includes requirements for: (1) quality assurance measures applied to the qualification activities, (2) documentation to support the qualification, and (3) documentation to provide the information needed for applying the qualified PLC platform to a specific application. The specifications are designed to encompass a broad range of safety applications; however, qualifying a particular platform for a different range of applications can be accomplished by appropriate adjustments to the requirements

  14. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (French Edition)

    International Nuclear Information System (INIS)

    2017-01-01

    This publication establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. A review of Safety Requirements publications was commenced in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan. The review revealed no significant areas of weakness and resulted in just a small set of amendments to strengthen the requirements and facilitate their implementation, which are contained in the present publication.

  15. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Russian Edition)

    International Nuclear Information System (INIS)

    2016-01-01

    This publication establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. A review of Safety Requirements publications was commenced in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan. The review revealed no significant areas of weakness and resulted in just a small set of amendments to strengthen the requirements and facilitate their implementation, which are contained in the present publication.

  16. Safety of Nuclear Power Plants: Commissioning and Operation. Specific Safety Requirements (French Edition)

    International Nuclear Information System (INIS)

    2016-01-01

    This publication describes the requirements to be met to ensure the safe operation of nuclear power plants. It takes into account developments in areas such as long term operation of nuclear power plants, plant ageing, periodic safety review, probabilistic safety analysis and risk informed decision making processes. In addition, the requirements are governed by, and must apply, the safety objective and safety principles that are established in the IAEA Safety Standards Series No. SF-1, Fundamental Safety Principles. A review of Safety Requirements publications was commenced in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan. The review revealed no significant areas of weakness and resulted in just a small set of amendments to strengthen the requirements and facilitate their implementation, which are contained in the present publication.

  17. Safety of Nuclear Power Plants: Commissioning and Operation. Specific Safety Requirements

    International Nuclear Information System (INIS)

    2016-01-01

    This publication describes the requirements to be met to ensure the safe operation of nuclear power plants. It takes into account developments in areas such as long term operation of nuclear power plants, plant ageing, periodic safety review, probabilistic safety analysis and risk informed decision making processes. In addition, the requirements are governed by, and must apply, the safety objective and safety principles that are established in the IAEA Safety Standards Series No. SF-1, Fundamental Safety Principles. A review of Safety Requirements publications was commenced in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan. The review revealed no significant areas of weakness and resulted in just a small set of amendments to strengthen the requirements and facilitate their implementation, which are contained in the present publication

  18. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Arabic Edition)

    International Nuclear Information System (INIS)

    2017-01-01

    This publication establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. A review of Safety Requirements publications was commenced in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan. The review revealed no significant areas of weakness and resulted in just a small set of amendments to strengthen the requirements and facilitate their implementation, which are contained in the present publication.

  19. Oxidation state specific generation of arsines from methylated arsenicals based on L-cysteine treatment in buffered media for speciation analysis by hydride generation-automated cryotrapping-gas chromatography-atomic absorption spectrometry with the multiatomizer

    Energy Technology Data Exchange (ETDEWEB)

    Matousek, Tomas [Institute of Analytical Chemistry of the ASCR, v.v.i., Videnska 1083, 14220 Prague (Czech Republic)], E-mail: matousek@biomed.cas.cz; Hernandez-Zavala, Araceli [Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7310 (United States); Svoboda, Milan; Langrova, Lenka [Institute of Analytical Chemistry of the ASCR, v.v.i., Videnska 1083, 14220 Prague (Czech Republic); Charles University, Faculty of Science, Albertov 8, 12840 Prague 2 (Czech Republic); Adair, Blakely M. [Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Drobna, Zuzana [Department of Nutrition, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7461 (United States); Thomas, David J. [Pharmacokinetics Branch, Experimental Toxicology Division, National Health and Environmental Effects Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711 (United States); Styblo, Miroslav [Center for Environmental Medicine, Asthma, and Lung Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7310 (United States); Department of Nutrition, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7461 (United States); Dedina, Jiri [Institute of Analytical Chemistry of the ASCR, v.v.i., Videnska 1083, 14220 Prague (Czech Republic)

    2008-03-15

    An automated system for hydride generation-cryotrapping-gas chromatography-atomic absorption spectrometry with the multiatomizer is described. Arsines are preconcentrated and separated in a Chromosorb filled U-tube. An automated cryotrapping unit, employing nitrogen gas formed upon heating in the detection phase for the displacement of the cooling liquid nitrogen, has been developed. The conditions for separation of arsines in a Chromosorb filled U-tube have been optimized. A complete separation of signals from arsine, methylarsine, dimethylarsine, and trimethylarsine has been achieved within a 60 s reading window. The limits of detection for methylated arsenicals tested were 4 ng l{sup -1}. Selective hydride generation is applied for the oxidation state specific speciation analysis of inorganic and methylated arsenicals. The arsines are generated either exclusively from trivalent or from both tri- and pentavalent inorganic and methylated arsenicals depending on the presence of L-cysteine as a prereductant and/or reaction modifier. A TRIS buffer reaction medium is proposed to overcome narrow optimum concentration range observed for the L-cysteine modified reaction in HCl medium. The system provides uniform peak area sensitivity for all As species. Consequently, the calibration with a single form of As is possible. This method permits a high-throughput speciation analysis of metabolites of inorganic arsenic in relatively complex biological matrices such as cell culture systems without sample pretreatment, thus preserving the distribution of tri- and pentavalent species.

  20. The Specific Direction Requirement for Aiding and Abetting: A Call for Revisiting Comparative Criminal Law

    DEFF Research Database (Denmark)

    Aksenova, Marina

    2015-01-01

    . After this judgment, the prosecution filed a motion to reconsider the acquittal in Perišić, which the Appeals Chamber denied. In sum, these developments diluted and mischaracterized the standard of aiding and abetting. Accordingly, this article has two purposes. First, it demonstrates......The ‘specific direction’ saga has been dominating the jurisprudence of the ICTY for nearly two years, and the end is yet to be seen. The story centers on the correct interpretation of liability for aiding and abetting, while, at the same time, exposing broader concerns of international criminal law....... The saga started with unexpected acquittals of Perišić, Stanišić, and Simatović due to a lack of specific direction in their aid and assistance towards specific offences. More specifically, the tribunal found that the traditional test - the provision of aid with the awareness that it would have...

  1. 49 CFR 178.320 - General requirements applicable to all DOT specification cargo tank motor vehicles.

    Science.gov (United States)

    2010-10-01

    ... removed from the motor vehicle; and (3) Is not fabricated under a specification for cylinders... determine leak tightness of the cargo tank when testing with pneumatic pressure. Internal self-closing stop...

  2. Waste Receiving and Processing Facility Module 1 Data Management System software requirements specification

    International Nuclear Information System (INIS)

    Rosnick, C.K.

    1996-01-01

    This document provides the software requirements for Waste Receiving and Processing (WRAP) Module 1 Data Management System (DMS). The DMS is one of the plant computer systems for the new WRAP 1 facility (Project W-0126). The DMS will collect, store and report data required to certify the low level waste (LLW) and transuranic (TRU) waste items processed at WRAP 1 as acceptable for shipment, storage, or disposal

  3. Waste Receiving and Processing Facility Module 1 Data Management System Software Requirements Specification

    International Nuclear Information System (INIS)

    Brann, E.C. II.

    1994-01-01

    This document provides the software requirements for Waste Receiving and Processing (WRAP) Module 1 Data Management System (DMS). The DMS is one of the plant computer systems for the new WRAP 1 facility (Project W-026). The DMS will collect, store and report data required to certify the low level waste (LLW) and transuranic (TRU) waste items processed at WRAP 1 as acceptable for shipment, storage, or disposal

  4. Waste Receiving and Processing Facility Module 1 Data Management System Software Requirements Specification

    Energy Technology Data Exchange (ETDEWEB)

    Brann, E.C. II

    1994-09-09

    This document provides the software requirements for Waste Receiving and Processing (WRAP) Module 1 Data Management System (DMS). The DMS is one of the plant computer systems for the new WRAP 1 facility (Project W-026). The DMS will collect, store and report data required to certify the low level waste (LLW) and transuranic (TRU) waste items processed at WRAP 1 as acceptable for shipment, storage, or disposal.

  5. m6A-Driver: Identifying Context-Specific mRNA m6A Methylation-Driven Gene Interaction Networks

    OpenAIRE

    Zhang, Song-Yao; Zhang, Shao-Wu; Liu, Lian; Meng, Jia; Huang, Yufei

    2016-01-01

    As the most prevalent mammalian mRNA epigenetic modification, N6-methyladenosine (m6A) has been shown to possess important post-transcriptional regulatory functions. However, the regulatory mechanisms and functional circuits of m6A are still largely elusive. To help unveil the regulatory circuitry mediated by mRNA m6A methylation, we develop here m6A-Driver, an algorithm for predicting m6A-driven genes and associated networks, whose functional interactions are likely to be actively modulated ...

  6. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    International Nuclear Information System (INIS)

    Brell, Marta; Ibáñez, Javier; Tortosa, Avelina

    2011-01-01

    The DNA repair protein O 6 -Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably

  7. O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Ibáñez Javier

    2011-01-01

    Full Text Available Abstract Background The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP the most commonly used for promoter methylation study, while immunohistochemistry (IHC has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. Methods A computer-aided search of MEDLINE (1950-October 2009, EBSCO (1966-October 2009 and EMBASE (1974-October 2009 was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Results Of 254 studies identified as eligible for full-text review, 52 (20.5% met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Conclusions Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably.

  8. Safety of Nuclear Power Plants: Commissioning and Operation. Specific Safety Requirements (Arabic Edition)

    International Nuclear Information System (INIS)

    2017-01-01

    This publication is a revision of IAEA Safety Standards Series No. NS-R-2, Safety of Nuclear Power Plants: Operation, and has been extended to cover the commissioning stage. It describes the requirements to be met to ensure the safe commissioning, operation, and transition from operation to decommissioning of nuclear power plants. Over recent years there have been developments in areas such as long term operation of nuclear power plants, plant ageing, periodic safety review, probabilistic safety analysis review and risk informed decision making processes. It became necessary to revise the IAEA’s Safety Requirements in these areas and to correct and/or improve the publication on the basis of feedback from its application by both the IAEA and its Member States. In addition, the requirements are governed by, and must apply, the safety objective and safety principles that are established in the IAEA Safety Standards Series No. SF-1, Fundamental Safety Principles. A review of Safety Requirements publications, initiated in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan, revealed no significant areas of weakness but resulted in a small set of amendments to strengthen the requirements and facilitate their implementation. These are contained in the present publication.

  9. Safety of Nuclear Power Plants: Commissioning and Operation. Specific Safety Requirements

    International Nuclear Information System (INIS)

    2017-01-01

    This publication is a revision of IAEA Safety Standards Series No. NS-R-2, Safety of Nuclear Power Plants: Operation, and has been extended to cover the commissioning stage. It describes the requirements to be met to ensure the safe commissioning, operation, and transition from operation to decommissioning of nuclear power plants. Over recent years there have been developments in areas such as long term operation of nuclear power plants, plant ageing, periodic safety review, probabilistic safety analysis review and risk informed decision making processes. It became necessary to revise the IAEA’s Safety Requirements in these areas and to correct and/or improve the publication on the basis of feedback from its application by both the IAEA and its Member States. In addition, the requirements are governed by, and must apply, the safety objective and safety principles that are established in the IAEA Safety Standards Series No. SF-1, Fundamental Safety Principles. A review of Safety Requirements publications, initiated in 2011 following the accident in the Fukushima Daiichi nuclear power plant in Japan, revealed no significant areas of weakness but resulted in a small set of amendments to strengthen the requirements and facilitate their implementation. These are contained in the present publication.

  10. TECHNICAL BASIS FOR VENTILATION REQUIREMENTS IN TANK FARMS OPERATING SPECIFICATIONS DOCUMENTS

    Energy Technology Data Exchange (ETDEWEB)

    BERGLIN, E J

    2003-06-23

    This report provides the technical basis for high efficiency particulate air filter (HEPA) for Hanford tank farm ventilation systems (sometimes known as heating, ventilation and air conditioning [HVAC]) to support limits defined in Process Engineering Operating Specification Documents (OSDs). This technical basis included a review of older technical basis and provides clarifications, as necessary, to technical basis limit revisions or justification. This document provides an updated technical basis for tank farm ventilation systems related to Operation Specification Documents (OSDs) for double-shell tanks (DSTs), single-shell tanks (SSTs), double-contained receiver tanks (DCRTs), catch tanks, and various other miscellaneous facilities.

  11. Tethys: The Marine and Hydrokinetic Technology Environmental Impacts Knowledge Management System -- Requirements Specification -- Version 1.0

    Energy Technology Data Exchange (ETDEWEB)

    Butner, R. Scott; Snowden-Swan, Lesley J.; Ellis, Peter C.

    2010-11-09

    The marine and hydrokinetic (MHK) environmental impacts knowledge management system (KMS), dubbed Tethys after the mythical Greek goddess of the seas, is being developed for the U.S. Department of Energy (DOE) Office of Energy Efficiency and Renewable Energy Wind and Hydropower Technologies Program (WHTP) by Pacific Northwest National Laboratory (PNNL). This requirements specification establishes the essential capabilities required of Tethys and clarifies for WHTP and the Tethys development team the results that must be achieved by the system.

  12. 24 CFR 200.945 - Supplementary specific requirements under the HUD building product standards and certification...

    Science.gov (United States)

    2010-04-01

    ... under the HUD building product standards and certification program for carpet. 200.945 Section 200.945... requirements under the HUD building product standards and certification program for carpet. (a) Applicable.... (b) Labeling. Under the procedures set forth in § 200.935(d)(6) concerning labeling of a product, the...

  13. Risk-Based Confidentiality Requirements Specification for Outsourced IT Systems (Extended Version)

    NARCIS (Netherlands)

    Morali, A.; Wieringa, Roelf J.

    2010-01-01

    Today, companies are required to be in control of their IT assets, and to provide proof of this in the form of independent IT audit reports. However, many companies have outsourced various parts of their IT systems to other companies, which potentially threatens the control they have of their IT

  14. 40 CFR 270.17 - Specific part B information requirements for surface impoundments.

    Science.gov (United States)

    2010-07-01

    ... migration of any hazardous constituents into the ground water or surface water at any future time; (2) The....19 of this chapter; (5) Proposed action leakage rate, with rationale, if required under § 264.222 of..., including the double liner system, leak detection system, cover system, and appurtenances for control of...

  15. 40 CFR 60.2095 - What site-specific documentation is required?

    Science.gov (United States)

    2010-07-01

    .... (5) Procedures for operating the incinerator and associated air pollution control systems within the... incinerator operating limits. (7) Reporting and recordkeeping procedures. (8) The waste management plan... required? 60.2095 Section 60.2095 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR...

  16. Formal Functional Test Designs: Bridging the Gap Between Test Requirements and Test Specifications

    Science.gov (United States)

    Hops, Jonathan

    1993-01-01

    This presentation describes the testing life cycle, the purpose of the test design phase, and test design methods and gives an example application. Also included is a description of Test Representation Language (TRL), a summary of the language, and an example of an application of TRL. A sample test requirement and sample test design are included.

  17. 49 CFR 180.407 - Requirements for test and inspection of specification cargo tanks.

    Science.gov (United States)

    2010-10-01

    ... may not prevent detection of leaks or damage the venting devices and must be removed immediately after... PIPELINE AND HAZARDOUS MATERIALS SAFETY ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS... percent of the required set pressure, and must reseat to a leak-tight condition at no less than 90 percent...

  18. The requirements and specific features related to power supply in telecommunications of Ukraine

    Energy Technology Data Exchange (ETDEWEB)

    Lubashenko, T. [Inst. Giprosvijaz, Kiev (Ukraine); Gordon, A. [ProElectro Ltd., Kiev (Ukraine)

    2000-07-01

    The article deals with the description of trends of power supplies in the telecommunication sector. The basic normative documents regarding the design of the power supplies are pointed out. The requirements concerning the design of the power supplies are outlined. The peculiarities of the power supplies in Ukrainian telecommunications are given. (orig.)

  19. NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNF AIP3/A20

    NARCIS (Netherlands)

    Drennan, M.B. (Michael B.); Govindarajan, S. (Srinath); Verheugen, E. (Eveline); Coquet, J.M. (Jonathan M.); Staal, J. (Jens); McGuire, C. (Conor); T. Taghon (Tom); G. Leclercq (Georges); R. Beyaert (Rudi); G. van Loo (Geert); B.N.M. Lambrecht (Bart); Elewaut, D. (Dirk)

    2016-01-01

    textabstractNatural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage specification and differentiation remain poorly understood. Here, we demonstrate that the

  20. 40 CFR 270.25 - Specific part B information requirements for equipment.

    Science.gov (United States)

    2010-07-01

    ..., boiler, process heater, condenser, or carbon adsorption system and chooses to use test data to determine....1033(j). (3) A design analysis, specifications, drawings, schematics, and piping and instrumentation... Administrator that present basic control device information. The design analysis shall address the vent stream...

  1. Proliferation requirements of cytomegalovirus-specific, effector-type human CD8+ T cells

    NARCIS (Netherlands)

    van Leeuwen, Ester M.; Gamadia, Laila E.; Baars, Paul A.; Remmerswaal, Ester B.; ten Berge, Ineke J.; van Lier, René A.

    2002-01-01

    Two prototypic types of virus-specific CD8(+) T cells can be found in latently infected individuals: CD45R0(+)CD27(+)CCR7(-) effector-memory, and CD45RA(+)CD27(-)CCR7(-) effector-type cells. It has recently been implied that CD45RA(+)CD27(-)CCR7(-) T cells are terminally differentiated effector

  2. Specification of requirements for the virtual environment for reactor applications simulation environment

    International Nuclear Information System (INIS)

    Hess, S. M.; Pytel, M.

    2012-01-01

    In 2010, the United States Dept. of Energy initiated a research and development effort to develop modern modeling and simulation methods that could utilize high performance computing capabilities to address issues important to nuclear power plant operation, safety and sustainability. To respond to this need, a consortium of national laboratories, academic institutions and industry partners (the Consortium for Advanced Simulation of Light Water Reactors - CASL) was formed to develop an integrated Virtual Environment for Reactor Applications (VERA) modeling and simulation capability. A critical element for the success of the CASL research and development effort was the development of an integrated set of overarching requirements that provides guidance in the planning, development, and management of the VERA modeling and simulation software. These requirements also provide a mechanism from which the needs of a broad array of external CASL stakeholders (e.g. reactor / fuel vendors, plant owner / operators, regulatory personnel, etc.) can be identified and integrated into the VERA development plans. This paper presents an overview of the initial set of requirements contained within the VERA Requirements Document (VRD) that currently is being used to govern development of the VERA software within the CASL program. The complex interdisciplinary nature of these requirements together with a multi-physics coupling approach to realize a core simulator capability pose a challenge to how the VRD should be derived and subsequently revised to accommodate the needs of different stakeholders. Thus, the VRD is viewed as an evolving document that will be updated periodically to reflect the changing needs of identified CASL stakeholders and lessons learned during the progress of the CASL modeling and simulation program. (authors)

  3. Interleukin-1 is required for cancer eradication mediated by tumor-specific Th1 cells.

    Science.gov (United States)

    Haabeth, Ole Audun Werner; Lorvik, Kristina Berg; Yagita, Hideo; Bogen, Bjarne; Corthay, Alexandre

    The role of inflammation in cancer is controversial as both tumor-promoting and tumor-suppressive aspects of inflammation have been reported. In particular, it has been shown that pro-inflammatory cytokines, like interleukin-1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor α (TNFα), may either promote or suppress cancer. However, the cellular and molecular basis underlying these opposing outcomes remains enigmatic. Using mouse models for myeloma and lymphoma, we have recently reported that inflammation driven by tumor-specific T helper 1 (Th1) cells conferred protection against B-cell cancer and that interferon-γ (IFN-γ) was essential for this process. Here, we have investigated the contribution of several inflammatory mediators. Myeloma eradication by Th1 cells was not affected by inhibition of TNF-α, TNF-related weak inducer of apoptosis (TWEAK), or TNF-related apoptosis-inducing ligand (TRAIL). In contrast, cancer elimination by tumor-specific Th1 cells was severely impaired by the in vivo neutralization of both IL-1α and IL-1β (collectively named IL-1) with IL-1 receptor antagonist (IL-1Ra). The antitumor functions of tumor-specific Th1 cells and tumor-infiltrating macrophages were both affected by IL-1 neutralization. Secretion of the Th1-derived cytokines IL-2 and IFN-γ at the incipient tumor site was severely reduced by IL-1 blockade. Moreover, IL-1 was shown to synergize with IFN-γ for induction of tumoricidal activity in tumor-infiltrating macrophages. This synergy between IL-1 and IFN-γ may explain how inflammation, when driven by tumor-specific Th1 cells, represses rather than promotes cancer. Collectively, the data reveal a central role of inflammation, and more specifically of the canonical pro-inflammatory cytokine IL-1, in enhancing Th1-mediated immunity against cancer.

  4. Expression of MUC17 is regulated by HIF1α-mediated hypoxic responses and requires a methylation-free hypoxia responsible element in pancreatic cancer.

    Directory of Open Access Journals (Sweden)

    Sho Kitamoto

    Full Text Available MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs; however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α-dependent pathway in some pancreatic cancer cells (e.g., AsPC1, whereas other pancreatic cancer cells (e.g., BxPC3 exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5'-RCGTG-3', a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2'-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells.

  5. The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana.

    Science.gov (United States)

    Hancock, C Nathan; Kent, Lia; McClure, Bruce A

    2005-09-01

    S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.

  6. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Science.gov (United States)

    Lu, Chenggang; Fuller, Margaret T

    2015-12-01

    Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  7. Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

    Directory of Open Access Journals (Sweden)

    Chenggang Lu

    2015-12-01

    Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

  8. CRISP. Requirements Specifications of Intelligent ICT Simulation Tools for Power Applications

    Energy Technology Data Exchange (ETDEWEB)

    Warmer, C.J.; Kester, J.C.P.; Kamphuis, I.G. [ECN Energy in the Built Environment and Networks, Petten (Netherlands); Carlsson, P [EnerSearch, Malmoe (Sweden); Fontela, M. [Laboratory of Electrical Engineering LEG, Grenoble (France); Gustavsson, R. [Blekinge Institute of Technology BTH, Karlskrona (Sweden)

    2003-10-15

    This report, deliverable D2.1 in the CRISP project, serves as a preparation report for the development of simulation tools and prototype software which will be developed in forthcoming stages of the CRISP project. Application areas for these simulations are: fault detection and diagnosis, supply and demand matching and intelligent load shedding. The context in which these applications function is the power network with a high degree of distributed generation, including renewables. In order to control a so called distributed grid we can benefit from a high level of distributed control and intelligence. This requires, on top of the power system network, an information and communication network.. We argue that such a network should be seen as an enabler of distributed control and intelligence. The applications, through which control and intelligence is implemented, then form a third network layer, the service oriented network. Building upon this three-layered network model we derive in this report the requirements for a simulation tool and experiments which study new techniques for fault detection and diagnostics and for simulation tools and experiments implementing intelligent load shedding and supply and demand matching scenarios. We also look at future implementation of these services within the three-layered network model and the requirements that follow for the core information and communication network and for the service oriented network. These requirements, supported by the studies performed in the CRISP Workpackage 1, serve as a basis for development of the simulation tools in the tasks 2.2 to 2.4.

  9. CRISP. Requirements Specifications of Intelligent ICT Simulation Tools for Power Applications

    International Nuclear Information System (INIS)

    Warmer, C.J.; Kester, J.C.P.; Kamphuis, I.G.; Carlsson, P; Fontela, M.; Gustavsson, R.

    2003-10-01

    This report, deliverable D2.1 in the CRISP project, serves as a preparation report for the development of simulation tools and prototype software which will be developed in forthcoming stages of the CRISP project. Application areas for these simulations are: fault detection and diagnosis, supply and demand matching and intelligent load shedding. The context in which these applications function is the power network with a high degree of distributed generation, including renewables. In order to control a so called distributed grid we can benefit from a high level of distributed control and intelligence. This requires, on top of the power system network, an information and communication network.. We argue that such a network should be seen as an enabler of distributed control and intelligence. The applications, through which control and intelligence is implemented, then form a third network layer, the service oriented network. Building upon this three-layered network model we derive in this report the requirements for a simulation tool and experiments which study new techniques for fault detection and diagnostics and for simulation tools and experiments implementing intelligent load shedding and supply and demand matching scenarios. We also look at future implementation of these services within the three-layered network model and the requirements that follow for the core information and communication network and for the service oriented network. These requirements, supported by the studies performed in the CRISP Workpackage 1, serve as a basis for development of the simulation tools in the tasks 2.2 to 2.4

  10. Cell-specific differences in the requirements for translation quality control

    DEFF Research Database (Denmark)

    Reynolds, Noah M; Ling, Jiqiang; Roy, Hervé

    2010-01-01

    Protein synthesis has an overall error rate of approximately 10(-4) for each mRNA codon translated. The fidelity of translation is mainly determined by two events: synthesis of cognate amino acid:tRNA pairs by aminoacyl-tRNA synthetases (aaRSs) and accurate selection of aminoacyl-tRNAs (aa-tRNAs)...... divergent requirements for quality control in different cell compartments and suggest that the limits of translational accuracy may be largely determined by cellular physiology....

  11. Specification and time required for the application of a lime-based render inside historic buildings

    Directory of Open Access Journals (Sweden)

    Vasco Peixoto de Freitas

    2008-01-01

    Full Text Available Intervention in ancient buildings with historical and architectural value requires traditional techniques, such as the use of lime mortars for internal and external wall renderings. In order to ensure the desired performance, these rendering mortars must be rigorously specified and quality controls have to be performed during application. The choice of mortar composition should take account of factors such as compatibility with the substrate, mechanical requirements and water behaviour. The construction schedule, which used to be considered a second order variable, nowadays plays a decisive role in the selection of the rendering technique, given its effects upon costs. How should lime-based mortars be specified? How much time is required for the application and curing of a lime-based render? This paper reflects upon the feasibility of using traditional lime mortars in three-layer renders inside churches and monasteries under adverse hygrothermal conditions and when time is critical. A case study is presented in which internal lime mortar renderings were applied in a church in Northern Portugal, where the very high relative humidity meant that several months were necessary before the drying process was complete.

  12. Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

    Science.gov (United States)

    Zhu, Wei; Pao, Gerald M; Satoh, Akira; Cummings, Gillian; Monaghan, James R; Harkins, Timothy T; Bryant, Susan V; Voss, S Randal; Gardiner, David M; Hunter, Tony

    2013-01-01

    The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration – wound healing, cellular dedifferentiation and blastemal formation – will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration. PMID:22841627

  13. Sex-specific signaling in the blood-brain barrier is required for male courtship in Drosophila.

    Directory of Open Access Journals (Sweden)

    Valbona Hoxha

    Full Text Available Soluble circulating proteins play an important role in the regulation of mating behavior in Drosophila melanogaster. However, how these factors signal through the blood-brain barrier (bbb to interact with the sex-specific brain circuits that control courtship is unknown. Here we show that male identity of the blood-brain barrier is necessary and that male-specific factors in the bbb are physiologically required for normal male courtship behavior. Feminization of the bbb of adult males significantly reduces male courtship. We show that the bbb-specific G-protein coupled receptor moody and bbb-specific Go signaling in adult males are necessary for normal courtship. These data identify sex-specific factors and signaling processes in the bbb as important regulators of male mating behavior.

  14. 47 CFR 95.1119 - Specific requirements for wireless medical telemetry devices operating in the 608-614 MHz band.

    Science.gov (United States)

    2010-10-01

    ... devices operating in the 608-614 MHz band. For a wireless medical telemetry device operating within the... 47 Telecommunication 5 2010-10-01 2010-10-01 false Specific requirements for wireless medical telemetry devices operating in the 608-614 MHz band. 95.1119 Section 95.1119 Telecommunication FEDERAL...

  15. 47 CFR 95.1121 - Specific requirements for wireless medical telemetry devices operating in the 1395-1400 and 1427...

    Science.gov (United States)

    2010-10-01

    ... wireless medical telemetry devices operating in the 1395-1400 and 1427-1432 MHz bands. Due to the critical... 47 Telecommunication 5 2010-10-01 2010-10-01 false Specific requirements for wireless medical telemetry devices operating in the 1395-1400 and 1427-1432 MHz bands. 95.1121 Section 95.1121...

  16. 40 CFR Table C-1 to Subpart C of... - Test Concentration Ranges, Number of Measurements Required, and Maximum Discrepancy Specification

    Science.gov (United States)

    2010-07-01

    ... Measurements Required, and Maximum Discrepancy Specification C Table C-1 to Subpart C of Part 53 Protection of... Reference Methods Pt. 53, Subpt. C, Table C-1 Table C-1 to Subpart C of Part 53—Test Concentration Ranges..., June 22, 2010, table C-1 to subpart C was revised, effective Aug. 23, 2010. For the convenience of the...

  17. 2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    Science.gov (United States)

    Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

    2012-01-01

    RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

  18. Isozyme-specific enzyme inhibitors. 14. 5'(R)-C-[(L-homocystein-S-yl)methyl]adenosine 5'-(beta,gamma-imidotriphosphate), a potent inhibitor of rat methionine adenosyltransferases.

    Science.gov (United States)

    Kappler, F; Vrudhula, V M; Hampton, A

    1987-09-01

    The title compound is a covalent adduct of L-methionine (Met) and beta,gamma-imido-ATP. In its synthesis the N-Boc derivative of 5'(R)-C-(aminomethyl)-N6-benzoyl-5'-O-tosyl-2',3'-O- isopropylidenadenosine was converted by the successive actions of CF3CO2H and HNO2 into the corresponding 5'(R)-C-hydroxymethyl derivative. Treatment of this with disodium L-homocysteinate led to attack of sulfur at C6', apparently via a 5',6'-epoxide, and to total stereoselective inversion at C5' to furnish, after debenzoylation, 5'(R)-C-(L-homocystein-S-ylmethyl)-2',3'-O-isopropylidene ade nosine. The 5' configuration was established by conversion of this into the known 5'(S)-C-methyl-2',3'-O-isopropylidene adenosine with Raney nickel. The alpha-amino acid residue was protected as an N-Boc methyl ester, after which the 5'-hydroxyl was phosphorylated with benzyl phosphate and dicyclohexylcarbodiimide. The phosphoanhydride bond with inorganic imidodiphosphate was then created by established methods. Finally, blocking groups were removed under conditions that gave the desired adduct with no racemization of its L-methionine residue. It was a potent inhibitor [KM(ATP)/Ki = 1080; KM(Met)/Ki = 7.7] of the M-2 (normal tissue) form of rat methionine adenosyltransferase and of the M-T (hepatoma tissue) form [KM(ATP)/Ki = 670; KM(Met)/Ki = 22]. Inhibitions were competitive with respect to ATP or to L-methionine, indicating a dual substrate site mode of binding to the enzyme forms.

  19. PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

    Science.gov (United States)

    Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

    2016-11-15

    Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. The Image Quality Translator – A Way to Support Specification of Imaging Requirements

    DEFF Research Database (Denmark)

    Kejser, Ulla Bøgvad; Bech, Mogens

    2015-01-01

    Archives, libraries, and museums run numerous imaging projects to digitize physical works and collections of cultural heritage. This study presents a tool called the 'Image Quality Translator' that is being designed at the Royal Library to support the planning of digitization projects and to make...... the process of specifying and controlling imaging requirements more efficient. The tool seeks to translate between the language used by collection managers and curators to express needs for image quality, and the more technical terms and metrics used by imaging experts and photographers to express...

  1. Requirements and prototype for supporting the planning of patient specific thermal ablation interventions

    International Nuclear Information System (INIS)

    Schramm, W.

    2010-01-01

    Background Thermal ablation is the process of destroying pathological tissue by either high temperatures of approximately 105 o C as achieved in radiofrequency ablation or low temperatures of approximately - 40 o C as used in cryotherapy. Ablations are widely used in clinical practice and provide a safe and generally well tolerated minimal invasive treatment if surgery is not an option. Thermal ablations are usually performed under image guidance, either by ultrasound, CT or MR. Even though ablations are widely used, very little textbook knowledge is available. Because of the treatment complexity there is a need for a well defined process which can be followed by an experienced radiologist as well as an inexperienced one. There is also a need for a planning platform which is capable of supporting the physician in planning the intervention on the basis of the patient's anatomy. For additional benefit this platform should also provide the means for estimating the final coagulation zone by simulations based on the patient's anatomy. The most widely used method to simulate the extend of a coagulation zone is by the usage of finite element analysis (FEA). FEA uses a defined geometry with the physical properties of the tissue and the ablation modality to create a model which can then be solved to make estimations about the extend of the final coagulation zone. Method and Results To deal with the problem of ablation knowledge being only available in distributed form, a workflow was abstracted and translated into diagrams. These workflow diagrams visualize the required steps and decisions when performing thermal ablations. The workflow is split into a planning, applicator placement, ablation and result evaluation phase. The information gained from this knowledge is then used to define the requirements for a platform which is capable of helping the physician when performing the ablation. In the next step I examined the possibility to increase an ablation's coagulation zone

  2. Surgical PACS for the digital operating room. Systems engineering and specification of user requirements.

    Science.gov (United States)

    Korb, Werner; Bohn, Stefan; Burgert, Oliver; Dietz, Andreas; Jacobs, Stephan; Falk, Volkmar; Meixensberger, Jürgen; Strauss, Gero; Trantakis, Christos; Lemke, Heinz U

    2006-01-01

    For better integration of surgical assist systems into the operating room, a common communication and processing plattform that is based on the users needs is needed. The development of such a system, a Surgical Picture Aquisition and Communication System (S-PACS), according the systems engineering cycle is oulined in this paper. The first two steps (concept and specification) for the engineering of the S-PACS are discussed.A method for the systematic integration of the users needs', the Quality Function Deployment (QFD), is presented. The properties of QFD for the underlying problem and first results are discussed. Finally, this leads to a first definition of an S-PACS system.

  3. Layer specific and general requirements for ERK/MAPK signaling in the developing neocortex

    Science.gov (United States)

    Xing, Lei; Larsen, Rylan S; Bjorklund, George Reed; Li, Xiaoyan; Wu, Yaohong; Philpot, Benjamin D; Snider, William D; Newbern, Jason M

    2016-01-01

    Aberrant signaling through the Raf/MEK/ERK (ERK/MAPK) pathway causes pathology in a family of neurodevelopmental disorders known as 'RASopathies' and is implicated in autism pathogenesis. Here, we have determined the functions of ERK/MAPK signaling in developing neocortical excitatory neurons. Our data reveal a critical requirement for ERK/MAPK signaling in the morphological development and survival of large Ctip2+ neurons in layer 5. Loss of Map2k1/2 (Mek1/2) led to deficits in corticospinal tract formation and subsequent corticospinal neuron apoptosis. ERK/MAPK hyperactivation also led to reduced corticospinal axon elongation, but was associated with enhanced arborization. ERK/MAPK signaling was dispensable for axonal outgrowth of layer 2/3 callosal neurons. However, Map2k1/2 deletion led to reduced expression of Arc and enhanced intrinsic excitability in both layers 2/3 and 5, in addition to imbalanced synaptic excitation and inhibition. These data demonstrate selective requirements for ERK/MAPK signaling in layer 5 circuit development and general effects on cortical pyramidal neuron excitability. DOI: http://dx.doi.org/10.7554/eLife.11123.001 PMID:26848828

  4. NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20.

    Science.gov (United States)

    Drennan, Michael B; Govindarajan, Srinath; Verheugen, Eveline; Coquet, Jonathan M; Staal, Jens; McGuire, Conor; Taghon, Tom; Leclercq, Georges; Beyaert, Rudi; van Loo, Geert; Lambrecht, Bart N; Elewaut, Dirk

    2016-09-19

    Natural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage specification and differentiation remain poorly understood. Here, we demonstrate that the ubiquitin-modifying enzyme TNFAIP3/A20, an upstream regulator of T cell receptor (TCR) signaling in T cells, is an essential cell-intrinsic regulator of NKT differentiation. A20 is differentially expressed during NKT cell development, regulates NKT cell maturation, and specifically controls the differentiation and survival of NKT1 and NKT2, but not NKT17, sublineages. Remaining A20-deficient NKT1 and NKT2 thymocytes are hyperactivated in vivo and secrete elevated levels of Th1 and Th2 cytokines after TCR ligation in vitro. Defective NKT development was restored by compound deficiency of MALT1, a key downstream component of TCR signaling in T cells. These findings therefore show that negative regulation of TCR signaling during NKT development controls the differentiation and survival of NKT1 and NKT2 cells. © 2016 Drennan et al.

  5. Requirements and Specifications for a Simplified, Low Pressure Medium Sized PWR

    International Nuclear Information System (INIS)

    Nisan, S.; Raymond, P.; Gautier, G-M.; Pignatel, J-F.

    1998-01-01

    This paper summarises part of our on-going investigations on the possible introduction of nuclear energy in developing countries or countries with low capacity electrical grids. These investigations are principally concerned with future PWR developments and basically try to search for plausible and economic answers to the three difficult questions that each nuclear technology exporting country faces today: 1)- how to compensate the apparent loss of economic competitiveness, related to the scaling effect, of a small or medium sized reactor? 2)- how to reconcile the introduction of nuclear energy on the large scale with the two major preoccupations of nuclear safety and nuclear proliferation? 3)- how to demonstrate that the proposed concept(s) can effectively meet the safety objectives of the requirements for future reactors in Europe and in other countries?

  6. SELECTED REQUIREMENTS OF INTEGRATED MANAGEMENT SYSTEMS BASED ON PAS 99 SPECIFICATION

    Directory of Open Access Journals (Sweden)

    Paweł Nowicki

    2013-03-01

    Full Text Available The aim this research was to analyze the ways of integration of management systems in food sector. The study involved the documentation, audits, corrective and preventive actions and management's review phases described in the specification PAS 99, which is one of common elements of integrated management systems. Four organizations were selected for the study. The organizations had introduced and certified at least two standardized management systems. It was assumed that the investigated organizations should have implemented the HACCP system. Studies were conducted as a case study. The employees responsible for the functioning of management systems were interviewed in all four organizations. The study was conducted in the form of in-depth interviews based on pre-prepared script. The scenario was developed based on the PAS 99 guideline. The process of integration of management systems implemented in the studied companies reveals the full compliance of an integrated management system with PASS 99 in the policy area.

  7. Hox genes require homothorax and extradenticle for body wall identity specification but not for appendage identity specification during metamorphosis of Tribolium castaneum.

    Science.gov (United States)

    Smith, Frank W; Jockusch, Elizabeth L

    2014-11-01

    The establishment of segment identity is a key developmental process that allows for divergence along the anteroposterior body axis in arthropods. In Drosophila, the identity of a segment is determined by the complement of Hox genes it expresses. In many contexts, Hox transcription factors require the protein products of extradenticle (exd) and homothorax (hth) as cofactors to perform their identity specification functions. In holometabolous insects, segment identity may be specified twice, during embryogenesis and metamorphosis. To glean insight into the relationship between embryonic and metamorphic segmental identity specification, we have compared these processes in the flour beetle Tribolium castaneum, which develops ventral appendages during embryogenesis that later metamorphose into adult appendages with distinct morphologies. At metamorphosis, comparisons of RNAi phenotypes indicate that Hox genes function jointly with Tc-hth and Tc-exd to specify several region-specific aspects of the adult body wall. On the other hand, Hox genes specify appendage identities along the anteroposterior axis independently of Tc-hth/Tc-exd and Tc-hth/Tc-exd specify proximal vs. distal identity within appendages independently of Hox genes during this stage. During embryogenesis, Tc-hth and Tc-exd play a broad role in the segmentation process and are required for specification of body wall identities in the thorax; however, contrasting with results from other species, we did not obtain homeotic transformations of embryonic appendages in response to Tc-hth or Tc-exd RNAi. In general, the homeotic effects of interference with the function of Hox genes and Tc-hth/Tc-exd during metamorphosis did not match predictions based on embryonic roles of these genes. Comparing metamorphic patterning in T. castaneum to embryonic and post-embryonic development in hemimetabolous insects suggests that holometabolous metamorphosis combines patterning processes of both late embryogenesis and

  8. Safety of Nuclear Power Plants: Design. Specific Safety Requirements (Russian Edition); Bezopasnost' atomnykh ehlektrostantsij: proektirovanie. Konkretnye trebovaniya bezopasnosti

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2012-04-15

    This publication is a revision of Safety Requirements No. NS-R-1, Safety of Nuclear Power Plants: Design. It establishes requirements applicable to the design of nuclear power plants and elaborates on the safety objective, safety principles and concepts that provide the basis for deriving the safety requirements that must be met for the design of a nuclear power plant. It will be useful for organizations involved in the design, manufacture, construction, modification, maintenance, operation and decommissioning of nuclear power plants, as well as for regulatory bodies. Contents: 1. Introduction; 2. Applying the safety principles and concepts; 3. Management of safety in design; 4. Principal technical requirements; 5. General plant design; 6. Design of specific plant systems.

  9. Quantifying sex, race, and age specific differences in bone microstructure requires measurement of anatomically equivalent regions.

    Science.gov (United States)

    Ghasem-Zadeh, Ali; Burghardt, Andrew; Wang, Xiao-Fang; Iuliano, Sandra; Bonaretti, Serena; Bui, Minh; Zebaze, Roger; Seeman, Ego

    2017-08-01

    Individuals differ in forearm length. As microstructure differs along the radius, we hypothesized that errors may occur when sexual and racial dimorphisms are quantified at a fixed distance from the radio-carpal joint. Microstructure was quantified ex vivo in 18 cadaveric radii using high resolution peripheral quantitative computed tomography and in vivo in 158 Asian and Caucasian women and men at a fixed region of interest (ROI), a corrected ROI positioned at 4.5-6% of forearm length and using the fixed ROI adjusted for cross sectional area (CSA), forearm length or height. Secular effects of age were assessed by comparing 38 younger and 33 older women. Ex vivo, similar amounts of bone mass fashioned adjacent cross sections. Larger distal cross sections had thinner porous cortices of lower matrix mineral density (MMD), a larger medullary CSA and higher trabecular density. Smaller proximal cross-sections had thicker less porous cortices of higher MMD, a small medullary canal with little trabecular bone. Taller persons had more distally positioned fixed ROIs which moved proximally when corrected. Shorter persons had more proximally positioned fixed ROIs which moved distally when corrected, so dimorphisms lessened. In the corrected ROIs, in Caucasians, women had 0.6 SD higher porosity and 0.6 SD lower trabecular density than men (pmicrostructure requires measurement of anatomically equivalent regions. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

    Science.gov (United States)

    Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

    2015-08-01

    Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  11. Donor-specific antibodies require preactivated immune system to harm renal transplant.

    Science.gov (United States)

    Süsal, Caner; Döhler, Bernd; Ruhenstroth, Andrea; Morath, Christian; Slavcev, Antonij; Fehr, Thomas; Wagner, Eric; Krüger, Bernd; Rees, Margaret; Balen, Sanja; Živčić-Ćosić, Stela; Norman, Douglas J; Kuypers, Dirk; Emonds, Marie-Paule; Pisarski, Przemyslaw; Bösmüller, Claudia; Weimer, Rolf; Mytilineos, Joannis; Scherer, Sabine; Tran, Thuong H; Gombos, Petra; Schemmer, Peter; Zeier, Martin; Opelz, Gerhard

    2016-07-01

    It is an unresolved issue why some kidney transplant recipients with pretransplant donor-specific HLA antibodies (DSA) show a high transplant failure rate, whereas in other patients DSA do not harm the graft. We investigated whether help from preactivated T-cells might be necessary for DSA to exert a deleterious effect. The impact of pretransplant DSA and immune activation marker soluble CD30 (sCD30) on 3-year graft survival was analyzed in 385 presensitized kidney transplant recipients. A deleterious influence of pretransplant DSA on graft survival was evident only in patients who were positive for the immune activation marker sCD30. In the absence of sCD30 positivity, 3-year graft survival was virtually identical in patients with or without DSA (83.1±3.9% and 84.3±2.8%, P=0.81). A strikingly lower 3-year graft survival rate of 62.1±6.4% was observed in patients who were both sCD30 and DSA positive (HR 2.92, PsCD30 negative. Pretransplant DSA have a significantly deleterious impact on graft survival only in the presence of high pretransplant levels of the activation marker sCD30. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Characterization of Dnmt1 Binding and DNA Methylation on Nucleosomes and Nucleosomal Arrays.

    Directory of Open Access Journals (Sweden)

    Anna Schrader

    Full Text Available The packaging of DNA into nucleosomes and the organisation into higher order structures of chromatin limits the access of sequence specific DNA binding factors to DNA. In cells, DNA methylation is preferentially occuring in the linker region of nucleosomes, suggesting a structural impact of chromatin on DNA methylation. These observations raise the question whether DNA methyltransferases are capable to recognize the nucleosomal substrates and to modify the packaged DNA. Here, we performed a detailed analysis of nucleosome binding and nucleosomal DNA methylation by the maintenance DNA methyltransferase Dnmt1. Our binding studies show that Dnmt1 has a DNA length sensing activity, binding cooperatively to DNA, and requiring a minimal DNA length of 20 bp. Dnmt1 needs linker DNA to bind to nucleosomes and most efficiently recognizes nucleosomes with symmetric DNA linkers. Footprinting experiments reveal that Dnmt1 binds to both DNA linkers exiting the nucleosome core. The binding pattern correlates with the efficient methylation of DNA linkers. However, the enzyme lacks the ability to methylate nucleosomal CpG sites on mononucleosomes and nucleosomal arrays, unless chromatin remodeling enzymes create a dynamic chromatin state. In addition, our results show that Dnmt1 functionally interacts with specific chromatin remodeling enzymes to enable complete methylation of hemi-methylated DNA in chromatin.

  13. Stage specific requirement of platelet-derived growth factor receptor-α in embryonic development.

    Directory of Open Access Journals (Sweden)

    Chen Qian

    Full Text Available Platelet-derived growth factor receptor alpha (PDGFRα is a cell-surface receptor tyrosine kinase for platelet-derived growth factors. Correct timing and level of Pdgfra expression is crucial for embryo development, and deletion of Pdgfra caused developmental defects of multiple endoderm and mesoderm derived structures, resulting in a complex phenotypes including orofacial cleft, spina bifida, rib deformities, and omphalocele in mice. However, it is not clear if deletion of Pdgfra at different embryonic stages differentially affects these structures.To address the temporal requirement of Pdgfra in embryonic development.We have deleted the Pdgfra in Pdgfra-expressing tissues at different embryonic stages in mice, examined and quantified the developmental anomalies.Current study showed that (i conditional deletion of Pdgfra at different embryonic days (between E7.5 and E10.5 resulted in orofacial cleft, spina bifida, rib cage deformities, and omphalocele, and (ii the day of Pdgfra deletion influenced the combinations, incidence and severities of these anomalies. Deletion of Pdgfra caused apoptosis of Pdgfra-expressing tissues, and developmental defects of their derivatives.Orofacial cleft, spina bifida and omphalocele are among the commonest skeletal and abdominal wall defects of newborns, but their genetic etiologies are largely unknown. The remarkable resemblance of our conditional Pdgfra knockout embryos to theses human congenital anomalies, suggesting that dysregulated PDGFRA expression could cause these anomalies in human. Future work should aim at defining (a the regulatory elements for the expression of the human PDGFRA during embryonic development, and (b if mutations / sequence variations of these regulatory elements cause these anomalies.

  14. A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

    Directory of Open Access Journals (Sweden)

    M. Lauren Donnelly

    2017-01-01

    Full Text Available Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use transmembrane germinant receptors to trigger germination, C. difficile uses the pseudoprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hydrolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family proteins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG, a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies.

  15. Improving software requirements specification for safety-related systems using the fault tree developed by an object-based method

    International Nuclear Information System (INIS)

    Cepin, M.; Mavko, B.

    1998-01-01

    A modification of the fault tree analysis is presented. The new fault tree integrates structural and behavioral models of a system. Information on the system structure is captured in the name of each gate and basic event of the fault tree. Information on the system behavior is captured in their description. Behavior is expressed using the axiomatic notation based on first order predicate logic. The new fault tree is a useful model for analysis and improvement of software requirements specification. The benefit of such improvements is reduced probability of failures in specification, which in turn results in increased reliability of the software.(author)

  16. Specific enkephalin-degrading aminopeptidase activity in the HPT and HPO axes of rats with breast cancer induced by N-methyl nitrosourea.

    Science.gov (United States)

    Carrera, María del Pilar; Ramírez-Expósito, María Jesús; Valenzuela, María Teresa; García, María Jesús; Mayas, María Dolores; Arias de Saavedra, José Manuel; Sánchez, Rafael; Pérez, María del Carmen; Martínez-Martos, José Manuel

    2005-01-15

    State and function of breast depend on an endocrinological balance, the upsetting of which can be a factor favorable to the development of cancer. Enkephalins (ENK) have been considered as a particular form of adaptation to defense to the organism against neoplastic processes. However, ENK may modify the endocrine functions of glands such as the ovary or the thyroid through the hypothalamus-pituitary axis, acting direct or indirectly as endocrine, paracrine or autocrine stimulatory growth factors. The present work analyses enkephalin-degrading tyrosyl aminopeptidase (EDA) activity in the hypothalamus-pituitary-thyroid (HPT) and hypothalamus-pituitary-ovary (HPO) axes in a rat model of breast cancer induced by N-methyl-nitrosourea (NMU) to state the relationship between ENK levels modification through EDA activity at different neuroendocrine levels and breast cancer. Results obtained show a decrease in EDA activity in hypothalamus, anterior and posterior pituitary, thyroid and ovary, suggesting increased levels of ENK in all these locations. These ENK may induce breast cancer cell growth and progression not only at breast level, but also acting at several neuroendocrine levels such as the HPT and HPO axes, inducing an unbalance of several other hormones, which could also facilitate the progression of cancer as an undesirable concomitant effect.

  17. PARTICLE, a Triplex-Forming Long ncRNA, Regulates Locus-Specific Methylation in Response to Low-Dose Irradiation

    Directory of Open Access Journals (Sweden)

    Valerie Bríd O’Leary

    2015-04-01

    Full Text Available Exposure to low-dose irradiation causes transiently elevated expression of the long ncRNA PARTICLE (gene PARTICLE, promoter of MAT2A-antisense radiation-induced circulating lncRNA. PARTICLE affords both a cytosolic scaffold for the tumor suppressor methionine adenosyltransferase (MAT2A and a nuclear genetic platform for transcriptional repression. In situ hybridization discloses that PARTICLE and MAT2A associate together following irradiation. Bromouridine tracing and presence in exosomes indicate intercellular transport, and this is supported by ex vivo data from radiotherapy-treated patients. Surface plasmon resonance indicates that PARTICLE forms a DNA-lncRNA triplex upstream of a MAT2A promoter CpG island. We show that PARTICLE represses MAT2A via methylation and demonstrate that the radiation-induced PARTICLE interacts with the transcription-repressive complex proteins G9a and SUZ12 (subunit of PRC2. The interplay of PARTICLE with MAT2A implicates this lncRNA in intercellular communication and as a recruitment platform for gene-silencing machineries through triplex formation in response to irradiation.

  18. Modifying effects of lemongrass essential oil on specific tissue response to the carcinogen N-methyl-N-nitrosurea in female BALB/c mice.

    Science.gov (United States)

    Bidinotto, Lucas T; Costa, Celso A R A; Costa, Mirtes; Rodrigues, Maria A M; Barbisan, Luís F

    2012-02-01

    Lemongrass (Cymbopogon citratus Stapf) essential oil has been used worldwide because of its ethnobotanical and medicinal usefulness. Regarding its medicinal usefulness, the present study evaluated the beneficial effects of lemongrass essential oil (LGEO) oral treatment on cell proliferation and apoptosis events and on early development of hyperplastic lesions in the mammary gland, colon, and urinary bladder induced by N-methyl-N-nitrosourea (MNU) in female BALB/c mice. The animals were allocated into three groups: G1, treated with LGEO vehicle for 5 weeks (five times per week); G2, treated with LGEO vehicle as for G1 and MNU (two injections each of 30 mg/kg of body weight at weeks 3 and 5); and G3, treated with LGEO (five times each with 500 mg/kg of body weight per week) and MNU as for G2. Twenty-four hours after the last MNU application, all animals were euthanized, and mammary glands, colon, and urinary bladder were collected for histological and immunohistochemical analysis. LGEO oral treatment significantly changed the indexes of apoptosis and/or cellular proliferation for the tissues analyzed. In particular, the treatment reduced the incidence of hyperplastic lesions and increased apoptosis in mammary epithelial cells. This increment in the apoptosis response may be related to a favorable balance in Bcl-2/Bax immunoreactivity in mammary epithelial cells. These findings indicate that LGEO presented a protective role against early MNU-induced mammary gland alterations in BALB/c mice.

  19. Definition of Specific Functions and Procedural Skills Required by Cuban Specialists in Intensive Care and Emergency Medicine.

    Science.gov (United States)

    Véliz, Pedro L; Berra, Esperanza M; Jorna, Ana R

    2015-07-01

    INTRODUCTION Medical specialties' core curricula should take into account functions to be carried out, positions to be filled and populations to be served. The functions in the professional profile for specialty training of Cuban intensive care and emergency medicine specialists do not include all the activities that they actually perform in professional practice. OBJECTIVE Define the specific functions and procedural skills required of Cuban specialists in intensive care and emergency medicine. METHODS The study was conducted from April 2011 to September 2013. A three-stage methodological strategy was designed using qualitative techniques. By purposive maximum variation sampling, 82 professionals were selected. Documentary analysis and key informant criteria were used in the first stage. Two expert groups were formed in the second stage: one used various group techniques (focus group, oral and written brainstorming) and the second used a three-round Delphi method. In the final stage, a third group of experts was questioned in semistructured in-depth interviews, and a two-round Delphi method was employed to assess priorities. RESULTS Ultimately, 78 specific functions were defined: 47 (60.3%) patient care, 16 (20.5%) managerial, 6 (7.7%) teaching, and 9 (11.5%) research. Thirty-one procedural skills were identified. The specific functions and procedural skills defined relate to the profession's requirements in clinical care of the critically ill, management of patient services, teaching and research at the specialist's different occupational levels. CONCLUSIONS The specific functions and procedural skills required of intensive care and emergency medicine specialists were precisely identified by a scientific method. This product is key to improving the quality of teaching, research, administration and patient care in this specialty in Cuba. The specific functions and procedural skills identified are theoretical, practical, methodological and social contributions to

  20. DNA sequence explains seemingly disordered methylation levels in partially methylated domains of Mammalian genomes.

    Directory of Open Access Journals (Sweden)

    Dimos Gaidatzis

    2014-02-01

    Full Text Available For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs, recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%-40% of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS. Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R(2 of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features.

  1. Development of guidance on applications of regulatory requirements for low specific activity materials and surface contaminated objects

    International Nuclear Information System (INIS)

    Pope, R.B.; Easton, E.P.; Shankman, S.F.

    1997-01-01

    In 1985, the International Atomic Energy Agency issued revised regulations for the safe transport of radioactive material. Significant among the changes were major revisions to requirements for Low Specific Activity (LSA) material and Surface Contaminated Objects (SCOs). In preparation for the adoption of these requirements into regulations in the United States, it became apparent that guidance on how to apply these requirements, clarifying technical uncertainties and ensuring proper implementation, would be needed both by the regulators and those regulated. Thus, the US Department of Transportation and the US Nuclear Regulatory Commission, with the assistance of staff from Oak Ridge National Laboratory, are preparing regulatory guidance for LSA material and SCO transport. The guidance will present examples of acceptable methods for demonstrating compliance with the revised rules. Ideas being investigated for inclusion in the pending guidance are discussed in this paper. Under current plans, the guidance will be issued for public comment prior to final issuance of the guidance in 1997

  2. Development of guidance on applications of regulatory requirements for low specific activity materials and surface contaminated objects

    International Nuclear Information System (INIS)

    Pope, R.B.; Easton, E.P.; Shankman, S.F.; Boyle, R.W.

    1997-01-01

    In 1985, the International Atomic Energy Agency issued revised regulations for the safe transport of radioactive material. Significant among the changes were major revisions to requirements for Low Specific Activity (LSA) material and Surface Contaminated Objects (SCOs). In preparation for the adoption of these requirements into regulations in the United States, it became apparent that guidance on how to apply these requirements, clarifying technical uncertainties and ensuring proper implementation, would be needed both by the regulators and those regulated. Thus, the US Department of Transportation and the U.S. Nuclear Regulatory Commission, with the assistance of staff from Oak Ridge National Laboratory, are preparing regulatory guidance for LSA material and SCO transport. The guidance will present examples of acceptable methods for demonstrating compliance with the revised rules. Ideas being investigated for inclusion in the pending guidance are discussed in this paper. Under current plans, the guidance will be issued for public comment prior to final issue of the guidance in 1997. (Author)

  3. Methylation in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Regina M. Santella

    2007-02-01

    Full Text Available

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

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

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

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

  4. PRMT5-Mediated Methylation of NF-κB p65 at Arg174 Is Required for Endothelial CXCL11 Gene Induction in Response to TNF-α and IFN-γ Costimulation.

    Directory of Open Access Journals (Sweden)

    Daniel P Harris

    Full Text Available Inflammatory agonists differentially activate gene expression of the chemokine family of proteins in endothelial cells (EC. TNF is a weak inducer of the chemokine CXCL11, while TNF and IFN-γ costimulation results in potent CXCL11 induction. The molecular mechanisms underlying TNF plus IFN-γ-mediated CXCL11 induction are not fully understood. We have previously reported that the protein arginine methyltransferase PRMT5 catalyzes symmetrical dimethylation of the NF-κB subunit p65 in EC at multiple arginine residues. Methylation of Arg30 and Arg35 on p65 is critical for TNF induction of CXCL10 in EC. Here we show that PRMT5-mediated methylation of p65 at Arg174 is required for induction of CXCL11 when EC are costimulated with TNF and IFN-γ. Knockdown of PRMT5 by RNAi reduced CXCL11 mRNA and protein levels in costimulated cells. Reconstitution of p65 Arg174Ala or Arg174Lys mutants into EC that were depleted of endogenous p65 blunted TNF plus IFN-γ-mediated CXCL11 induction. Mass spectrometric analyses showed that p65 Arg174 arginine methylation is enhanced by TNF plus IFN-γ costimulation, and is catalyzed by PRMT5. Chromatin immunoprecipitation assays (ChIP demonstrated that PRMT5 is necessary for p65 association with the CXCL11 promoter in response to TNF plus IFN-γ. Further, reconstitution of p65 Arg174Lys mutant in EC abrogated this p65 association with the CXCL11 promoter. Finally, ChIP and Re-ChIP assays revealed that symmetrical dimethylarginine-containing proteins complexed with the CXCL11 promoter were diminished in p65 Arg174Lys-reconstituted EC stimulated with TNF and IFN-γ. In total, these results indicate that PRMT5-mediated p65 methylation at Arg174 is essential for TNF plus IFN-γ-mediated CXCL11 gene induction. We therefore suggest that the use of recently developed small molecule inhibitors of PRMT5 may present a therapeutic approach to moderating chronic inflammatory pathologies.

  5. The origin and fate of 4-methyl steroid hydrocarbons. I. Diagenesis of 4-methyl sterenes

    Science.gov (United States)

    Wolff, George A.; Lamb, Neil A.; Maxwell, James R.

    1986-03-01

    Treatment of 4-methylcholest-4-ene under mild acid conditions at low temperatures gives chemical evidence for certain features seen in the distributions of sedimentary 4-methyl steroid hydrocarbons, and further indicates that many low temperature diagenetic reactions of steroids are explicable in terms of acid catalysed rearrangements. Specifically, the results provide: (i) Indirect evidence that the 4-ene skeleton is a key intermediate in the dehydration of 4-methyl stanols in sediments. (ii) An explanation for the distribution of 4-methyl sterenes and A-nor sterenes in the lacustrine Messel shale (Eocene). (iii) An explanation for the presence of 4β-methyl steranes in relatively immature sedimentary rocks, despite the precursor stanols having the 4α-methyl configuration. With increasing maturity in the Paris Basin shales (Lower Toarcian), the less stable 4β-methyl steranes decrease gradually in abundance relative to their 4α-methyl counterparts, at a rate fairly similar to the change in pristane stereochemistry.

  6. Evolution of DNA Methylation across Insects.

    Science.gov (United States)

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

    2017-03-01

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

  7. Computer system requirements specification for 101-SY hydrogen mitigation test project data acquisition and control system (DACS-1)

    International Nuclear Information System (INIS)

    McNeece, S.G.; Truitt, R.W.

    1994-01-01

    The system requirements specification for SY-101 hydrogen mitigation test project (HMTP) data acquisition and control system (DACS-1) documents the system requirements for the DACS-1 project. The purpose of the DACS is to provide data acquisition and control capabilities for the hydrogen mitigation testing of Tank SY-101. Mitigation testing uses a pump immersed in the waste, directed at varying angles and operated at different speeds and time durations. Tank and supporting instrumentation is brought into the DACS to monitor the status of the tank and to provide information on the effectiveness of the mitigation test. Instrumentation is also provided for closed loop control of the pump operation. DACS is also capable for being expanded to control and monitor other mitigation testing. The intended audience for the computer system requirements specification includes the SY-101 hydrogen mitigation test data acquisition and control system designers: analysts, programmers, instrument engineers, operators, maintainers. It is intended for the data users: tank farm operations, mitigation test engineers, the Test Review Group (TRG), data management support staff, data analysis, Hanford data stewards, and external reviewers

  8. Identification of regioisomers of methylated kaempferol and quercetin by ultra high performance liquid chromatography quadrupole time-of-flight (UHPLC–QTOF) tandem mass spectrometry combined with diagnostic fragmentation pattern analysis

    International Nuclear Information System (INIS)

    Ma, Chengying; Lv, Haipeng; Zhang, Xinzhong; Chen, Zongmao; Shi, Jiang; Lu, Meiling; Lin, Zhi

    2013-01-01

    Highlights: •Found methane elimination is position-specific for methylated flavonols. •Found retro Diels–Alder fragments retained methoxy at original ring of flavonols. •Proposed a diagnostic pattern for discriminating regioisomers of flavonols. •Identified the specificity of three novel flavonol O-methyltransferases. •Identified six biologically active compounds and four new compounds. -- Abstract: The O-methylation of active flavonoids can enhance their antiallergic, anticancerous, and cardioprotective effects depending on the methylation position. Thus, it is biologically and pharmacologically important to differentiate methylated flavonoid regioisomers. In this study, we examined the regioisomers of methylated kaempferol and quercetin using ultra high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry. The methyl groups on the flavonoids can generally be cleaved as methyl radicals in a position-independent manner. We found that methyl groups can be cleaved as methane. If there are protons adjacent the methoxy on the flavonol rings, intra-molecule proton transfer can occur via collision-induced dissociation, and one molecule of methane can then be eliminated. The remaining charged fragment ([M+H−CH 4 ] + ) reflects the adjacent structure and is specific to the methoxy position. Furthermore, the retro Diels–Alder (RDA) fragmentation of methylated flavonols can generate fragments with the methoxy at the original methylated ring. Combining the position-specific [M+H−CH 4 ] + fragment with the RDA fragments provides a diagnostic pattern for rapidly identifying methylated regioisomeric flavonols. Along with their retention behaviour, we have successfully identified ten regioisomers of methylated kaempferol and quercetin, which include six compounds previously reported in plants and shown to be biologically active. The developed approach is sensitive, rapid, reliable, and requires few standard compounds. It is highly

  9. A novel method to quantify local CpG methylation density by regional methylation elongation assay on microarray

    Directory of Open Access Journals (Sweden)

    Qiao Yingjuan

    2008-01-01

    Full Text Available Abstract Background DNA methylation based techniques are important tools in both clinical diagnostics and therapeutics. But most of these methods only analyze a few CpG sites in a target region. Indeed, difference of site-specific methylation may also lead to a change of methylation density in many cases, and it has been found that the density of methylation is more important than methylation of single CpG site for gene silencing. Results We have developed a novel approach for quantitative analysis of CpG methylation density on the basis of microarray-based hybridization and incorporation of Cy5-dCTP into the Cy3 labeled target DNA by using Taq DNA Polymerase on microarray. The quantification is achieved by measuring Cy5/Cy3 signal ratio which is proportional to methylation density. This methylation-sensitive technique, termed RMEAM (regional methylation elongation assay on microarray, provides several advantages over existing methods used for methylation analysis. It can determine an exact methylation density of the given region, and has potential of high throughput. We demonstrate a use of this method in determining the methylation density of the promoter region of the tumor-related gene MLH1, TERT and MGMT in colorectal carcinoma patients. Conclusion This technique allows for quantitative analysis of regional methylation density, which is the representative of all allelic methylation patterns in the sample. The results show that this technique has the characteristics of simplicity, rapidness, specificity and high-throughput.

  10. Developmental differences in posttranslational calmodulin methylation in pea plants

    International Nuclear Information System (INIS)

    Oh, Sukheung; Roberts, D.M.

    1990-01-01

    A calmodulin-N-methyltransferase was used to analyze the degree of lysine-115 methylation of pea calmodulin. Calmodulin was isolated from segments of developing roots of young etiolated and green pea plants and was tested for its ability to be methylated by the calmodulin methyltransferase in the presence of 3 H-methyl-S-adenosylmethionine. Calmodulin methylation levels were lower in apical root segments and in the young lateral roots compared with the mature, differentiated root tissues. The methylation of these calmodulin samples occurs specifically at lysine 115 since site-directed mutants of calmodulin with substitutions at this position were not methylated and competitively inhibited methylation. The present findings, combined with previous data showing differences in NAD kinase activation by methylated and unmethylated calmodulins, raise the possibility that posttranslational methylation could affect calmodulin action

  11. Improving the action requirements of technical specifications: A risk-comparison of continued operation and plant shutdown

    Energy Technology Data Exchange (ETDEWEB)

    Kim, I.S.; Samanta, P.K. [Brookhaven National Lab., Upton, NY (United States); Mankamo, T.

    1995-04-01

    When the systems needed to remove decay heat are inoperable or degraded, the risk of shutting down the plant may be comparable to, or even higher than, that of continuing power operation with the equipment inoperable while giving priority to repairs. This concern arises because the plant may not have sufficient capability for removing decay heat during the shutdown. However, Technical Specifications (TSs) often require {open_quotes}immediate{close_quotes} shutdown of the plant. In this paper, we present risk-based analyses of the various operational policy alternatives available in such situations, with an example application to the standby service water (SSW) system of a BWR. These analyses can be used to define risk-effective requirements for those standby safety systems under discussion.

  12. Improving the action requirements of technical specifications: A risk-comparison of continued operation and plant shutdown

    International Nuclear Information System (INIS)

    Kim, I.S.; Samanta, P.K.

    1994-01-01

    When the systems needed to remove decay heat are inoperable or degraded, the risk of shutting down the plant may be comparable to, or even higher than, that of continuing power operation with the equipment inoperable while giving priority to repairs. This concern arises because the plant may not have sufficient capability for removing decay heat during the shutdown. However, Technical Specifications (TSs) often require ''immediate'' shutdown of the plant. In this paper, the authors present risk-based analyses of the various operational policy alternatives available in such situations, with an example application to the standby service water (SSW) system of a BWR. These analyses can be used to define risk-effective requirements for those standby safety systems under discussion

  13. An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

    Directory of Open Access Journals (Sweden)

    Marcela V Maus

    2016-01-01

    Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

  14. The specific radioimmunoassay in pharmacokinetics: potency, requirements and development for routine use as illustrated with an assay for Pirenzepin

    International Nuclear Information System (INIS)

    Bozler, G.

    1977-01-01

    Requirements for using RIA in pharmacokinetics are specified. An assay system developed for Pirenzepin was used as example to illustrate the general strategy striving mainly for specificity, sensitivity and reliability. The type of error in the final data is considered. It is a widespread assumption that weak crossreactions with metabolites result in a 'relative' error and, therefore, don't have relevance for the determination of the parent drug. In contrast, it was shown for biological samples that such weak crossreactivity gives rise to a concentration independent (absolute) error, which is analogous to that caused by a 'blank value' in chemical analysis. (orig.) [de

  15. Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    DEFF Research Database (Denmark)

    Milani, Lili; Lundmark, Anders; Nordlund, Jessica

    2008-01-01

    To identify genes that are regulated by cis-acting functional elements in acute lymphoblastic leukemia (ALL) we determined the allele-specific expression (ASE) levels of 2, 529 genes by genotyping a genome-wide panel of single nucleotide polymorphisms in RNA and DNA from bone marrow and blood...

  16. Methylation analysis of polysaccharides: Technical advice.

    Science.gov (United States)

    Sims, Ian M; Carnachan, Susan M; Bell, Tracey J; Hinkley, Simon F R

    2018-05-15

    Glycosyl linkage (methylation) analysis is used widely for the structural determination of oligo- and poly-saccharides. The procedure involves derivatisation of the individual component sugars of a polysaccharide to partially methylated alditol acetates which are analysed and quantified by gas chromatography-mass spectrometry. The linkage positions for each component sugar can be determined by correctly identifying the partially methylated alditol acetates. Although the methods are well established, there are many technical aspects to this procedure and both careful attention to detail and considerable experience are required to achieve a successful methylation analysis and to correctly interpret the data generated. The aim of this article is to provide the technical details and critical procedural steps necessary for a successful methylation analysis and to assist researchers (a) with interpreting data correctly and (b) in providing the comprehensive data required for reviewers to fully assess the work. Copyright © 2018 Elsevier Ltd. All rights reserved.

  17. Automata learning algorithms and processes for providing more complete systems requirements specification by scenario generation, CSP-based syntax-oriented model construction, and R2D2C system requirements transformation

    Science.gov (United States)

    Hinchey, Michael G. (Inventor); Margaria, Tiziana (Inventor); Rash, James L. (Inventor); Rouff, Christopher A. (Inventor); Steffen, Bernard (Inventor)

    2010-01-01

    Systems, methods and apparatus are provided through which in some embodiments, automata learning algorithms and techniques are implemented to generate a more complete set of scenarios for requirements based programming. More specifically, a CSP-based, syntax-oriented model construction, which requires the support of a theorem prover, is complemented by model extrapolation, via automata learning. This may support the systematic completion of the requirements, the nature of the requirement being partial, which provides focus on the most prominent scenarios. This may generalize requirement skeletons by extrapolation and may indicate by way of automatically generated traces where the requirement specification is too loose and additional information is required.

  18. Clinical Utility of promoter methylation of the tumor suppressor ...

    African Journals Online (AJOL)

    Aim: Aim is to examine the potential usefulness of blood based methylation specific polymerase chain reaction (MSP) of methylated DKK3 and RASSF1A genes in early detection of breast cancer. Method: Methylation status of DKK3 and RASSF1 was investigated in forty breast cancer patients, twenty fibroadenoma patients ...

  19. Poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA): a bioactive copolymer for specific targeting to folate receptor-positive cancer cells.

    Science.gov (United States)

    Chen, Yin; Cao, Wenbin; Zhou, Junli; Pidhatika, Bidhari; Xiong, Bin; Huang, Lu; Tian, Qian; Shu, Yiwei; Wen, Weijia; Hsing, I-Ming; Wu, Hongkai

    2015-02-04

    In this study, we present the preparation, characterization and application of a novel bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), which has a specific interaction with folate receptor (FR)-positive cancer cells. Glass surface immobilized with PLL-g-PMOXA-c-FA was demonstrated to be adhesive to FR-positive cancer cells (HeLa, JEG-3) while nonadhesive to FR-negative ones (MCF-7, HepG2) in 3 h. The specific interaction between conjugated FA on the substrate and FRs on the cells could hardly be inhibited unless a high concentration (5 mM) of free FA was used due to the multivalent nature of it. The FA functionality ratio of the copolymer on the substrate had a significant influence on the adhesion of HeLa cells, and our experiments revealed that the affinity of the substrate to the cells declined dramatically with the decrease of functionality ratio. This was believed to be caused by the polydispersity of PMOXA tethers, as supported by GPC and ToF-SIMS data. As a proof of concept in the application of our material, we demonstrated successful recovery of HeLa cells from mixture with MCF-7 (1:100) on the copolymer-coated glass, and our results showed that both high sensitivity (95.6 ± 13.3%) and specificity (24.3 ± 8.6%) were achieved.

  20. DNA Methylation as a Biomarker for Body Fluid Identification

    Directory of Open Access Journals (Sweden)

    Rania Gomaa

    2017-12-01

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

  1. Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

    Directory of Open Access Journals (Sweden)

    Simon E. Vidal

    2014-10-01

    Full Text Available The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs. To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

  2. Genome-wide methylation profiling identifies an essential role of reactive oxygen species in pediatric glioblastoma multiforme and validates a methylome specific for H3 histone family 3A with absence of G-CIMP/isocitrate dehydrogenase 1 mutation.

    Science.gov (United States)

    Jha, Prerana; Pia Patric, Irene Rosita; Shukla, Sudhanshu; Pathak, Pankaj; Pal, Jagriti; Sharma, Vikas; Thinagararanjan, Sivaarumugam; Santosh, Vani; Suri, Vaishali; Sharma, Mehar Chand; Arivazhagan, Arimappamagan; Suri, Ashish; Gupta, Deepak; Somasundaram, Kumaravel; Sarkar, Chitra

    2014-12-01

    Pediatric glioblastoma multiforme (GBM) is rare, and there is a single study, a seminal discovery showing association of histone H3.3 and isocitrate dehydrogenase (IDH)1 mutation with a DNA methylation signature. The present study aims to validate these findings in an independent cohort of pediatric GBM, compare it with adult GBM, and evaluate the involvement of important functionally altered pathways. Genome-wide methylation profiling of 21 pediatric GBM cases was done and compared with adult GBM data (GSE22867). We performed gene mutation analysis of IDH1 and H3 histone family 3A (H3F3A), status evaluation of glioma cytosine-phosphate-guanine island methylator phenotype (G-CIMP), and Gene Ontology analysis. Experimental evaluation of reactive oxygen species (ROS) association was also done. Distinct differences were noted between methylomes of pediatric and adult GBM. Pediatric GBM was characterized by 94 hypermethylated and 1206 hypomethylated cytosine-phosphate-guanine (CpG) islands, with 3 distinct clusters, having a trend to prognostic correlation. Interestingly, none of the pediatric GBM cases showed G-CIMP/IDH1 mutation. Gene Ontology analysis identified ROS association in pediatric GBM, which was experimentally validated. H3F3A mutants (36.4%; all K27M) harbored distinct methylomes and showed enrichment of processes related to neuronal development, differentiation, and cell-fate commitment. Our study confirms that pediatric GBM has a distinct methylome compared with that of adults. Presence of distinct clusters and an H3F3A mutation-specific methylome indicate existence of epigenetic subgroups within pediatric GBM. Absence of IDH1/G-CIMP status further indicates that findings in adult GBM cannot be simply extrapolated to pediatric GBM and that there is a strong need for identification of separate prognostic markers. A possible role of ROS in pediatric GBM pathogenesis is demonstrated for the first time and needs further evaluation. © The Author(s) 2014

  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. Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform.

    Directory of Open Access Journals (Sweden)

    Sara A Bumgardner

    Full Text Available Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785, L. acidophilus (strain NCFM, or NCFM-derived mutants-NCK2025 and NCK2031-elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2, the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2 receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1 Gag or membrane proximal external region (MPER, we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.

  5. Parameterization of disorder predictors for large-scale applications requiring high specificity by using an extended benchmark dataset

    Directory of Open Access Journals (Sweden)

    Eisenhaber Frank

    2010-02-01

    Full Text Available Abstract Background Algorithms designed to predict protein disorder play an important role in structural and functional genomics, as disordered regions have been reported to participate in important cellular processes. Consequently, several methods with different underlying principles for disorder prediction have been independently developed by various groups. For assessing their usability in automated workflows, we are interested in identifying parameter settings and threshold selections, under which the performance of these predictors becomes directly comparable. Results First, we derived a new benchmark set that accounts for different flavours of disorder complemented with a similar amount of order annotation derived for the same protein set. We show that, using the recommended default parameters, the programs tested are producing a wide range of predictions at different levels of specificity and sensitivity. We identify settings, in which the different predictors have the same false positive rate. We assess conditions when sets of predictors can be run together to derive consensus or complementary predictions. This is useful in the framework of proteome-wide applications where high specificity is required such as in our in-house sequence analysis pipeline and the ANNIE webserver. Conclusions This work identifies parameter settings and thresholds for a selection of disorder predictors to produce comparable results at a desired level of specificity over a newly derived benchmark dataset that accounts equally for ordered and disordered regions of different lengths.

  6. A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

    Science.gov (United States)

    Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

    2012-06-01

    Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  8. An 11bp region with stem formation potential is essential for de novo DNA methylation of the RPS element.

    Directory of Open Access Journals (Sweden)

    Matthew Gentry

    Full Text Available The initiation of DNA methylation in Arabidopsis is controlled by the RNA-directed DNA methylation (RdDM pathway that uses 24nt siRNAs to recruit de novo methyltransferase DRM2 to the target site. We previously described the REPETITIVE PETUNIA SEQUENCE (RPS fragment that acts as a hot spot for de novo methylation, for which it requires the cooperative activity of all three methyltransferases MET1, CMT3 and DRM2, but not the RdDM pathway. RPS contains two identical 11nt elements in inverted orientation, interrupted by a 18nt spacer, which resembles the features of a stemloop structure. The analysis of deletion/substitution derivatives of this region showed that deletion of one 11nt element RPS is sufficient to eliminate de novo methylation of RPS. In addition, deletion of a 10nt region directly adjacent to one of the 11nt elements, significantly reduced de novo methylation. When both 11nt regions were replaced by two 11nt elements with altered DNA sequence but unchanged inverted repeat homology, DNA methylation was not affected, indicating that de novo methylation was not targeted to a specific DNA sequence element. These data suggest that de novo DNA methylation is attracted by a secondary structure to which the two 11nt elements contribute, and that the adjacent 10nt region influences the stability of this structure. This resembles the recognition of structural features by DNA methyltransferases in animals and suggests that similar mechanisms exist in plants.

  9. Methionine kinetics and balance at the 1985 FAO/WHO/UNU intake requirement in adult men studied with L-[2H3-methyl-1-13C]methionine as a tracer

    International Nuclear Information System (INIS)

    Young, V.R.; Wagner, D.A.; Burini, R.; Storch, K.J.

    1991-01-01

    The upper range of the requirement for methionine plus cystine in healthy adults was proposed in 1985 by FAO/WHO/UNU to be 13 mg.kg body wt-1.d-1. To explore the validity of this estimate, five healthy, young adult men were given for 7 d a diet based on an L-amino acid mixture supplying 13 mg methionine.kg-1.d-1 (87 mumol.kg-1.d-1) without cystine. Constant intravenous infusions of L-[2H3-methyl-1-13C]methionine were given on days 5 and 7 while subjects were in the fed and postabsorptive states, respectively. Estimates were made of methionine oxidation, and daily methionine balance was derived from the intake-oxidation data. For the five subjects, methionine balances were -0.9, +0.7, +3.5, -3.1, and -3.8 mg kg-1.d-1, or -6, +5, +23, -21, and -26 mumol.kg-1.d-1. These findings lead to the conclusion that the upper range of the requirement for methionine plus cystine probably exceeds 13 mg.kg-1.d-1 in healthy young adults. The implications of this conclusion for establishing an appropriate amount of sulfur amino acids in an amino acid requirement pattern for adults is discussed

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

    Directory of Open Access Journals (Sweden)

    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.

  11. Alloantigen-specific suppressor T cells are not inhibited by cyclosporin A, but do require IL 2 for activation

    International Nuclear Information System (INIS)

    Bucy, R.P.

    1986-01-01

    Alloantigen-specific suppressor T cells are activated from normal murine spleen cells in mixed lymphocyte reactions (MLR). These T cells are radioresistant and suppress the activation of cytotoxic T lymphocytes (CTL) in second primary MLR cultures. This report demonstrates that cyclosporin A (CsA) blocks the activation of these suppressor cells at a dose of 1 microgram/ml. However, reconstitution of CsA blocked cultures with IL 2 restores the activation of the suppressor T cells, but fails to significantly restore the activation of CTL in these same cultures. This differential activation requirement was used to establish T cell lines that demonstrate enriched suppressor cell activity but depletion of CTL activity. These findings are discussed in terms of the mechanism of action of CsA in these distinct T cell subsets and the relevance to models of allograft unresponsiveness

  12. Technical Specification action statements requiring shutdown. A risk perspective with application to the RHR/SSW systems of a BWR

    Energy Technology Data Exchange (ETDEWEB)

    Mankamo, T. [Avaplan Oy, Espoo (Finland); Kim, I.S.; Samanta, P.K. [Brookhaven National Lab., Upton, NY (United States)

    1993-11-01

    When safety systems fail during power operation, the limiting conditions for operation (LCOs) and associated action statements of technical specifications typically require that the plant be shut down within the limits of allowed outage time (AOT). However, when a system needed to remove decay heat, such as the residual heat removal (RHR) system, is inoperable or degraded, shutting down the plant may not necessarily be preferable, from a risk perspective, to continuing power operation over a usual repair time, giving priority to the repairs. The risk impact of the basic operational alternatives, i.e., continued operation or shutdown, was evaluated for failures in the RHR and standby service water (SSW) systems of a boiling-water reactor (BWR) nuclear power plant. A complete or partial failure of the SSW system fails or degrades not only the RHR system but other front-line safety systems supported by the SSW system. This report presents the methodology to evaluate the risk impact of LCOs and associated AOT; the results of risk evaluation from its application to the RHR and SSW systems of a BWR; the findings from the risk-sensitivity analyses to identify alternative operational policies; and the major insights and recommendations to improve the technical specifications action statements.

  13. Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

    Science.gov (United States)

    Kim, Hyun-Jun; Kwon, Hye-Rim; Bae, Chang-Dae; Park, Joobae; Hong, Kyung U

    2010-05-15

    During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

  14. Aberrantly methylated DNA as a biomarker in breast cancer.

    Science.gov (United States)

    Kristiansen, Søren; Jørgensen, Lars M; Guldberg, Per; Sölétormos, György

    2013-01-01

    Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

  15. FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

    DEFF Research Database (Denmark)

    Dikopoltsev, Elena; Foltyn, Veronika N; Zehl, Martin

    2014-01-01

    d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little......, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association...... is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex...

  16. Profilin is required for viral morphogenesis, syncytium formation, and cell-specific stress fiber induction by respiratory syncytial virus

    Directory of Open Access Journals (Sweden)

    Barik Sailen

    2003-05-01

    Full Text Available Abstract Background Actin is required for the gene expression and morphogenesis of respiratory syncytial virus (RSV, a clinically important Pneumovirus of the Paramyxoviridae family. In HEp-2 cells, RSV infection also induces actin stress fibers, which may be important in the immunopathology of the RSV disease. Profilin, a major regulator of actin polymerization, stimulates viral transcription in vitro. Thus, we tested the role of profilin in RSV growth and RSV-actin interactions in cultured cells (ex vivo. Results We tested three cell lines: HEp-2 (human, A549 (human, and L2 (rat. In all three, RSV grew well and produced fused cells (syncytium, and two RSV proteins, namely, the phosphoprotein P and the nucleocapsid protein N, associated with profilin. In contrast, induction of actin stress fibers by RSV occurred in HEp-2 and L2 cells, but not in A549. Knockdown of profilin by RNA interference had a small effect on viral macromolecule synthesis but strongly inhibited maturation of progeny virions, cell fusion, and induction of stress fibers. Conclusions Profilin plays a cardinal role in RSV-mediated cell fusion and viral maturation. In contrast, interaction of profilin with the viral transcriptional proteins P and N may only nominally activate viral RNA-dependent RNA polymerase. Stress fiber formation is a cell-specific response to infection, requiring profilin and perhaps other signaling molecules that are absent in certain cell lines. Stress fibers per se play no role in RSV replication in cell culture. Clearly, the cellular architecture controls multiple steps of host-RSV interaction, some of which are regulated by profilin.

  17. An engineered split M.HhaI-zinc finger fusion lacks the intended methyltransferase specificity

    International Nuclear Information System (INIS)

    Meister, Glenna E.; Chandrasegaran, Srinivasan; Ostermeier, Marc

    2008-01-01

    The ability to site-specifically methylate DNA in vivo would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of diseases. Natural DNA methyltransferases lack the specificity required for these applications. Nomura and Barbas [W. Nomura, C.F. Barbas 3rd, In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase, J. Am. Chem. Soc. 129 (2007) 8676-8677] have reported that an engineered DNA methyltransferase comprised of fragments of M.HhaI methyltransferase and zinc finger proteins has very high specificity for the chosen target site. Our analysis of this engineered enzyme shows that the fusion protein methylates target and non-target sites with similar efficiency

  18. Maternal intake of methyl-group donors affects DNA methylation of metabolic genes in infants.

    Science.gov (United States)

    Pauwels, Sara; Ghosh, Manosij; Duca, Radu Corneliu; Bekaert, Bram; Freson, Kathleen; Huybrechts, Inge; Langie, Sabine A S; Koppen, Gudrun; Devlieger, Roland; Godderis, Lode

    2017-01-01

    Maternal nutrition during pregnancy and infant nutrition in the early postnatal period (lactation) are critically involved in the development and health of the newborn infant. The Maternal Nutrition and Offspring's Epigenome (MANOE) study was set up to assess the effect of maternal methyl-group donor intake (choline, betaine, folate, methionine) on infant DNA methylation. Maternal intake of dietary methyl-group donors was assessed using a food-frequency questionnaire (FFQ). Before and during pregnancy, we evaluated maternal methyl-group donor intake through diet and supplementation (folic acid) in relation to gene-specific ( IGF2 DMR, DNMT1 , LEP , RXRA ) buccal epithelial cell DNA methylation in 6 months old infants ( n  = 114) via pyrosequencing. In the early postnatal period, we determined the effect of maternal choline intake during lactation (in mothers who breast-fed for at least 3 months) on gene-specific buccal DNA methylation ( n  = 65). Maternal dietary and supplemental intake of methyl-group donors (folate, betaine, folic acid), only in the periconception period, was associated with buccal cell DNA methylation in genes related to growth ( IGF2 DMR), metabolism ( RXRA ), and appetite control ( LEP ). A negative association was found between maternal folate and folic acid intake before pregnancy and infant LEP (slope = -1.233, 95% CI -2.342; -0.125, p  = 0.0298) and IGF2 DMR methylation (slope = -0.706, 95% CI -1.242; -0.107, p  = 0.0101), respectively. Positive associations were observed for maternal betaine (slope = 0.875, 95% CI 0.118; 1.633, p  = 0.0241) and folate (slope = 0.685, 95% CI 0.245; 1.125, p  = 0.0027) intake before pregnancy and RXRA methylation. Buccal DNMT1 methylation in the infant was negatively associated with maternal methyl-group donor intake in the first and second trimester of pregnancy and negatively in the third trimester. We found no clear association between maternal choline intake

  19. Synthesis of [14α-methyl-3H]-24,25-dihydrolanosterol

    International Nuclear Information System (INIS)

    DeKeczer, S.; Kertesz, D.; Parnes, H.

    1993-01-01

    We describe the first synthesis of isomerically pure title compound (6) at high specific activity. This required the development of a convenient, regiospecific synthesis of the δ 8(9) -15-ketone and subsequent alkylation with methyl-[ 3 H 3 ]iodide. A key step in our procedure was the use of an electrochemical reduction of the intermediate [14α-methyl- 3 H]-15-oxo-dihydrolanosterol. This process was effected cleanly and in high yield to give (6), an important assay tool in the search for cholesterol lowering agents. This approach was found to be significantly superior to the Wolff-Kishner reduction of the corresponding 3-benzoate. (Author)

  20. Specific Sirt1 Activator-mediated Improvement in Glucose Homeostasis Requires Sirt1-Independent Activation of AMPK

    Directory of Open Access Journals (Sweden)

    Sung-Jun Park

    2017-04-01

    Full Text Available The specific Sirt1 activator SRT1720 increases mitochondrial function in skeletal muscle, presumably by activating Sirt1. However, Sirt1 gain of function does not increase mitochondrial function, which raises a question about the central role of Sirt1 in SRT1720 action. Moreover, it is believed that the metabolic effects of SRT1720 occur independently of AMP-activated protein kinase (AMPK, an important metabolic regulator that increases mitochondrial function. Here, we show that SRT1720 activates AMPK in a Sirt1-independent manner and SRT1720 activates AMPK by inhibiting a cAMP degrading phosphodiesterase (PDE in a competitive manner. Inhibiting the cAMP effector protein Epac prevents SRT1720 from activating AMPK or Sirt1 in myotubes. Moreover, SRT1720 does not increase mitochondrial function or improve glucose tolerance in AMPKα2 knockout mice. Interestingly, weight loss induced by SRT1720 is not sufficient to improve glucose tolerance. Therefore, contrary to current belief, the metabolic effects produced by SRT1720 require AMPK, which can be activated independently of Sirt1.

  1. Adolescents with type 1 diabetes mellitus and attention-deficit/hyperactivity disorder require specific support from healthcare professionals.

    Science.gov (United States)

    Lindblad, Ida; Engström, Ann-Charlotte; Nylander, Charlotte; Fernell, Elisabeth

    2017-12-01

    Managing type 1 diabetes mellitus requires efficient cognitive and executive skills, and adolescents who have attention-deficit/hyperactivity disorder (ADHD) may face specific challenges. This study explored young people's experiences of diabetes treatment and care. In a population-based study, comprising 175 patients aged 5-16 years with type 1 diabetes mellitus in two Swedish counties, we found that eight also met criteria for ADHD. Six of these, aged 14.5-16 years, participated 2013-2014 in interviews that targeted aspects of their diabetes treatment. Conducted by two psychologists, these used the inductive qualitative, semi-structured interview format. The two boys and four girls all reported difficulties in creating routines for their diabetes treatment and that problems were aggravated during stress. They had been criticised by their parents and the diabetes team when their blood levels indicated inadequate diabetes control. They requested ongoing information, involvement of their friends, group meetings and easy access to the healthcare system during difficult times. Patients with type 1 diabetes mellitus and concomitant ADHD faced problems with their diabetes management, especially during stressful situations. Diabetes care provision should pay particular attention to patients with co-existing neuropsychiatric and neurodevelopmental disorders such as ADHD. ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

  2. Ubiquitin-specific protease 5 is required for the efficient repair of DNA double-strand breaks.

    Directory of Open Access Journals (Sweden)

    Satoshi Nakajima

    Full Text Available During the DNA damage response (DDR, ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5, a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.

  3. The Nanos3-3'UTR is required for germ cell specific NANOS3 expression in mouse embryos.

    Directory of Open Access Journals (Sweden)

    Hitomi Suzuki

    Full Text Available BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo.

  4. METHOD FOR SECURITY SPECIFICATION SOFTWARE REQUIREMENTS AS A MEANS FOR IMPLEMENTING A SOFTWARE DEVELOPMENT PROCESS SECURE - MERSEC

    Directory of Open Access Journals (Sweden)

    Castro Mecías, L.T.

    2015-06-01

    Full Text Available Often security incidents that have the object or use the software as a means of causing serious damage and legal, economic consequences, etc. Results of a survey by Kaspersky Lab reflectvulnerabilities in software are the main cause of security incidents in enterprises, the report shows that 85% of them have reported security incidents and vulnerabilities in software are the main reason is further estimated that incidents can cause significant losses estimated from 50,000 to $ 649.000. (1 In this regard academic and industry research focuses on proposals based on reducing vulnerabilities and failures of technology, with a positive influence on how the software is developed. A development process for improved safety practices and should include activities from the initial phases of the software; so that security needs are identified, manage risk and appropriate measures are implemented. This article discusses a method of analysis, acquisition and requirements specification of the software safety analysis on the basis of various proposals and deficiencies identified from participant observation in software development teams. Experiments performed using the proposed yields positive results regarding the reduction of security vulnerabilities and compliance with the safety objectives of the software.

  5. Methylation status of the APC and RASSF1A promoter in cell-free circulating DNA and its prognostic role in patients with colorectal cancer.

    Science.gov (United States)

    Matthaios, Dimitrios; Balgkouranidou, Ioanna; Karayiannakis, Anastasios; Bolanaki, Helen; Xenidis, Nikolaos; Amarantidis, Kyriakos; Chelis, Leonidas; Romanidis, Konstantinos; Chatzaki, Aikaterini; Lianidou, Evi; Trypsianis, Grigorios; Kakolyris, Stylianos

    2016-07-01

    DNA methylation is the most frequent epigenetic alteration. Using methylation-specific polymerase chain reaction (MSP), the methylation status of the adenomatous polyposis coli ( APC ) and Ras association domain family 1 isoform A ( RASSF1A ) genes was examined in cell-free circulating DNA from 155 plasma samples obtained from patients with early and advanced colorectal cancer (CRC). APC and RASSF1A hypermethylation was frequently observed in both early and advanced disease, and was significantly associated with a poorer disease outcome. The methylation status of the APC and RASSF1A promoters was investigated in cell-free DNA of patients with CRC. Using MSP, the promoter methylation status of APC and RASSF1A was examined in 155 blood samples obtained from patients with CRC, 88 of whom had operable CRC (oCRC) and 67 had metastatic CRC (mCRC). The frequency of APC methylation in patients with oCRC was 33%. Methylated APC promoter was significantly associated with older age (P=0.012), higher stage (P=0.014) and methylated RASSF1A status (P=0.050). The frequency of APC methylation in patients with mCRC was 53.7%. In these patients, APC methylation was significantly associated with methylated RASSF1A status (P=0.016). The frequency of RASSF1A methylation in patients with oCRC was 25%. Methylated RASSF1A in oCRC was significantly associated with higher stage (P=0.021). The frequency of RASSF1A methylation in mCRC was 44.8%. Methylated RASSF1A in mCRC was associated with moderate differentiation (P=0.012), high levels of carcinoembryonic antigen (P=0.023) and methylated APC status (P=0.016). Patients with an unmethylated APC gene had better survival in both early (81±5 vs. 27±4 months, PAPC . Patients with an unmethylated RASSF1A gene had better survival in both early (71±6 vs. 46±8 months, PAPC and RASSF1A promoter methylation status and survival may be indicative of a prognostic role for these genes in CRC, which requires additional testing in larger studies.

  6. Conceptual waste package interim product specifications and data requirements for disposal of borosilicate glass defense high-level waste forms in salt geologic repositories

    International Nuclear Information System (INIS)

    1983-06-01

    The conceptual waste package interim product specifications and data requirements presented are applicable specifically to the normal borosilicate glass product of the Defense Waste Processing Facility (DWPF). They provide preliminary numerical values for the defense high-level waste form parameters and properties identified in the waste form performance specification for geologic isolation in salt repositories. Subject areas treated include containment and isolation, operational period safety, criticality control, waste form/production canister identification, and waste package performance testing requirements. This document was generated for use in the development of conceptual waste package designs in salt. It will be revised as additional data, analyses, and regulatory requirements become available

  7. Epigenetic control of viral life-cycle by a DNA-methylation dependent transcription factor.

    Directory of Open Access Journals (Sweden)

    Kirsty Flower

    Full Text Available Epstein-Barr virus (EBV encoded transcription factor Zta (BZLF1, ZEBRA, EB1 is the prototype of a class of transcription factor (including C/EBPalpha that interact with CpG-containing DNA response elements in a methylation-dependent manner. The EBV genome undergoes a biphasic methylation cycle; it is extensively methylated during viral latency but is reset to an unmethylated state following viral lytic replication. Zta is expressed transiently following infection and again during the switch between latency and lytic replication. The requirement for CpG-methylation at critical Zta response elements (ZREs has been proposed to regulate EBV replication, specifically it could aid the activation of viral lytic gene expression from silenced promoters on the methylated genome during latency in addition to preventing full lytic reactivation from the non-methylated EBV genome immediately following infection. We developed a computational approach to predict the location of ZREs which we experimentally assessed using in vitro and in vivo DNA association assays. A remarkably different binding motif is apparent for the CpG and non-CpG ZREs. Computational prediction of the location of these binding motifs in EBV revealed that the majority of lytic cycle genes have at least one and many have multiple copies of methylation-dependent CpG ZREs within their promoters. This suggests that the abundance of Zta protein coupled with the methylation status of the EBV genome act together to co-ordinate the expression of lytic cycle genes at the majority of EBV promoters.

  8. Regulation and function of DNA methylation in plants and animals

    KAUST Repository

    He, Xinjian

    2011-02-15

    DNA methylation is an important epigenetic mark involved in diverse biological processes. In plants, DNA methylation can be established through the RNA-directed DNA methylation pathway, an RNA interference pathway for transcriptional gene silencing (TGS), which requires 24-nt small interfering RNAs. In mammals, de novo DNA methylation occurs primarily at two developmental stages: during early embryogenesis and during gametogenesis. While it is not clear whether establishment of DNA methylation patterns in mammals involves RNA interference in general, de novo DNA methylation and suppression of transposons in germ cells require 24-32-nt piwi-interacting small RNAs. DNA methylation status is dynamically regulated by DNA methylation and demethylation reactions. In plants, active DNA demethylation relies on the repressor of silencing 1 family of bifunctional DNA glycosylases, which remove the 5-methylcytosine base and then cleave the DNA backbone at the abasic site, initiating a base excision repair (BER) pathway. In animals, multiple mechanisms of active DNA demethylation have been proposed, including a deaminase- and DNA glycosylase-initiated BER pathway. New information concerning the effects of various histone modifications on the establishment and maintenance of DNA methylation has broadened our understanding of the regulation of DNA methylation. The function of DNA methylation in plants and animals is also discussed in this review. © 2011 IBCB, SIBS, CAS All rights reserved.

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

    African Journals Online (AJOL)

    etchie

    2013-02-20

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

  10. Quantitative DNA methylation analysis of candidate genes in cervical cancer.

    Directory of Open Access Journals (Sweden)

    Erin M Siegel

    Full Text Available Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2. A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003. Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

  11. The conjugation-specific Die5 protein is required for development of the somatic nucleus in both Paramecium and Tetrahymena.

    Science.gov (United States)

    Matsuda, Atsushi; Shieh, Annie Wan-Yi; Chalker, Douglas L; Forney, James D

    2010-07-01

    Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena DeltaDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway.

  12. The Conjugation-Specific Die5 Protein Is Required for Development of the Somatic Nucleus in both Paramecium and Tetrahymena▿

    Science.gov (United States)

    Matsuda, Atsushi; Shieh, Annie Wan-Yi; Chalker, Douglas L.; Forney, James D.

    2010-01-01

    Development in ciliated protozoa involves extensive genome reorganization within differentiating macronuclei, which shapes the somatic genome of the next vegetative generation. Major events of macronuclear differentiation include excision of internal eliminated sequences (IESs), chromosome fragmentation, and genome amplification. Proteins required for these events include those with homology throughout eukaryotes as well as proteins apparently unique to ciliates. In this study, we identified the ciliate-specific Defective in IES Excision 5 (DIE5) genes of Paramecium tetraurelia (PtDIE5) and Tetrahymena thermophila (TtDIE5) as orthologs that encode nuclear proteins expressed exclusively during development. Abrogation of PtDie5 protein (PtDie5p) function by RNA interference (RNAi)-mediated silencing or TtDie5p by gene disruption resulted in the failure of developing macronuclei to differentiate into new somatic nuclei. Tetrahymena ΔDIE5 cells arrested late in development and failed to complete genome amplification, whereas RNAi-treated Paramecium cells highly amplified new macronuclear DNA before the failure in differentiation, findings that highlight clear differences in the biology of these distantly related species. Nevertheless, IES excision and chromosome fragmentation failed to occur in either ciliate, which strongly supports that Die5p is a critical player in these processes. In Tetrahymena, loss of zygotic expression during development was sufficient to block nuclear differentiation. This observation, together with the finding that knockdown of Die5p in Paramecium still allows genome amplification, indicates that this protein acts late in macronuclear development. Even though DNA rearrangements in these two ciliates look to be quite distinct, analysis of DIE5 establishes the action of a conserved mechanism within the genome reorganization pathway. PMID:20495055

  13. Methylation pathways in schizophrenia

    International Nuclear Information System (INIS)

    Sargent, T.W. III.

    1982-01-01

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

  14. Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Domber, E.A.

    1986-01-01

    These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

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

    International Nuclear Information System (INIS)

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

    2004-01-01

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

  16. Requirements of Employers for Young Specialists and Issues of Their Training at the Educational Institutions: Regional Specificity of Moscow

    Directory of Open Access Journals (Sweden)

    Vyacheslav Nikolaevich Bobkov

    2016-12-01

    Full Text Available The subject-matter of the article is the study of the peculiarities of the youth labour market in such a specific region of Russia as the city of Moscow. The topic of the work is connected to the adaptation of young specialists training system to the regional requirements of the employers of Moscow. The main hypothesis of the research assumes that Moscow is the atypical region of Russia and it is inexpedient to apply criteria and methodological approaches, which are standard for our country, to its labour market. As a method of the research, a selective survey of key employers of Moscow and respondents aged from 14 till 30 years has been used. The data of the sociological survey conducted by the authors have allowed to establish the following features of the researched region: a respectively low level of youth unemployment compared with the whole Russia; focusing of employers on such features of personnel as a good communicative skills learning ability, professional knowledge and competences. Moscow employers don’t pay significant attention to such characteristics of young specialists as language and computer skills, computer knowledge and the diploma of a prestigious educational institution. The main difficulties in finding employment for young specialists in the city of Moscow are: overestimated salary expectations; the weak professional training level and unwillingness, in fact, to work. As a result of the survey, the practical offers have been formulated. There are two directions of their application: the offers focused on the behaviour of the youth at a stage of their training and those offers focused on the increase of a practical component of the activity of professional educational institutions. The authors came to conclusions about the need of a deep orientation of educational institutions to the applied training for specialists, of practical workers for teaching special disciplines, the development by future specialists their

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

    Science.gov (United States)

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

    2011-08-15

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

  18. DNA methylation of candidate genes in peripheral blood from patients with type 2 diabetes or the metabolic syndrome.

    Science.gov (United States)

    van Otterdijk, Sanne D; Binder, Alexandra M; Szarc Vel Szic, Katarzyna; Schwald, Julia; Michels, Karin B

    2017-01-01

    The prevalence of type 2 diabetes (T2D) and the metabolic syndrome (MetS) is increasing and several studies suggested an involvement of DNA methylation in the development of these metabolic diseases. This study was designed to investigate if differential DNA methylation in blood can function as a biomarker for T2D and/or MetS. Pyrosequencing analyses were performed for the candidate genes KCNJ11, PPARγ, PDK4, KCNQ1, SCD1, PDX1, FTO and PEG3 in peripheral blood leukocytes (PBLs) from 25 patients diagnosed with only T2D, 9 patients diagnosed with T2D and MetS and 11 control subjects without any metabolic disorders. No significant differences in gene-specific methylation between patients and controls were observed, although a trend towards significance was observed for PEG3. Differential methylation was observed between the groups in 4 out of the 42 single CpG loci located in the promoters regions of the genes FTO, KCNJ11, PPARγ and PDK4. A trend towards a positive correlation was observed for PEG3 methylation with HDL cholesterol levels. Altered levels of DNA methylation in PBLs of specific loci might serve as a biomarker for T2D or MetS, although further investigation is required.

  19. DNA methylation of candidate genes in peripheral blood from patients with type 2 diabetes or the metabolic syndrome.

    Directory of Open Access Journals (Sweden)

    Sanne D van Otterdijk

    Full Text Available The prevalence of type 2 diabetes (T2D and the metabolic syndrome (MetS is increasing and several studies suggested an involvement of DNA methylation in the development of these metabolic diseases. This study was designed to investigate if differential DNA methylation in blood can function as a biomarker for T2D and/or MetS.Pyrosequencing analyses were performed for the candidate genes KCNJ11, PPARγ, PDK4, KCNQ1, SCD1, PDX1, FTO and PEG3 in peripheral blood leukocytes (PBLs from 25 patients diagnosed with only T2D, 9 patients diagnosed with T2D and MetS and 11 control subjects without any metabolic disorders.No significant differences in gene-specific methylation between patients and controls were observed, although a trend towards significance was observed for PEG3. Differential methylation was observed between the groups in 4 out of the 42 single CpG loci located in the promoters regions of the genes FTO, KCNJ11, PPARγ and PDK4. A trend towards a positive correlation was observed for PEG3 methylation with HDL cholesterol levels.Altered levels of DNA methylation in PBLs of specific loci might serve as a biomarker for T2D or MetS, although further investigation is required.

  20. Synthesis of no-carrier-added alpha-[11C]methyl-L-tryptophan

    International Nuclear Information System (INIS)

    Chaly, T.; Diksic, M.

    1988-01-01

    Described here is a synthesis of no-carrier-added alpha-[ 11 C]methyl-L-tryptophan based on alkylation with 11 CH 3 I of an anion generated by reacting the Schiff base of L-tryptophan methyl ester with di-isopropylamine. The synthesis requires approximately 30 min after the end of 11 CO 2 collection and gives alpha-[ 11 C]methyl-L-tryptophan in a 20-25% radiochemical yield calculated at the end of the synthesis and without correction for radioactive decay. The specific activity of the final radiopharmaceutical, measured at the end of the synthesis, was around 2000 Ci/mmol. Data confirming the stereospecificity of the synthesis are also presented

  1. Evidence for methyl group transfer between the methyl-accepting chemotaxis proteins in Bacillus subtilis

    International Nuclear Information System (INIS)

    Bedale, W.A.; Nettleton, D.O.; Sopata, C.S.; Thoelke, M.S.; Ordal, G.W.

    1988-01-01

    The authors present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro was strongly stimulated by the chemoattractant aspartate and thus may plan an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [ 3 H] methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2

  2. Specification of requirements for upgrades using digital instrument and control systems. Report prepared within the framework of the international working group on nuclear power plant control and instrumentation

    International Nuclear Information System (INIS)

    1999-01-01

    The need to develop good specifications of requirements for instrument and control (I and C) systems applies throughout the world and is becoming more and more important as more upgrades are planned. Better guidance on how to develop good requirements specifications would support safer, more effective and more economical refits and upgrades. The need for this was pointed out by the IAEA International Working Group on Nuclear Power Plant Control and Instrumentation (IWG-NPPCI). This report is the result of a series of advisory and consultants meetings held by the IAEA in 1997 and 1998 in Vienna. The scope of the activities described covers a methodology for the determination of requirements and the development of the necessary specifications and plans needed through the life-cycle of digital instrumentation and control systems. It is restricted to technical aspects and indicates subjects which should be included in specifications and plans at different phases

  3. 40 CFR 270.22 - Specific part B information requirements for boilers and industrial furnaces burning hazardous...

    Science.gov (United States)

    2010-07-01

    ... requirements for boilers and industrial furnaces burning hazardous waste. 270.22 Section 270.22 Protection of... requirements for boilers and industrial furnaces burning hazardous waste. When an owner or operator of a cement kiln, lightweight aggregate kiln, solid fuel boiler, liquid fuel boiler, or hydrochloric acid...

  4. Is methylation analysis of SFRP2, TFPI2, NDRG4, and BMP3 promoters suitable for colorectal cancer screening in the Korean population?

    Directory of Open Access Journals (Sweden)

    Soo-Kyung Park

    2017-10-01

    Full Text Available Background/Aims: Colorectal cancer (CRC screening using stool DNA was recently found to yield good detection rates. A multi-target stool DNA test (Cologuard®, Exact Sciences, including methylated genes has been recently approved by the U.S. Food and Drug Administration. The aim of this study was to validate these aberrantly methylated genes as stool-based DNA markers for detecting CRC and colorectal advanced adenoma (AA in the Korean population.Methods: A single-center study was conducted in 36 patients with AA; 35 patients with CRC; and 40 endoscopically diagnosed healthy controls using CRC screening colonoscopy. The methylation status of the SFRP2, TFPI2, NDRG4, and BMP3 promoters was investigated blindly using bisulfate-modified stool DNA obtained from 111 participants. Methylation status was investigated by methylation-specific polymerase chain reaction.Results: Methylated SFRP2, TFPI2, NDRG4, and BMP3 promoters were detected in 60.0%, 31.4%, 68.8%, and 40.0% of CRC samples and in 27.8%, 27.8%, 27.8%, and 33.3% of AA samples, respectively. The sensitivities obtained using 4 markers to detect CRC and AA were 94.3% and 72.2%, respectively. The specificity was 55.0%.Conclusions: Our results demonstrate that the SFRP2, TFPI2, NDRG4, and BMP3 promoter methylation analysis of stool sample DNA showed high sensitivity but low specificity for detecting CRC and AA. Because of the low specificity, 4 methylated markers might not be sufficient for CRC screening in the Korean population. Further large-scale studies are required to validate the methylation of these markers in the Asian population and to find new markers for the Asian population.

  5. Autonomous Integrated Receive System (AIRS) requirements definition. Volume 4: Functional specification for the prototype Automated Integrated Receive System (AIRS)

    Science.gov (United States)

    Chie, C. M.

    1984-01-01

    The functional requirements for the performance, design, and testing for the prototype Automated Integrated Receive System (AIRS) to be demonstrated for the TDRSS S-Band Single Access Return Link are presented.

  6. Methylated DNA Immunoprecipitation Analysis of Mammalian Endogenous Retroviruses.

    Science.gov (United States)

    Rebollo, Rita; Mager, Dixie L

    2016-01-01

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

  7. The Johnson Space Center Management Information Systems (JSCMIS). 1: Requirements Definition and Design Specifications for Versions 2.1 and 2.1.1. 2: Documented Test Scenario Environments. 3: Security Design and Specifications

    Science.gov (United States)

    1986-01-01

    The Johnson Space Center Management Information System (JSCMIS) is an interface to computer data bases at NASA Johnson which allows an authorized user to browse and retrieve information from a variety of sources with minimum effort. This issue gives requirements definition and design specifications for versions 2.1 and 2.1.1, along with documented test scenario environments, and security object design and specifications.

  8. DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

    2012-10-01

    DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

  9. Methyl donor supplementation alters cognitive performance and motivation in female offspring from high-fat diet-fed dams.

    Science.gov (United States)

    McKee, Sarah E; Grissom, Nicola M; Herdt, Christopher T; Reyes, Teresa M

    2017-06-01

    During gestation, fetal nutrition is entirely dependent on maternal diet. Maternal consumption of excess fat during pregnancy has been linked to an increased risk of neurologic disorders in offspring, including attention deficit/hyperactivity disorder, autism, and schizophrenia. In a mouse model, high-fat diet (HFD)-fed offspring have cognitive and executive function deficits as well as whole-genome DNA and promoter-specific hypomethylation in multiple brain regions. Dietary methyl donor supplementation during pregnancy or adulthood has been used to alter DNA methylation and behavior. Given that extensive brain development occurs during early postnatal life-particularly within the prefrontal cortex (PFC), a brain region critical for executive function-we examined whether early life methyl donor supplementation ( e.g., during adolescence) could ameliorate executive function deficits observed in offspring that were exposed to maternal HFD. By using operant testing, progressive ratio, and the PFC-dependent 5-choice serial reaction timed task (5-CSRTT), we determined that F1 female offspring (B6D2F1/J) from HFD-fed dams have decreased motivation (decreased progressive ratio breakpoint) and require a longer stimulus length to complete the 5-CSRTT task successfully, whereas early life methyl donor supplementation increased motivation and shortened the minimum stimulus length required for a correct response in the 5-CSRTT. Of interest, we found that expression of 2 chemokines, CCL2 and CXCL10, correlated with the median stimulus length in the 5-CSRTT. Furthermore, we found that acute adult supplementation of methyl donors increased motivation in HFD-fed offspring and those who previously received supplementation with methyl donors. These data point to early life as a sensitive time during which dietary methyl donor supplementation can alter PFC-dependent cognitive behaviors.-McKee, S. E., Grissom, N. M., Herdt, C. T., Reyes, T. M. Methyl donor supplementation alters

  10. Methyl donor supplementation alters cognitive performance and motivation in female offspring from high-fat diet–fed dams

    Science.gov (United States)

    McKee, Sarah E.; Grissom, Nicola M.; Herdt, Christopher T.; Reyes, Teresa M.

    2017-01-01

    During gestation, fetal nutrition is entirely dependent on maternal diet. Maternal consumption of excess fat during pregnancy has been linked to an increased risk of neurologic disorders in offspring, including attention deficit/hyperactivity disorder, autism, and schizophrenia. In a mouse model, high-fat diet (HFD)–fed offspring have cognitive and executive function deficits as well as whole-genome DNA and promoter-specific hypomethylation in multiple brain regions. Dietary methyl donor supplementation during pregnancy or adulthood has been used to alter DNA