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Sample records for combined bisulfite restriction

  1. Analysis of locus-specific changes in methylation patterns using a COBRA (combined bisulfite restriction analysis) assay.

    PubMed

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    DNA methylation is a major mechanism for the reversible control of gene expression, chromatin structure, and genome stability. Methylation analysis at a given locus allows one to evaluate levels of chromatin packaging, gene expression, and even homologous recombination. We have shown that the combined bisulfite restriction analysis (COBRA) assay makes it possible to analyze methylation levels at a defined locus. The major steps are: bisulfite conversion of nonmethylate cytosines to uracils, locus-specific PCR amplification of converted DNA, restriction digestion, and analysis of restriction patterns on the gel. Due to the availability of various restriction enzymes that have cytosines in the restriction recognition sequence, the assay allows analysis of various cytosines, including those potentially targeted for symmetrical and nonsymmetrical methylation.

  2. Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay.

    PubMed

    Hattori, Mitsu; Torii, Chiharu; Yagihashi, Tatsuhiko; Izumi, Kosuke; Suda, Naoto; Ohyama, Kimie; Takahashi, Takao; Moriyama, Keiji; Kosaki, Kenjiro

    2009-10-01

    Russell-Silver syndrome (RSS) is characterized by prenatal and postnatal growth retardation, triangular facies, and fifth-finger clinodactyly. Half of all patients with RSS have hypomethylation of the differentially methylated region of the H19 gene on chromosome 11p15.5. Hence, a quantitative methylation analysis of this region can be useful for the molecular diagnosis of RSS. However, conventional assays based on bisulfite clone sequencing are rather time and labor consuming and are not suitable for clinical use. In the present study, we investigated a possible method of quantitatively determining H19 hypomethylation in RSS patients using a combined bisulfite restriction analysis (COBRA)-denaturing high-performance liquid chromatography (DHPLC) assay; in this combined assay, polymerase chain reaction products amplified from the H19 differentially methylated region of bisulfite-treated genomic DNA were analyzed using a COBRA assay, which detects methylation-dependent sequence differences in the bisulfite-treated genomic DNA using a restriction enzyme analysis. We designed the assay so that a restriction enzyme (HinfI) would cut the methylated, but not the unmethylated, template. The molar ratio between the cut and uncut fragments was measured using DHPLC, and the construction of a calibration curve enabled the methylation index for the original genomic DNA to be estimated. An analysis of seven RSS patients using the COBRA-DHPLC assay demonstrated that three of the seven RSS patients had a low methylation index of around 10%. A comparison of the methylation indices obtained using COBRA-DHPLC and conventional bisulfite clone sequencing revealed an excellent intermethod agreement. In summary, we have developed a robust, rapid, and cost-effective COBRA-DHPLC-based screening system for RSS.

  3. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis.

    PubMed

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 degrees C or 65 degrees C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis. Copyright 2010 Elsevier Inc. All rights reserved.

  4. Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

    SciTech Connect

    Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro; Kawakami, Masanori; Kage, Hidenori; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2010-08-27

    Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonuclease I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.

  5. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  6. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  7. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  8. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Potassium bisulfite. 182.3616 Section 182.3616...) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Chemical Preservatives § 182.3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or explanation. This substance is generally...

  9. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  10. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  11. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations, restrictions, or...

  12. 21 CFR 182.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Potassium bisulfite. 182.3616 Section 182.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  13. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) (c) Limitations, restrictions, or...

  14. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or...

  15. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or...

  16. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or...

  17. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or...

  18. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or explanation....

  19. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or explanation....

  20. 21 CFR 582.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Sodium bisulfite. 582.3739 Section 582.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3739 Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or...

  1. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or explanation....

  2. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR... Sodium bisulfite. (a) Product. Sodium bisulfite. (b) (c) Limitations, restrictions, or explanation....

  3. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms

    PubMed Central

    Leakey, Tatiana I.; Zielinski, Jerzy; Siegfried, Rachel N.; Siegel, Eric R.; Fan, Chun-Yang; Cooney, Craig A.

    2008-01-01

    DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites. PMID:18480118

  4. A simple algorithm for quantifying DNA methylation levels on multiple independent CpG sites in bisulfite genomic sequencing electropherograms.

    PubMed

    Leakey, Tatiana I; Zielinski, Jerzy; Siegfried, Rachel N; Siegel, Eric R; Fan, Chun-Yang; Cooney, Craig A

    2008-06-01

    DNA methylation at cytosines is a widely studied epigenetic modification. Methylation is commonly detected using bisulfite modification of DNA followed by PCR and additional techniques such as restriction digestion or sequencing. These additional techniques are either laborious, require specialized equipment, or are not quantitative. Here we describe a simple algorithm that yields quantitative results from analysis of conventional four-dye-trace sequencing. We call this method Mquant and we compare it with the established laboratory method of combined bisulfite restriction assay (COBRA). This analysis of sequencing electropherograms provides a simple, easily applied method to quantify DNA methylation at specific CpG sites.

  5. Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.

    PubMed

    Garrett-Bakelman, Francine E; Sheridan, Caroline K; Kacmarczyk, Thadeous J; Ishii, Jennifer; Betel, Doron; Alonso, Alicia; Mason, Christopher E; Figueroa, Maria E; Melnick, Ari M

    2015-02-24

    DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

  6. 21 CFR 582.3616 - Potassium bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Potassium bisulfite. 582.3616 Section 582.3616 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL....3616 Potassium bisulfite. (a) Product. Potassium bisulfite. (b) [Reserved] (c) Limitations...

  7. A polymerase engineered for bisulfite sequencing

    PubMed Central

    Millar, Doug; Christova, Yonka; Holliger, Philipp

    2015-01-01

    Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains. PMID:26271989

  8. 21 CFR 182.3739 - Sodium bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Sodium bisulfite. 182.3739 Section 182.3739 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) SUBSTANCES GENERALLY RECOGNIZED AS SAFE Chemical Preservatives § 182.3739 Sodium bisulfite. (a) Product....

  9. Effects of oxygen and weak magnetic field on Fe(0)/bisulfite system: performance and mechanisms.

    PubMed

    Xiong, Xinmei; Gan, Jinhong; Zhan, Wei; Sun, Bo

    2016-08-01

    The performance and mechanisms of 4-nitrophenol (4-NP) degradation by the Fe(0)/bisulfite system were systematically investigated for the first time. The evidences presented in this study verified that O2 was a crucial factor that affected the mechanism of Fe(0)/bisulfite-driven 4-NP degradation. In the Fe(0)/bisulfite/O2 system, Fe(0) acted as a supplier of Fe(2+) to catalyze bisulfite oxidation that induced a chain reaction to produce reactive radicals for 4-NP degradation. While under N2 purging condition, bisulfite worked as a specified reductant that facilitated the transformation of Fe(3+) to nascent Fe(2+) ions, which principally accounted for the reductive removal of 4-NP. The application of a weak magnetic field (WMF) efficiently improved the removal rate of 4-NP and did not alter the mechanisms in both Fe(0)/bisulfite/O2 and Fe(0)/bisulfite/N2 processes. The secondary radicals, HO(·), SO4 (·-), and SO5 (·-), were considered as the most possible active oxidants contributing to the oxidative removal of 4-NP and even partial mineralization under an oxic condition. Compared with anoxic conditions, the performance removal of 4-NP by the WMF-Fe(0)/bisulfite/O2 system showed less pHini dependence. To facilitate the application of WMF-Fe(0)/bisulfite/O2 technology in real practice, premagnetization of Fe(0) was employed to combine with bisulfite/O2 and proved to be an effective and applicable method for 4-NP removal.

  10. Genome-wide bisulfite sensitivity profiling of yeast suggests bisulfite inhibits transcription.

    PubMed

    Segovia, Romulo; Mathew, Veena; Tam, Annie S; Stirling, Peter C

    2017-09-01

    Bisulfite, in the form of sodium bisulfite or metabisulfite, is used commercially as a food preservative. Bisulfite is used in the laboratory as a single-stranded DNA mutagen in epigenomic analyses of DNA methylation. Recently it has also been used on whole yeast cells to induce mutations in exposed single-stranded regions in vivo. To understand the effects of bisulfite on live cells we conducted a genome-wide screen for bisulfite sensitive mutants in yeast. Screening the deletion mutant array, and collections of essential gene mutants we define a genetic network of bisulfite sensitive mutants. Validation of screen hits revealed hyper-sensitivity of transcription and RNA processing mutants, rather than DNA repair pathways and follow-up analyses support a role in perturbation of RNA transactions. We propose a model in which bisulfite-modified nucleotides may interfere with transcription or RNA metabolism when used in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Alcohol and Sleep Restriction Combined Reduces Vigilant Attention, Whereas Sleep Restriction Alone Enhances Distractibility

    PubMed Central

    Lee, James; Manousakis, Jessica; Fielding, Joanne; Anderson, Clare

    2015-01-01

    Study Objectives: Alcohol and sleep loss are leading causes of motor vehicle crashes, whereby attention failure is a core causal factor. Despite a plethora of data describing the effect of alcohol and sleep loss on vigilant attention, little is known about their effect on voluntary and involuntary visual attention processes. Design: Repeated-measures, counterbalanced design. Setting: Controlled laboratory setting. Participants: Sixteen young (18–27 y; M = 21.90 ± 0.60 y) healthy males. Interventions: Participants completed an attention test battery during the afternoon (13:00–14:00) under four counterbalanced conditions: (1) baseline; (2) alcohol (0.05% breath alcohol concentration); (3) sleep restriction (02:00–07:00); and (4) alcohol/sleep restriction combined. This test battery included a Psychomotor Vigilance Task (PVT) as a measure of vigilant attention, and two ocular motor tasks—visually guided and antisaccade—to measure the involuntary and voluntary allocation of visual attention. Measurements and Results: Only the combined condition led to reductions in vigilant attention characterized by slower mean reaction time, fastest 10% responses, and increased number of lapses (P < 0.05) on the PVT. In addition, the combined condition led to a slowing in the voluntary allocation of attention as reflected by increased antisaccade latencies (P < 0.05). Sleep restriction alone however increased both antisaccade inhibitory errors [45.8% errors versus < 28.4% all others; P < 0.001] and the involuntary allocation of attention, as reflected by faster visually guided latencies (177.7 msec versus > 185.0 msec all others) to a peripheral target (P < 0.05). Conclusions: Our data reveal specific signatures for sleep related attention failure: the voluntary allocation of attention is impaired, whereas the involuntary allocation of attention is enhanced. This provides key evidence for the role of distraction in attention failure during sleep loss. Citation: Lee J

  12. Alcohol and sleep restriction combined reduces vigilant attention, whereas sleep restriction alone enhances distractibility.

    PubMed

    Lee, James; Manousakis, Jessica; Fielding, Joanne; Anderson, Clare

    2015-05-01

    Alcohol and sleep loss are leading causes of motor vehicle crashes, whereby attention failure is a core causal factor. Despite a plethora of data describing the effect of alcohol and sleep loss on vigilant attention, little is known about their effect on voluntary and involuntary visual attention processes. Repeated-measures, counterbalanced design. Controlled laboratory setting. Sixteen young (18-27 y; M = 21.90 ± 0.60 y) healthy males. Participants completed an attention test battery during the afternoon (13:00-14:00) under four counterbalanced conditions: (1) baseline; (2) alcohol (0.05% breath alcohol concentration); (3) sleep restriction (02:00-07:00); and (4) alcohol/sleep restriction combined. This test battery included a Psychomotor Vigilance Task (PVT) as a measure of vigilant attention, and two ocular motor tasks-visually guided and antisaccade-to measure the involuntary and voluntary allocation of visual attention. Only the combined condition led to reductions in vigilant attention characterized by slower mean reaction time, fastest 10% responses, and increased number of lapses (P < 0.05) on the PVT. In addition, the combined condition led to a slowing in the voluntary allocation of attention as reflected by increased antisaccade latencies (P < 0.05). Sleep restriction alone however increased both antisaccade inhibitory errors [45.8% errors versus < 28.4% all others; P < 0.001] and the involuntary allocation of attention, as reflected by faster visually guided latencies (177.7 msec versus > 185.0 msec all others) to a peripheral target (P < 0.05). Our data reveal specific signatures for sleep related attention failure: the voluntary allocation of attention is impaired, whereas the involuntary allocation of attention is enhanced. This provides key evidence for the role of distraction in attention failure during sleep loss. © 2015 Associated Professional Sleep Societies, LLC.

  13. Soil Cations Influence Bryophyte Susceptibility to Bisulfite

    PubMed Central

    BHARALI, BHAGAWAN; BATES, JEFFREY W.

    2002-01-01

    The hypothesis that metal ions absorbed by bryophytes from the underlying soil may ameliorate adverse effects of SO2 was investigated in the terricolous moss species Pleurozium schreberi (Brid.) Mitt. and Rhytidiadelphus triquetrus (Hedw.) Warnst. Dilute sodium bisulfite solutions (equivalent to dissolved SO2) were applied to shoots isolated from soil or in contact with artificial substrata. Marked inhibition of net photosynthesis was observed within 2 h of treatment with 0·3 mm bisulfite in both mosses. Progressive recovery of net photosynthesis occurred 2–8 h after bisulfite treatment, although the extent of this depended on the concentration and pH of the solution. When R. triquetrus and P. schreberi were grown on artificial substrata (calcareous, acid‐mineral or acid‐organic) with weekly bisulfite applications, the only significant effect was poorer growth of P. schreberi receiving bisulfite on the calcareous and acid‐organic substrata. In both species, growth on the calcareous substratum led to increased concentrations of exchangeable Ca2+, whereas exchangeable Fe3+ concentrations increased following growth on the acid‐mineral soil. In another experiment the two mosses were pre‐treated with either Ca2+ or Fe3+ before incubation with bisulfite. In P. schreberi, the depression of net photosynthetic rate caused by bisulfite was ameliorated from 33 to 64 % of the control by pre‐treatment with Fe3+, but it was unaffected by Ca2+ pre‐treatment. In R. triquetrus, the amelioration caused by Fe3+ pre‐treatment was from 16 to 60 % of the control, but pre‐treatment with Ca2+ gave a greater amelioration, to 75 % of the control value. The responses are discussed in terms of soil preferences of the mosses and possible underlying bisulfite amelioration mechanisms. PMID:12234145

  14. Saliva indices track hypohydration during 48h of fluid restriction or combined fluid and energy restriction.

    PubMed

    Oliver, Samuel J; Laing, Stewart J; Wilson, Sally; Bilzon, James L J; Walsh, Neil P

    2008-10-01

    To investigate whether unstimulated whole saliva flow rate (UFR) and osmolality (Sosm) track changes in hydration status during 48h of restricted fluid intake (RF) or combined fluid and energy restriction (RF+RE). Following the 48h periods, UFR and Sosm were assessed after acute exercise dehydration and rehydration. Thirteen healthy males completed three trials in a randomised order: control (CON) where participants received their estimated energy (12,154+/-230kJ/d: mean+/-S.E.M) and fluid (3912+/-140ml/d) requirements, RF trial where participants received their energy requirements and 193+/-19ml/d water to drink (total fluid 960+/-15ml/d) and RF+RE where participants received 1214+/-25kJ/d and 962+/-16ml/d. After 48h, participants completed 30min of maximal exercise followed by rehydration (0-2h) and refeeding (2-6h). At 48h body mass loss exceeded 3% on RF and RF+RE. UFR decreased during 48h on RF (510+/-122 to 169+/-37microl/min) and RF+RE (452+/-92 to 265+/-53microl/min) and was lower than CON at 48h (441+/-90microl/min: P<0.05). Sosm increased during 48h on RF (54+/-3 to 73+/-5mOsmol/kg) and RF+RE (52+/-3 to 68+/-5mOsmol/kg) and was greater than CON at 48h (52+/-2mOsmol/kg: P<0.05). Unlike UFR, Sosm identified the additional hypohydration associated with exercise (P<0.05) and returned to within 0h values with rehydration. Sosm, and to a lesser extent UFR, track hydration status during a 48h period of RF or RF+RE and after subsequent exercise and rehydration.

  15. SMAP: a streamlined methylation analysis pipeline for bisulfite sequencing.

    PubMed

    Gao, Shengjie; Zou, Dan; Mao, Likai; Zhou, Quan; Jia, Wenlong; Huang, Yi; Zhao, Shancen; Chen, Gang; Wu, Song; Li, Dongdong; Xia, Fei; Chen, Huafeng; Chen, Maoshan; Ørntoft, Torben F; Bolund, Lars; Sørensen, Karina D

    2015-01-01

    DNA methylation has important roles in the regulation of gene expression and cellular specification. Reduced representation bisulfite sequencing (RRBS) has prevailed in methylation studies due to its cost-effectiveness and single-base resolution. The rapid accumulation of RRBS data demands well designed analytical tools. To streamline the data processing of DNA methylation from multiple RRBS samples, we present a flexible pipeline named SMAP, whose features include: (i) handling of single-and/or paired-end diverse bisulfite sequencing data with reduced false-positive rates in differentially methylated regions; (ii) detection of allele-specific methylation events with improved algorithms; (iii) a built-in pipeline for detection of novel single nucleotide polymorphisms (SNPs); (iv) support of multiple user-defined restriction enzymes; (v) conduction of all methylation analyses in a single-step operation when well configured. Simulation and experimental data validated the high accuracy of SMAP for SNP detection and methylation identification. Most analyses required in methylation studies (such as estimation of methylation levels, differentially methylated cytosine groups, and allele-specific methylation regions) can be executed readily with SMAP. All raw data from diverse samples could be processed in parallel and 'packetized' streams. A simple user guide to the methylation applications is also provided.

  16. Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

    PubMed Central

    Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L.; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M.

    2017-01-01

    Abstract Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. PMID:28126923

  17. Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells.

    PubMed

    Han, Lin; Wu, Hua-Jun; Zhu, Haiying; Kim, Kun-Yong; Marjani, Sadie L; Riester, Markus; Euskirchen, Ghia; Zi, Xiaoyuan; Yang, Jennifer; Han, Jasper; Snyder, Michael; Park, In-Hyun; Irizarry, Rafael; Weissman, Sherman M; Michor, Franziska; Fan, Rong; Pan, Xinghua

    2017-06-02

    Conventional DNA bisulfite sequencing has been extended to single cell level, but the coverage consistency is insufficient for parallel comparison. Here we report a novel method for genome-wide CpG island (CGI) methylation sequencing for single cells (scCGI-seq), combining methylation-sensitive restriction enzyme digestion and multiple displacement amplification for selective detection of methylated CGIs. We applied this method to analyzing single cells from two types of hematopoietic cells, K562 and GM12878 and small populations of fibroblasts and induced pluripotent stem cells. The method detected 21 798 CGIs (76% of all CGIs) per cell, and the number of CGIs consistently detected from all 16 profiled single cells was 20 864 (72.7%), with 12 961 promoters covered. This coverage represents a substantial improvement over results obtained using single cell reduced representation bisulfite sequencing, with a 66-fold increase in the fraction of consistently profiled CGIs across individual cells. Single cells of the same type were more similar to each other than to other types, but also displayed epigenetic heterogeneity. The method was further validated by comparing the CpG methylation pattern, methylation profile of CGIs/promoters and repeat regions and 41 classes of known regulatory markers to the ENCODE data. Although not every minor methylation differences between cells are detectable, scCGI-seq provides a solid tool for unsupervised stratification of a heterogeneous cell population. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. DNA methylation detection: bisulfite genomic sequencing analysis.

    PubMed

    Li, Yuanyuan; Tollefsbol, Trygve O

    2011-01-01

    DNA methylation, which most commonly occurs at the C5 position of cytosines within CpG dinucleotides, plays a pivotal role in many biological procedures such as gene expression, embryonic development, cellular proliferation, differentiation, and chromosome stability. Aberrant DNA methylation is often associated with loss of DNA homeostasis and genomic instability leading to the development of human diseases such as cancer. The importance of DNA methylation creates an urgent demand for effective methods with high sensitivity and reliability to explore innovative diagnostic and therapeutic strategies. Bisulfite genomic sequencing developed by Frommer and colleagues was recognized as a revolution in DNA methylation analysis based on conversion of genomic DNA by using sodium bisulfite. Besides various merits of the bisulfite genomic sequencing method such as being highly qualitative and quantitative, it serves as a fundamental principle to many derived methods to better interpret the mystery of DNA methylation. Here, we present a protocol currently frequently used in our laboratory that has proven to yield optimal outcomes. We also discuss the potential technical problems and troubleshooting notes for a variety of applications in this field.

  19. MethyQA: a pipeline for bisulfite-treated methylation sequencing quality assessment

    PubMed Central

    2013-01-01

    Background DNA methylation is an epigenetic event that adds a methyl-group to the 5’ cytosine. This epigenetic modification can significantly affect gene expression in both normal and diseased cells. Hence, it is important to study methylation signals at the single cytosine site level, which is now possible utilizing bisulfite conversion technique (i.e., converting unmethylated Cs to Us and then to Ts after PCR amplification) and next generation sequencing (NGS) technologies. Despite the advances of NGS technologies, certain quality issues remain. Some of the more prevalent quality issues involve low per-base sequencing quality at the 3’ end, PCR amplification bias, and bisulfite conversion rates. Therefore, it is important to conduct quality assessment before downstream analysis. To the best of our knowledge, no existing software packages can generally assess the quality of methylation sequencing data generated based on different bisulfite-treated protocols. Results To conduct the quality assessment of bisulfite methylation sequencing data, we have developed a pipeline named MethyQA. MethyQA combines currently available open-source software packages with our own custom programs written in Perl and R. The pipeline can provide quality assessment results for tens of millions of reads in under an hour. The novelty of our pipeline lies in its examination of bisulfite conversion rates and of the DNA sequence structure of regions that have different conversion rates or coverage. Conclusions MethyQA is a new software package that provides users with a unique insight into the methylation sequencing data they are researching. It allows the users to determine the quality of their data and better prepares them to address the research questions that lie ahead. Due to the speed and efficiency at which MethyQA operates, it will become an important tool for studies dealing with bisulfite methylation sequencing data. PMID:23968174

  20. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  1. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  2. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  3. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  4. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  5. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.620 Menadione dimethylpyrimidinol bisulfite. The food additive... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620...

  6. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF ANIMALS Food Additive Listing § 573.625 Menadione nicotinamide bisulfite. The food additive may be safely... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Menadione nicotinamide bisulfite. 573.625 Section...

  7. Inactivation of glutaraldehyde by reaction with sodium bisulfite.

    PubMed

    Jordan, S L; Russo, M R; Blessing, R L; Theis, A B

    1996-02-23

    The microbiocidal activity of glutaraldehyde was inactivated by reaction with sodium bisulfite via formation of a proposed glutaraldehyde-bisulfite complex. High-performance liquid chromatography (HPLC) analysis of 2% (0.2M) alkaline glutaraldehyde indicated complete loss of glutaraldehyde at a 2.2:1 molar ratio of sodium bisulfite to glutaraldehyde. Neither 1.7% (0.17 M) sodium bisulfite alone nor the glutaraldehyde-bisulfite complex was microbiocidal when tested against Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, and Polybac Polyseed BOD seed inoculum. Bacterial inhibition tests indicated that the glutaraldehyde-sodium bisulfite complex had no effect on the growth of sewage microorganisms at concentrations as high as 50-100 ppm (5 x 10(-4)-1 x 10(-3) M), with an IC50 of 230-440 ppm (2.3 x 10(-3)-4.4 x 10(-3) M), based on glutaraldehyde concentration. A 28-close bottle test showed a 5-d biodegradation of 48% and 51%, and a 15-d biodegradation of 57% and 63% for 3:1 and 2.2:1 bisulfite to glutaraldehyde molar ratios, respectively. Acute aquatic toxicity testing with Daphnia magna demonstrated an LC50 of 41-109 ppm (4.1 x 10(-4)-10.9 x 10(-4) M) and a no-observed-effect concentration (NOEC) of 16 ppm (1.6 x 10(-4) M) for the proposed glutaraldehyde-bisulfite complex (based on glutaraldehyde concentration), approximately 10-fold higher than found for glutaraldehyde alone, indicating that the proposed glutaraldehyde-bisulfite complex is less toxic to the environment than glutaraldehyde.

  8. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing

    PubMed Central

    Leontiou, Chrysanthia A.; Hadjidaniel, Michael D.; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C.

    2015-01-01

    Introduction Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Methods Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. Results The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Conclusion Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed

  9. Bisulfite Conversion of DNA: Performance Comparison of Different Kits and Methylation Quantitation of Epigenetic Biomarkers that Have the Potential to Be Used in Non-Invasive Prenatal Testing.

    PubMed

    Leontiou, Chrysanthia A; Hadjidaniel, Michael D; Mina, Petros; Antoniou, Pavlos; Ioannides, Marios; Patsalis, Philippos C

    2015-01-01

    Epigenetic alterations, including DNA methylation, play an important role in the regulation of gene expression. Several methods exist for evaluating DNA methylation, but bisulfite sequencing remains the gold standard by which base-pair resolution of CpG methylation is achieved. The challenge of the method is that the desired outcome (conversion of unmethylated cytosines) positively correlates with the undesired side effects (DNA degradation and inappropriate conversion), thus several commercial kits try to adjust a balance between the two. The aim of this study was to compare the performance of four bisulfite conversion kits [Premium Bisulfite kit (Diagenode), EpiTect Bisulfite kit (Qiagen), MethylEdge Bisulfite Conversion System (Promega) and BisulFlash DNA Modification kit (Epigentek)] regarding conversion efficiency, DNA degradation and conversion specificity. Performance was tested by combining fully methylated and fully unmethylated λ-DNA controls in a series of spikes by means of Sanger sequencing (0%, 25%, 50% and 100% methylated spikes) and Next-Generation Sequencing (0%, 3%, 5%, 7%, 10%, 25%, 50% and 100% methylated spikes). We also studied the methylation status of two of our previously published differentially methylated regions (DMRs) at base resolution by using spikes of chorionic villus sample in whole blood. The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode). The DMRs, EP6 and EP10, were confirmed to be hypermethylated in the CVS and hypomethylated in whole blood. Our findings indicate that the MethylEdge Bisulfite Conversion System (Promega) was shown to have the best performance among the kits. In addition, the methylation level of two of our DMRs, EP6 and EP10, was confirmed. Finally, we showed that bisulfite

  10. Chlorophyll destruction in the presence of bisulfite. [Spinach oleracea

    SciTech Connect

    Peiser, G.; Yang, S.F.

    1985-01-01

    Chlorophyll extracted from Spinacia oleracea leaves as well as purified chlorophyll a in ethanolic solutions (pH 4) was rapidly destroyed, as measured by a decrease in absorbance, in the presence of sodium bisulfite, oxygen and light. Omission of sodium bisulfite, oxygen or light resulted in negligible destruction. The light requirement could be partially substituted by addition of manganous sulfate and destruction was further stimulated in the presence of linoleic acid. Hydroquinone, a free radical scavenger, inhibited both light and Mn/sup 2 +/-mediated (dark) destruction. These results suggest that free radicals produced during the aerobic oxidation of bisulfite are involved in the destruction of chlorophyll. When /sub 35/S-bisulfite was used, two labeled degradation products of chlorophyll were observed.

  11. Metabolic Consequences in Humans of Prolonged Sleep Restriction Combined with Circadian Disruption

    PubMed Central

    Buxton, Orfeu M.; Cain, Sean W.; O’Connor, Shawn P.; Porter, James H.; Duffy, Jeanne F.; Wang, Wei; Czeisler, Charles A.; Shea, Steven A.

    2013-01-01

    Epidemiological studies link short sleep and circadian disruption with risk of metabolic syndrome and diabetes. We tested the hypotheses that prolonged sleep restriction with concurrent circadian disruption, as can occur with shift work, impairs glucose regulation and metabolism. Healthy adults spent >5 weeks in controlled laboratory conditions including: sleep extension (baseline), 3-week sleep restriction (5.6 h sleep/24 h) combined with circadian disruption (recurring 28-h ‘days’), and 9-day recovery sleep with circadian re-entrainment. Prolonged sleep restriction with concurrent circadian disruption significantly decreased resting metabolic rate, and increased postprandial plasma via inadequate pancreatic beta cell responsivity; these normalized with 9 days of recovery sleep and stable circadian reentrainment. Thus, in humans, prolonged sleep restriction with concurrent circadian disruption alters metabolism and could increase risk of obesity and diabetes. PMID:22496545

  12. Combined CMV- and HSV-1 brainstem encephalitis restricted to medulla oblongata.

    PubMed

    Katchanov, J; Branding, G; Stocker, H

    2014-04-15

    We report a very rare case of a combined CMV- and HSV-1 isolated brainstem encephalitis restricted to medulla oblongata in a patient with advanced HIV disease. Neither limbic nor general ventricular involvement was detected on neuroimaging. The case highlights the importance of testing for HSV-1 and CMV in HIV-infected patients presenting with an isolated brainstem syndrome.

  13. Combining Restricted Grazing and Nitrification Inhibitors to Reduce Nitrogen Leaching on New Zealand Dairy Farms.

    PubMed

    Romera, Alvaro J; Cichota, Rogerio; Beukes, Pierre C; Gregorini, Pablo; Snow, Val O; Vogeler, Iris

    2017-01-01

    Intensification of pastoral dairy systems often means more nitrogen (N) leaching. A number of mitigation strategies have been proposed to reduce or reverse this trend. The main strategies focus on reducing the urinary N load onto pastures or reducing the rate of nitrification once the urine has been deposited. Restricted grazing is an example of the former and the use of nitrification inhibitors an example of the latter. A relevant concern is the cost effectiveness of these strategies, independently and jointly. To address this concern, we employed a modeling approach to estimate N leaching with and without the use of these mitigation options from a typical grazing dairy farm in New Zealand. Three restricted grazing options were modeled with and without a nitrification inhibitor (dicyandiamide, DCD) and the results were compared with a baseline farm (no restricted grazing, no inhibitor). Applying DCD twice a year, closely following the cows after an autumn and winter grazing round, has the potential to reduce annualized and farm-scale N leaching by ∼12%, whereas restricted grazing had leaching reductions ranging from 23 to 32%, depending on the timing of restricted grazing. Combining the two strategies resulted in leaching reductions of 31 to 40%. The abatement cost per kilogram of N leaching reduction was NZ$50 with DCD, NZ$32 to 37 for restricted grazing, and NZ$40 to 46 when the two were combined. For the range analyzed, all treatments indicated similar cost per percentage unit of mitigated N leaching, demonstrating that restricted grazing and nitrification inhibitors can be effective when used concurrently. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

  14. Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on Circulating Estrogens and IGF-I in Premenopausal Women

    DTIC Science & Technology

    2005-08-01

    1-0361 TITLE: Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on Circulating Estrogens and...COVERED (From - To) 17 SEP 2001 - 16 SEP 2005 4. TITLE AND SUBTITLE Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on 5a...14. ABSTRACT This proposal entitled “Effects of moderate aerobic exercise combined with caloric restriction on circulating estrogens and IGF- 1 in

  15. Detoxification of Dissolved SO2 (Bisulfite) by Terricolous Mosses

    PubMed Central

    BHARALI, BHAGAWAN; BATES, JEFFREY W.

    2006-01-01

    • Background and Aims The widespread calcifuge moss Pleurozium schreberi is moderately tolerant of SO2, whereas Rhytidiadelphus triquetrus is limited to calcareous soils in regions of the UK that were strongly affected by SO2 pollution in the 20th century. The proposition that tolerance of SO2 by these terricolous mosses depends on metabolic detoxification of dissolved bisulfite was investigated. • Methods The capacities of the two mosses to accelerate loss of bisulfite from aqueous solutions of NaHSO3 were studied using DTNB [5, 5-dithio-(2-nitrobenzoic acid)] to assay bisulfite, and HPLC to assay sulfate in the incubation solutions. Incubations were performed for different durations, in the presence and absence of light, at a range of solution pH values, in the presence of metabolic inhibitors and with altered moss apoplastic Ca2+ and Fe3+ levels. • Key Results Bisulfite disappearance was markedly stimulated in the light and twice as great for R. triquetrus as for P. schreberi. DCMU, an inhibitor of photosynthetic electron chain transport, significantly reduced bisufite loss. • Conclusions Bisulfite (SO2) tolerance in these terricolous mosses involves extracellular oxidation using metabolic (photo-oxidative) energy, passive oxidation by adsorbed Fe3+ (only available to the calcifuge) and probably also internal metabolic detoxification. PMID:16319108

  16. Chlorophyll Destruction by the Bisulfite-Oxygen System 1

    PubMed Central

    Peiser, Galen D.; Yang, Shang Fa

    1977-01-01

    Destruction of chlorophyll, as determined by the loss in absorbance at 665 nm, occurred in two in vitro systems in the presence of bisulfite in 76% ethanol. The first system required light and O2 in addition to bisulfite and exhibited an optimum pH of 4. Chlorophyll functioned as a photosensitizer and there was little chlorophyll destruction occurring above pH 5. With 286 μeinsteins m−2 irradiation, approximately 80% of the chlorophyll was destroyed in three minutes. In the second system, chlorophyll destruction in the presence of bisulfite occurred in the dark and required Mn2+, O2, and glycine. Destruction of chlorophyll in this system was much more rapid than in the light system with approximately 70% destruction occurring in two seconds. In both systems, chlorophyll destruction was linked to bisulfite oxidation. The free radical scavengers hydroquinone, butylated hydroxytoluene, 1,2-dihydroxybenzene-3,5-disulfonic acid, and α-tocopherol were effective in inhibiting the destruction of chlorophyll in both systems. The singlet O2 scavengers, 2,5-dimethylfuran and 1,3-diphenylisobenzofuran, were ineffective inhibitors and β-carotene only slightly effective when tested in the light system. The evidence suggests that in these two systems chlorophyll was destroyed by free radicals, probably superoxide radical, which was produced during the aerobic oxidation of bisulfite. PMID:16660075

  17. Combined effect of gender and caloric restriction on liver proteomic expression profile.

    PubMed

    Valle, Adamo; Silvestri, Elena; Moreno, Maria; Chambery, Angela; Oliver, Jordi; Roca, Pilar; Goglia, Fernando

    2008-07-01

    We analyzed the combined effect of gender and CR on protein expression profile in liver. We identified 27 differentially expressed proteins involved in several cellular functions such as substrate metabolism, antioxidant systems, stress response, iron homeostasis and cardiovascular protection. This study reveals new cellular pathways liable to be similarly regulated in females and calorie restricted rats and which could be related with the greater longevity in these animals.

  18. Combined effects of short-term calorie restriction and exercise on insulin action in normal rats.

    PubMed

    Jiang, H Y; Koike, T; Li, P; Wang, Z H; Kawata, Y; Oshida, Y

    2010-12-01

    The present study examined the effect of combination of short-term calorie restriction (CR) and moderate exercise on insulin action in normal rats. Rats were divided randomly into 4 groups: ad libitum, sedentary (A-Sed); calorie restriction, sedentary (CR-Sed); ad libitum, exercise (A-Ex); and calorie restriction, exercise (CR-Ex). Rats in the exercise groups were run on a rodent treadmill. Rats in the CR groups were fed every alternate day. Oral glucose tolerance test (OGTT) showed improvements in both CR-Sed and A-Ex groups compared with the A-Sed group; no further improvement in glucose tolerance was observed in the CR-Ex group. In contrast, glucose infusion rates (GIRs) determined by the hyperinsulinemic-euglycemic clamp method indicated that the GIR of the CR and exercise combination was significantly better than that of the sole intervention of CR or exercise. There was no difference in the levels of fasting glucose, insulin, or high-molecular weight forms of adiponectin among the 4 groups. Protein expression of GLUT-4 in the skeletal muscle increased by exercise, but not by CR. Our findings indicate that the combination of exercise and CR may be effective in enhancing insulin sensitivity at the skeletal muscle in normal subjects. © Georg Thieme Verlag KG Stuttgart · New York.

  19. BisQC: an operational pipeline for multiplexed bisulfite sequencing

    PubMed Central

    2014-01-01

    Background Bisulfite sequencing is the most efficient single nucleotide resolution method for analysis of methylation status at whole genome scale, but improved quality control metrics are needed to better standardize experiments. Results We describe BisQC, a step-by-step method for multiplexed bisulfite-converted DNA library construction, pooling, spike-in content, and bioinformatics. We demonstrate technical improvements for library preparation and bioinformatic analyses that can be done in standard laboratories. We find that decoupling amplification of bisulfite converted (bis) DNA from the indexing reaction is an advantage, specifically in reducing total PCR cycle number and pre-selecting high quality bis-libraries. We also introduce a progressive PCR method for optimal library amplification and size-selection. At the sequencing stage, we thoroughly test the benefits of pooling non-bis DNA library with bis-libraries and find that BisSeq libraries can be pooled with a high proportion of non-bis DNA libraries with minimal impact on BisSeq output. For informatics analysis, we propose a series of optimization steps including the utilization of the mitochondrial genome as a QC standard, and we assess the validity of using duplicate reads for coverage statistics. Conclusion We demonstrate several quality control checkpoints at the library preparation, pre-sequencing, post-sequencing, and post-alignment stages, which should prove useful in determining sample and processing quality. We also determine that including a significant portion of non-bisulfite converted DNA with bisulfite converted DNA has a minimal impact on usable bisulfite read output. PMID:24734894

  20. Fungi Associated with Softening of Bisulfite-Brined Cherries.

    PubMed

    Lewis, J C; Pierson, C F; Powers, M J

    1963-03-01

    Softening of sound, calcium bisulfite-brined cherries was induced fairly quickly by brining them with cherries rotted by Aspergillus niger, Cytospora leucostoma, and Penicillium expansum, but not with cherries rotted by a variety of other microorganisms, including Alternaria sp., Aspergillus oryzae, Aureobasidium pullulans, Botrytis cinerea, Cladosporium sp., Mucor racemosus, Rhizopus stolonifer, and Sclerotinia fructicola. Rapid softening was correlated with the presence of a bisulfite-stable polygalacturonase, as demonstrated by a cup-plate test. A survey of naturally rotted cherries suggests the involvement of a bark-canker fungus, C. leucostoma, in softening of commercially brined cherries in the Pacific Northwest.

  1. Adverse metabolic consequences in humans of prolonged sleep restriction combined with circadian disruption.

    PubMed

    Buxton, Orfeu M; Cain, Sean W; O'Connor, Shawn P; Porter, James H; Duffy, Jeanne F; Wang, Wei; Czeisler, Charles A; Shea, Steven A

    2012-04-11

    Epidemiological studies link short sleep duration and circadian disruption with higher risk of metabolic syndrome and diabetes. We tested the hypotheses that prolonged sleep restriction with concurrent circadian disruption, as can occur in people performing shift work, impairs glucose regulation and metabolism. Healthy adults spent >5 weeks under controlled laboratory conditions in which they experienced an initial baseline segment of optimal sleep, 3 weeks of sleep restriction (5.6 hours of sleep per 24 hours) combined with circadian disruption (recurring 28-hour "days"), followed by 9 days of recovery sleep with circadian re-entrainment. Exposure to prolonged sleep restriction with concurrent circadian disruption, with measurements taken at the same circadian phase, decreased the participants' resting metabolic rate and increased plasma glucose concentrations after a meal, an effect resulting from inadequate pancreatic insulin secretion. These parameters normalized during the 9 days of recovery sleep and stable circadian re-entrainment. Thus, in humans, prolonged sleep restriction with concurrent circadian disruption alters metabolism and could increase the risk of obesity and diabetes.

  2. Combining Generative and Discriminative Representation Learning for Lung CT Analysis With Convolutional Restricted Boltzmann Machines.

    PubMed

    van Tulder, Gijs; de Bruijne, Marleen

    2016-05-01

    The choice of features greatly influences the performance of a tissue classification system. Despite this, many systems are built with standard, predefined filter banks that are not optimized for that particular application. Representation learning methods such as restricted Boltzmann machines may outperform these standard filter banks because they learn a feature description directly from the training data. Like many other representation learning methods, restricted Boltzmann machines are unsupervised and are trained with a generative learning objective; this allows them to learn representations from unlabeled data, but does not necessarily produce features that are optimal for classification. In this paper we propose the convolutional classification restricted Boltzmann machine, which combines a generative and a discriminative learning objective. This allows it to learn filters that are good both for describing the training data and for classification. We present experiments with feature learning for lung texture classification and airway detection in CT images. In both applications, a combination of learning objectives outperformed purely discriminative or generative learning, increasing, for instance, the lung tissue classification accuracy by 1 to 8 percentage points. This shows that discriminative learning can help an otherwise unsupervised feature learner to learn filters that are optimized for classification.

  3. A novel bisulfite-microfluidic temperature gradient capillary electrophoresis platform for highly sensitive detection of gene promoter methylation.

    PubMed

    Zhang, Huidan; Shan, Lianfeng; Wang, Xiaonan; Ma, Qian; Fang, Jin

    2013-04-15

    The hypermethylated tumor suppressor gene promoters are widely recognized as promising markers for cancer screening and ideal targets for cancer therapy, however, a major obstacle in their clinical study is highly sensitive screening. To address this limitation, we developed a novel bisulfite-microfluidic temperature gradient capillary electrophoresis (bisulfite-μTGCE) platform for gene methylation analysis by combining bisulfite treatment and slantwise radiative heating system-based μTGCE. Bisulfite-treated genomic DNA (gDNA) was amplified with universal primers for both methylated and unmethylated sequences, and introduced into glass microfluidic chip to perform electrophorectic separation under a continuous temperature gradient based on the formation of heteroduplexes. Eight CDKN2A promoter model fragments with different number and location of methylation sites were prepared and successfully analyzed according to their electrophoretic peak patterns, with high stability, picoliter-scale sample consumption, and significantly increased detection speed. The bisulfite-μTGCE could detect methylated gDNA with a detection limit of 7.5pg, and could distinguish as low as 0.1% methylation level in CDKN2A in an unmethylated background. Detection of seven colorectal cancer (CRC) cell lines with known and unknown methylation statuses of CDKN2A promoter and 20 tumor tissues derived from CRC patients demonstrated the capability of detecting hypermethylation in real-world samples. The wider adaptation of this platform was further supported by the detection of the CDKN2A and MLH1 promoters' methylation statuses in combination. This highly sensitive, fast, and low-consumption platform for methylation detection shows great potential for future clinical applications.

  4. 21 CFR 573.625 - Menadione nicotinamide bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Menadione nicotinamide bisulfite. 573.625 Section 573.625 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF...

  5. 21 CFR 573.620 - Menadione dimethylpyrimidinol bisulfite.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Menadione dimethylpyrimidinol bisulfite. 573.620 Section 573.620 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS FOOD ADDITIVES PERMITTED IN FEED AND DRINKING WATER OF...

  6. A Bayesian Assignment Method for Ambiguous Bisulfite Short Reads.

    PubMed

    Tran, Hong; Wu, Xiaowei; Tithi, Saima; Sun, Ming-an; Xie, Hehuang; Zhang, Liqing

    2016-01-01

    DNA methylation is an epigenetic modification critical for normal development and diseases. The determination of genome-wide DNA methylation at single-nucleotide resolution is made possible by sequencing bisulfite treated DNA with next generation high-throughput sequencing. However, aligning bisulfite short reads to a reference genome remains challenging as only a limited proportion of them (around 50-70%) can be aligned uniquely; a significant proportion, known as multireads, are mapped to multiple locations and thus discarded from downstream analyses, causing financial waste and biased methylation inference. To address this issue, we develop a Bayesian model that assigns multireads to their most likely locations based on the posterior probability derived from information hidden in uniquely aligned reads. Analyses of both simulated data and real hairpin bisulfite sequencing data show that our method can effectively assign approximately 70% of the multireads to their best locations with up to 90% accuracy, leading to a significant increase in the overall mapping efficiency. Moreover, the assignment model shows robust performance with low coverage depth, making it particularly attractive considering the prohibitive cost of bisulfite sequencing. Additionally, results show that longer reads help improve the performance of the assignment model. The assignment model is also robust to varying degrees of methylation and varying sequencing error rates. Finally, incorporating prior knowledge on mutation rate and context specific methylation level into the assignment model increases inference accuracy. The assignment model is implemented in the BAM-ABS package and freely available at https://github.com/zhanglabvt/BAM_ABS.

  7. Comparison and quantitative verification of mapping algorithms for whole genome bisulfite sequencing

    USDA-ARS?s Scientific Manuscript database

    Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitat...

  8. Combination of intermittent calorie restriction and eicosapentaenoic acid for inhibition of mammary tumors.

    PubMed

    Mizuno, Nancy K; Rogozina, Olga P; Seppanen, Christine M; Liao, D Joshua; Cleary, Margot P; Grossmann, Michael E

    2013-06-01

    There are a number of dietary interventions capable of inhibiting mammary tumorigenesis; however, the effectiveness of dietary combinations is largely unexplored. Here, we combined 2 interventions previously shown individually to inhibit mammary tumor development. The first was the use of the omega-3 fatty acid, eicosapentaenoic acid (EPA), and the second was the implementation of calorie restriction. MMTV-Her2/neu mice were used as a model for human breast cancers, which overexpress Her2/neu. Six groups of mice were enrolled. Half were fed a control (Con) diet with 10.1% fat calories from soy oil, whereas the other half consumed a diet with 72% fat calories from EPA. Within each diet, mice were further divided into ad libitum (AL), chronic calorie-restricted (CCR), or intermittent calorie-restricted (ICR) groups. Mammary tumor incidence was lowest in ICR-EPA (15%) and highest in AL-Con mice (87%), whereas AL-EPA, CCR-Con, CCR-EPA, and ICR-Con groups had mammary tumor incidence rates of 63%, 47%, 40%, and 59%, respectively. Survival was effected similarly by the interventions. Consumption of EPA dramatically reduced serum leptin (P < 0.02) and increased serum adiponectin in the AL-EPA mice compared with AL-Con mice (P < 0.001). Both CCR and ICR decreased serum leptin and insulin-like growth factor I (IGF-I) compared with AL mice but not compared with each other. These results illustrate that mammary tumor inhibition is significantly increased when ICR and EPA are combined as compared with either intervention alone. This response may be related to alterations in the balance of serum growth factors and adipokines.

  9. RRBS-analyser: a comprehensive web server for reduced representation bisulfite sequencing data analysis.

    PubMed

    Wang, Tao; Liu, Qi; Li, Xianfeng; Wang, Xiaobing; Li, Jinchen; Zhu, Xiaochun; Sun, Zhong Sheng; Wu, Jinyu

    2013-12-01

    In reduced representation bisulfite sequencing (RRBS), genomic DNA is digested with the restriction enzyme and then subjected to next-generation sequencing, which enables detection and quantification of DNA methylation at whole-genome scale with low cost. However, the data processing, interpretation, and analysis of the huge amounts of data generated pose a bioinformatics challenge. We developed RRBS-Analyser, a comprehensive genome-scale DNA methylation analysis server based on RRBS data. RRBS-Analyser can assess sequencing quality, generate detailed statistical information, align the bisulfite-treated short reads to reference genome, identify and annotate the methylcytosines (5mCs) and associate them with different genomic features in CG, CHG, and CHH content. RRBS-Analyser supports detection, annotation, and visualization of differentially methylated regions (DMRs) for multiple samples from nine reference organisms. Moreover, RRBS-Analyser provides researchers with detailed annotation of DMR-containing genes, which will greatly aid subsequent studies. The input of RRBS-Analyser can be raw FASTQ reads, generic SAM format, or self-defined format containing individual 5mC sites. RRBS-Analyser can be widely used by researchers wanting to unravel the complexities of DNA methylome in the epigenetic community. RRBS-Analyser is freely available at http://122.228.158.106/RRBSAnalyser/. © 2013 WILEY PERIODICALS, INC.

  10. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing.

    PubMed

    Yu, Miao; Ji, Lexiang; Neumann, Drexel A; Chung, Dae-Hwan; Groom, Joseph; Westpheling, Janet; He, Chuan; Schmitz, Robert J

    2015-12-02

    Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N(6)-methyladenine (6mA), 5-methylcytosine (5mC) and N(4)-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

  11. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing

    DOE PAGES

    Yu, Miao; Ji, Lexiang; Neumann, Drexel A.; ...

    2015-07-15

    Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly andmore » cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. Lastly, in combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.« less

  12. Detection of DNA Methylation by Whole-Genome Bisulfite Sequencing.

    PubMed

    Li, Qing; Hermanson, Peter J; Springer, Nathan M

    2018-01-01

    DNA methylation plays an important role in the regulation of the expression of transposons and genes. Various methods have been developed to assay DNA methylation levels. Bisulfite sequencing is considered to be the "gold standard" for single-base resolution measurement of DNA methylation levels. Coupled with next-generation sequencing, whole-genome bisulfite sequencing (WGBS) allows DNA methylation to be evaluated at a genome-wide scale. Here, we described a protocol for WGBS in plant species with large genomes. This protocol has been successfully applied to assay genome-wide DNA methylation levels in maize and barley. This protocol has also been successfully coupled with sequence capture technology to assay DNA methylation levels in a targeted set of genomic regions.

  13. [Effects of linggui zhugan decoction combined calorie restriction on the insulin resistance of model rats and mechanisms research].

    PubMed

    Wang, Yuan-yuan; Jin, Ming-hua; Ke, Bin; Li, Su-hua; Shen, Yong-zhi; Zhai, Jia-yu; Chen, Chun-yu; Qin, Jian

    2013-03-01

    To explore the effects of Linggui Zhugan Decoction (LZD) combined calorie restriction on fasting plasma glucose (FPG), the insulin resistance (IR), and the peroxisome proliferator-activated receptor gamma (PPAR-gamma) of IR model rats. Totally 48 male Wistar rats were randomly divided into the control group, the model group, the calorie restriction group, and the TCM + calorie restriction group, 12 in each group. Ordinary forage was given to those in the control group, and high fat diet was fed to those in the rest 3 groups for 12 weeks to establish the IR model. After successful modeling, rats in the control group and the model group were continually fed with the original farage for 4 days. The normal saline at the daily dose of 20 mL/kg was given to them by gastrogavage. The normal saline at the daily dose of 20 mL/kg was given to rats in the calorie restriction group by gastrogavage after 4-day calorie restriction. LZD at the daily dose of 20 mL/kg was given to rats in the TCM +calorie restriction group by gastrogavage after 4-day calorie restriction. The body weight, FPG, serum fasting insulin (FINS), insulin resistance index (IRI), and the protein expression of PPAR-y in the omental adipose tissue were compared. After 4-day calorie restriction, the body weight obviously decreased in the calorie restriction group and the TCM +calorie restriction group, when compared with the model group (P <0.01). There was no statistical difference between the former two groups (P >0.05). The FINS and IRI obviously decreased in the calorie restriction group (P <0.01, P <0.05). The FPG, FINS, and IRI significantly decreased in the TCM + calorie restriction group (P <0. 05, P <0.01). The protein expression of PPAR-gamma obviously decreased in the calorie restriction group and the TCM + calorie restriction group (P <0.01).The phlegm dampness state was alleviated, with more significant effects shown in the TCM + calorie restriction group. LZD combined calorie restriction could

  14. Combined renin-angiotensin system blockade and dietary sodium restriction impairs cardiomyocyte contractility.

    PubMed

    Trongtorsak, Petcharat; Morgan, Trefor O; Delbridge, Lea M D

    2003-12-01

    Blockade of the renin-angiotensin system (RAS) by combined angiotensin-converting enzyme inhibitor and angiotensin type 1 receptor (AT(1)) antagonist treatment with reduced dietary sodium intake produces suppression of cardiac growth and regression of cardiac hypertrophy. The purpose of this study was to investigate whether cardiac growth suppression by combined RAS blockade under conditions of dietary sodium restriction is associated with cardiomyocyte atrophy and contractile dysfunction and whether this intervention modifies cardiomyocyte inotropic responsiveness to angiotensin II (Ang II). Rats were fed a high (4% w/w) or low (0.2% w/w) NaCl diet for six days. Both groups were then given a combined intraperitoneal injection of perindopril (6 mg/kg/day) and losartan (10 mg/kg/day) with maintained dietary treatment for another seven days. At the end of the treatment period, animals were anaesthetised and their hearts were removed and weighed. Left ventricular cardiomyocytes were isolated by enzymatic dissociation and cell dimensions were evaluated. A line scan camera and digital imaging technique were used to assess cardiomyocyte contraction and inotropic responses to exogenous Ang II (10 to 10(-8) M). Dietary treatment alone had no effect on body growth, whereas combined RAS blockade suppressed somatic growth in the low sodium (LS) group, compared with the high sodium (HS) group. This growth suppression in the LS group was also evident in the heart at the organ and cellular level. Studies of cardiomyocyte contraction showed that myocytes from the LS group exhibited contractile instability and depression of contractile performance. Compared with the HS group, myocytes from the LS group showed a significant reduction in maximum cell shortening (6.40+0.17 vs. 7.32+0.16% resting length, p<0.05), and maximum rate of shortening (3.85+0.14 vs. 4.29+0.11 cell length/ms, p<0.05). Myocytes of the HS group exhibited negative inotropic responses to Ang II at all

  15. WBSA: web service for bisulfite sequencing data analysis.

    PubMed

    Liang, Fang; Tang, Bixia; Wang, Yanqing; Wang, Jianfeng; Yu, Caixia; Chen, Xu; Zhu, Junwei; Yan, Jiangwei; Zhao, Wenming; Li, Rujiao

    2014-01-01

    Whole-Genome Bisulfite Sequencing (WGBS) and genome-wide Reduced Representation Bisulfite Sequencing (RRBS) are widely used to study DNA methylation. However, data analysis is complicated, lengthy, and hampered by a lack of seamless analytical pipelines. To address these issues, we developed a convenient, stable, and efficient web service called Web Service for Bisulfite Sequencing Data Analysis (WBSA) to analyze bisulfate sequencing data. WBSA focuses on not only CpG methylation, which is the most common biochemical modification in eukaryotic DNA, but also non-CG methylation, which have been observed in plants, iPS cells, oocytes, neurons and stem cells of human. WBSA comprises three main modules as follows: WGBS data analysis, RRBS data analysis, and differentially methylated region (DMR) identification. The WGBS and RRBS modules execute read mapping, methylation site identification, annotation, and advanced analysis, whereas the DMR module identifies actual DMRs and annotates their correlations to genes. WBSA can be accessed and used without charge either online or local version. WBSA also includes the executables of the Portable Batch System (PBS) and standalone versions that can be downloaded from the website together with the installation instructions. WBSA is available at no charge for academic users at http://wbsa.big.ac.cn.

  16. WBSA: Web Service for Bisulfite Sequencing Data Analysis

    PubMed Central

    Wang, Yanqing; Wang, Jianfeng; Yu, Caixia; Chen, Xu; Zhu, Junwei; Yan, Jiangwei; Zhao, Wenming; Li, Rujiao

    2014-01-01

    Whole-Genome Bisulfite Sequencing (WGBS) and genome-wide Reduced Representation Bisulfite Sequencing (RRBS) are widely used to study DNA methylation. However, data analysis is complicated, lengthy, and hampered by a lack of seamless analytical pipelines. To address these issues, we developed a convenient, stable, and efficient web service called Web Service for Bisulfite Sequencing Data Analysis (WBSA) to analyze bisulfate sequencing data. WBSA focuses on not only CpG methylation, which is the most common biochemical modification in eukaryotic DNA, but also non-CG methylation, which have been observed in plants, iPS cells, oocytes, neurons and stem cells of human. WBSA comprises three main modules as follows: WGBS data analysis, RRBS data analysis, and differentially methylated region (DMR) identification. The WGBS and RRBS modules execute read mapping, methylation site identification, annotation, and advanced analysis, whereas the DMR module identifies actual DMRs and annotates their correlations to genes. WBSA can be accessed and used without charge either online or local version. WBSA also includes the executables of the Portable Batch System (PBS) and standalone versions that can be downloaded from the website together with the installation instructions. WBSA is available at no charge for academic users at http://wbsa.big.ac.cn. PMID:24497972

  17. Identification of differentially methylated regions using streptavidin bisulfite ligand methylation enrichment (SuBLiME), a new method to enrich for methylated DNA prior to deep bisulfite genomic sequencing

    PubMed Central

    Ross, Jason P.; Shaw, Jan M.; Molloy, Peter L.

    2013-01-01

    We have developed a method that enriches for methylated cytosines by capturing the fraction of bisulfite-treated DNA with unconverted cytosines. The method, called streptavidin bisulfite ligand methylation enrichment (SuBLiME), involves the specific labeling (using a biotin-labeled nucleotide ligand) of methylated cytosines in bisulfite-converted DNA. This step is then followed by affinity capture, using streptavidin-coupled magnetic beads. SuBLiME is highly adaptable and can be combined with deep sequencing library generation and/or genomic complexity-reduction. In this pilot study, we enriched methylated DNA from Csp6I-cut complexity-reduced genomes of colorectal cancer cell lines (HCT-116, HT-29 and SW-480) and normal blood leukocytes with the aim of discovering colorectal cancer biomarkers. Enriched libraries were sequenced with SOLiD-3 technology. In pairwise comparisons, we scored a total of 1,769 gene loci and 33 miRNA loci as differentially methylated between the cell lines and leukocytes. Of these, 516 loci were differently methylated in at least two promoter-proximal CpG sites over two discrete Csp6I fragments. Identified methylated gene loci were associated with anatomical development, differentiation and cell signaling. The data correlated with good agreement to a number of published colorectal cancer DNA methylation biomarkers and genomic data sets. SuBLiME is effective in the enrichment of methylated nucleic acid and in the detection of known and novel biomarkers. PMID:23257838

  18. Fractionation of oil palm empty fruit bunch by bisulfite pretreatment for the production of bioethanol and high value products.

    PubMed

    Tan, Liping; Wang, Meimei; Li, Xuezhi; Li, Hongxing; Zhao, Jian; Qu, Yinbo; Choo, Yuen May; Loh, Soh Kheang

    2016-01-01

    In this work, fractionation of empty fruit bunch (EFB) by bisulfite pretreatment was studied for the production of bioethanol and high value products to achieve biorefinery of EFB. EFB was fractionated to solid and liquor components by bisulfite process. The solid components were used for bioethanol production by quasi-simultaneous saccharification and fermentation. The liquor components were then converted to furfural by hydrolysis with sulfuric acid. Preliminary results showed that the concentration of furfural was highest at 18.8g/L with 0.75% sulfuric acid and reaction time of 25min. The conversion of xylose to furfural was 82.5%. Furthermore, we attempted to fractionate the liquor into hemicellulose sugars and lignin by different methods for producing potential chemicals, such as xylose, xylooligosaccharide, and lignosulfonate. Our research showed that the combination of bisulfite pretreatment and resin separation could effectively fractionate EFB components to produce bioethanol and other high value chemicals. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. The reaction of cytosine with bisulfite by base flipping from the duplex.

    PubMed

    Oka, Yoshimi; Peng, Tao; Takei, Fumie; Nakatani, Kazuhiko

    2008-01-01

    Methylation of cytosine at the C5-position in DNA plays a major role in epigenetic gene control. The detection of 5-methylcytosine was designed with the base flipping from the duplex using NC and CGG/CGG triad. Using bisulfite and hydroxylamine, the reaction of cytosine in 11-mer duplex produced the adduct of bisulfite and hydroxylamine, which was isolated by HPLC and identified by MALDI-TOF MS. The duplex containing (5m)C in (5m)CGG/(5m)CGG did not react with bisulfite and hydroxylamine under the same conditions. Further this method enables cytosine adjacent to mismatch guanine to react with bisulfite and hydroxylamine selectively.

  20. DNA methylation mapping by tag-modified bisulfite genomic sequencing.

    PubMed

    Han, Weiguo; Cauchi, Stephane; Herman, James G; Spivack, Simon D

    2006-08-01

    A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p<0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.

  1. Kinetics, mechanism and thermodynamics of bisulfite-aldehyde adduct formation

    SciTech Connect

    Olson, T.M.; Boyce, S.D.; Hoffmann, M.R.

    1986-04-01

    The kinetics and mechanism of bisulfite addition to benzaldehyde were studied at low pH in order to assess the importance of this reaction in stabilizing S(IV) in fog-, cloud-, and rainwater. Previously, the authors established that appreciable concentrations of the formaldehyde-bisulfite adduct (HMSA) are often present in fogwater. Measured HMSA concentrations in fogwater often do not fully account for observed excess S(IV) concentrations, however, so that other S(IV)-aldehyde adducts may be present. Reaction rates were determined by monitoring the disappearance of benzaldehyde by U.V. spectrophotometry under pseudo-first order conditions, (S(IV))/sub T/ >>(phi-CHO)/sub T/, in the pH range 0 - 4.4 at 25/sup 0/C. The equilibrium constant was determined by dissolving the sodium salt of the addition compound in a solution adjusted to pH 3.9, and measuring the absorbance of the equilibrated solution at 250 nm. A literature value of the extinction coefficient for benzaldehyde was used to calculate the concentration of free benzaldehyde. All solutions were prepared under an N/sub 2/ atmosphere using deoxygenated, deionized water and ionic strength was maintained at 1.0 M with sodium chloride.

  2. Application of microdroplet PCR for large-scale targeted bisulfite sequencing

    PubMed Central

    Komori, H. Kiyomi; LaMere, Sarah A.; Torkamani, Ali; Hart, G. Traver; Kotsopoulos, Steve; Warner, Jason; Samuels, Michael L.; Olson, Jeff; Head, Steven R.; Ordoukhanian, Phillip; Lee, Pauline L.; Link, Darren R.; Salomon, Daniel R.

    2011-01-01

    Cytosine methylation of DNA CpG dinucleotides in gene promoters is an epigenetic modification that regulates gene transcription. While many methods exist to interrogate methylation states, few current methods offer large-scale, targeted, single CpG resolution. We report an approach combining bisulfite treatment followed by microdroplet PCR with next-generation sequencing to assay the methylation state of 50 genes in the regions 1 kb upstream of and downstream from their transcription start sites. This method yielded 96% coverage of the targeted CpGs and demonstrated high correlation between CpG island (CGI) DNA methylation and transcriptional regulation. The method was scaled to interrogate the methylation status of 77,674 CpGs in the promoter regions of 2100 genes in primary CD4 T cells. The 2100 gene library yielded 97% coverage of all targeted CpGs and 99% of the target amplicons. PMID:21757609

  3. Effects of Moderate Aerobic Exercise Combined With Calorie Restriction on Circulating Estrogens and IGF-I in Premenopausal Women

    DTIC Science & Technology

    2004-10-01

    Kines 496c Kines 496c Kines 596c Kines 597i Kines 395b Scientific basis of Exercise for Older Adults Independent Study 3 Fitness Appraisal 4...AD Award Number: DAMD17-01-1-0360 TITLE: Effects of Moderate Aerobic Exercise Combined with Calorie Restriction on Circulating Estrogens and IGF...AND DATES COVERED Annual Summary (17 Sep 2003 - 16 Sep 2004) 4. TITLE AND SUBTITLE Effects of Moderate Aerobic Exercise Combined with

  4. A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements

    PubMed Central

    Yang, Allen S.; Estécio, Marcos R. H.; Doshi, Ketan; Kondo, Yutaka; Tajara, Eloiza H.; Issa, Jean-Pierre J.

    2004-01-01

    We report a method for studying global DNA methylation based on using bisulfite treatment of DNA and simultaneous PCR of multiple DNA repetitive elements, such as Alu elements and long interspersed nucleotide elements (LINE). The PCR product, which represents a pool of approximately 15 000 genomic loci, could be used for direct sequencing, selective restriction digestion or pyrosequencing, in order to quantitate DNA methylation. By restriction digestion or pyrosequencing, the assay was reproducible with a standard deviation of only 2% between assays. Using this method we found that almost two-thirds of the CpG methylation sites in Alu elements are mutated, but of the remaining methylation target sites, 87% were methylated. Due to the heavy methylation of repetitive elements, this assay was especially useful in detecting decreases in DNA methylation, and this assay was validated by examining cell lines treated with the methylation inhibitor 5-aza-2′deoxycytidine (DAC), where we found a 1–16% decrease in Alu element and 18–60% LINE methylation within 3 days of treatment. This method can be used as a surrogate marker of genome-wide methylation changes. In addition, it is less labor intensive and requires less DNA than previous methods of assessing global DNA methylation. PMID:14973332

  5. Ultrasound Evaluation of the Combined Effects of Thoracolumbar Fascia Injury and Movement Restriction in a Porcine Model.

    PubMed

    Bishop, James H; Fox, James R; Maple, Rhonda; Loretan, Caitlin; Badger, Gary J; Henry, Sharon M; Vizzard, Margaret A; Langevin, Helene M

    2016-01-01

    The persistence of back pain following acute back "sprains" is a serious public health problem with poorly understood pathophysiology. The recent finding that human subjects with chronic low back pain (LBP) have increased thickness and decreased mobility of the thoracolumbar fascia measured with ultrasound suggest that the fasciae of the back may be involved in LBP pathophysiology. This study used a porcine model to test the hypothesis that similar ultrasound findings can be produced experimentally in a porcine model by combining a local injury of fascia with movement restriction using a "hobble" device linking one foot to a chest harness for 8 weeks. Ultrasound measurements of thoracolumbar fascia thickness and shear plane mobility (shear strain) during passive hip flexion were made at the 8 week time point on the non-intervention side (injury and/or hobble). Injury alone caused both an increase in fascia thickness (p = .007) and a decrease in fascia shear strain on the non-injured side (p = .027). Movement restriction alone did not change fascia thickness but did decrease shear strain on the non-hobble side (p = .024). The combination of injury plus movement restriction had additive effects on reducing fascia mobility with a 52% reduction in shear strain compared with controls and a 28% reduction compared to movement restriction alone. These results suggest that a back injury involving fascia, even when healed, can affect the relative mobility of fascia layers away from the injured area, especially when movement is also restricted.

  6. Ultrasound Evaluation of the Combined Effects of Thoracolumbar Fascia Injury and Movement Restriction in a Porcine Model

    PubMed Central

    Bishop, James H.; Fox, James R.; Maple, Rhonda; Loretan, Caitlin; Badger, Gary J.; Henry, Sharon M.; Vizzard, Margaret A.; Langevin, Helene M.

    2016-01-01

    The persistence of back pain following acute back “sprains” is a serious public health problem with poorly understood pathophysiology. The recent finding that human subjects with chronic low back pain (LBP) have increased thickness and decreased mobility of the thoracolumbar fascia measured with ultrasound suggest that the fasciae of the back may be involved in LBP pathophysiology. This study used a porcine model to test the hypothesis that similar ultrasound findings can be produced experimentally in a porcine model by combining a local injury of fascia with movement restriction using a “hobble” device linking one foot to a chest harness for 8 weeks. Ultrasound measurements of thoracolumbar fascia thickness and shear plane mobility (shear strain) during passive hip flexion were made at the 8 week time point on the non-intervention side (injury and/or hobble). Injury alone caused both an increase in fascia thickness (p = .007) and a decrease in fascia shear strain on the non-injured side (p = .027). Movement restriction alone did not change fascia thickness but did decrease shear strain on the non-hobble side (p = .024). The combination of injury plus movement restriction had additive effects on reducing fascia mobility with a 52% reduction in shear strain compared with controls and a 28% reduction compared to movement restriction alone. These results suggest that a back injury involving fascia, even when healed, can affect the relative mobility of fascia layers away from the injured area, especially when movement is also restricted. PMID:26820883

  7. 40 CFR 415.540 - Applicability; description of the sodium bisulfite production subcategory.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 28 2010-07-01 2010-07-01 true Applicability; description of the sodium bisulfite production subcategory. 415.540 Section 415.540 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) EFFLUENT GUIDELINES AND STANDARDS INORGANIC CHEMICALS MANUFACTURING POINT SOURCE CATEGORY Sodium Bisulfite...

  8. Reversible Fluorescent Probe for Selective Detection and Cell Imaging of Oxidative Stress Indicator Bisulfite.

    PubMed

    Zhang, Yajiao; Guan, Lingmei; Yu, Huan; Yan, Yehan; Du, Libo; Liu, Yang; Sun, Mingtai; Huang, Dejian; Wang, Suhua

    2016-04-19

    In this paper, we report a benzothiazole-functionalized cyanine fluorescence probe and demonstrate that it is selectively reactive to bisulfite, an intermediate indicator for oxidative stress. The selective reaction can be monitored by distinct ratiometric fluorescence variation favorable for cell imaging and visualization. The original probe can be regenerated in high yield through the elimination of bisulfite from the product by peroxides such as hydrogen peroxide, accompanied by fluorescence turning on at 590 nm, showing a potential application for the detection of peroxides. We successfully applied this probe for fluorescence imaging of bisulfite in cancer cells (MCF-7) treated with bisulfite and hydrogen peroxide as well as a selective detection limit of 0.34 μM bisulfite in aqueous solution.

  9. The vasorelaxant effect and its mechanisms of sodium bisulfite as a sulfur dioxide donor.

    PubMed

    Meng, Ziqiang; Yang, Zhenhua; Li, Junling; Zhang, Quanxi

    2012-10-01

    To study the biological role of bisulfite on vascular contractility and its underlying cellular and molecular mechanisms, to explore whether bisulfite can be used as a sulfur dioxide (SO(2)) donor in the biological experiments, the vasorelaxant effects of sodium bisulfite and sodium sulfite on isolated rat thoracic aortic rings were compared; and the signal transduction pathways and the ion channels involved in the vascular effects of bisulfite were investigated. The results show that: (1) Sodium bisulfite relaxed rat thoracic aortic rings in a concentration-dependent manner (from 100 to 4000 μM); however, sodium sulfite at 500 and 1000 μM caused vasoconstriction, and only at higher concentrations (from 2000 to 4000 μM) it caused vasorelaxation in a concentration-dependent manner. (2) The vasorelaxation caused by the bisulfite at low concentrations (≤500 μM) was endothelium-dependent, but at high concentrations (≥1000 μM) it was endothelium-independent. (3) The vasorelaxation by the bisulfite at the low concentrations was partially mediated by the cGMP pathway and the vasorelaxation was related to big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, but not due to prostaglandin, protein kinase C (PKC) and cAMP pathways. (4) The vasorelaxation by the bisulfite at high concentrations was partially inhibited by tetraethylammonium (TEA) and glibenclamide, suggesting that the vasorelaxation was related to ATP-sensitive K(+) channel (K(ATP)) and L-type calcium-channel. These results led to the conclusion that bisulfite (HSO(3)(-)) might be a vasoactive factor and sodium bisulfite can be used as a SO(2) donor for the study of SO(2) biology.

  10. Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite-sequencing

    SciTech Connect

    Yu, Miao; Ji, Lexiang; Neumann, Drexel A.; Chung, Dae -Hwan; Groom, Joseph; Westpheling, Janet; He, Chuan; Schmitz, Robert J.

    2015-07-15

    Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N6-methyladenine (6mA), 5-methylcytosine (5mC) and N4-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. Lastly, in combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.

  11. Final report on the safety assessment of sodium sulfite, potassium sulfite, ammonium sulfite, sodium bisulfite, ammonium bisulfite, sodium metabisulfite and potassium metabisulfite.

    PubMed

    Nair, Bindu; Elmore, Amy R

    2003-01-01

    Sodium Sulfite, Ammonium Sulfite, Sodium Bisulfite, Potassium Bisulfite, Ammonium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite are inorganic salts that function as reducing agents in cosmetic formulations. All except Sodium Metabisulfite also function as hair-waving/straightening agents. In addition, Sodium Sulfite, Potassium Sulfite, Sodium Bisulfite, and Sodium Metabisulfite function as antioxidants. Although Ammonium Sulfite is not in current use, the others are widely used in hair care products. Sulfites that enter mammals via ingestion, inhalation, or injection are metabolized by sulfite oxidase to sulfate. In oral-dose animal toxicity studies, hyperplastic changes in the gastric mucosa were the most common findings at high doses. Ammonium Sulfite aerosol had an acute LC(50) of >400 mg/m(3) in guinea pigs. A single exposure to low concentrations of a Sodium Sulfite fine aerosol produced dose-related changes in the lung capacity parameters of guinea pigs. A 3-day exposure of rats to a Sodium Sulfite fine aerosol produced mild pulmonary edema and irritation of the tracheal epithelium. Severe epithelial changes were observed in dogs exposed for 290 days to 1 mg/m(3) of a Sodium Metabisulfite fine aerosol. These fine aerosols contained fine respirable particle sizes that are not found in cosmetic aerosols or pump sprays. None of the cosmetic product types, however, in which these ingredients are used are aerosolized. Sodium Bisulfite (tested at 38%) and Sodium Metabisulfite (undiluted) were not irritants to rabbits following occlusive exposures. Sodium Metabisulfite (tested at 50%) was irritating to guinea pigs following repeated exposure. In rats, Sodium Sulfite heptahydrate at large doses (up to 3.3 g/kg) produced fetal toxicity but not teratogenicity. Sodium Bisulfite, Sodium Metabisulfite, and Potassium Metabisulfite were not teratogenic for mice, rats, hamsters, or rabbits at doses up to 160 mg/kg. Generally, Sodium Sulfite, Sodium

  12. Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on Circulating Estrogens and IGF-I in Premenopausal Women

    DTIC Science & Technology

    2003-10-01

    kcals; P< 0.05 pre vs post in both groups). The combination of moderate exercise and diet produced significant weight loss in both groups (Low BMI...Kines 481W Scientific basis of 3 50 Exercise for Older Adults Kines 496C Independent Study 3 1 Spring 1998 Kines 456 Fitness Appraisal 4 96 Kines...N AD_ Award Number: DAMD17-01-1-0360 TITLE: Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on Circulating Estrogens and IGF-I

  13. Ratiometric fluorescent probe for rapid detection of bisulfite through 1,4-addition reaction in aqueous solution.

    PubMed

    Sun, Yue; Zhao, Dong; Fan, Shanwei; Duan, Lian; Li, Ruifeng

    2014-04-16

    A ratiometric fluorescent probe based on a positively charged benzo[e]indolium moiety for bisulfite is reported. The bisulfite underwent a 1,4-addition reaction with the C-4 atom in the ethylene group. This reaction resulted in a large emission wavelength shift (Δλ = 106 nm) and an observable fluorescent color change from orange to cyan. The reaction could be completed in 90 s in a PBS buffer solution and displayed high selectivity and sensitivity for bisulfite. A simple paper test strip system was developed to detect bisulfite rapidly. Probe 1 was used to detect bisulfite in real samples with good recovery.

  14. Combined transductional untargeting/retargeting and transcriptional restriction enhance adenovirus gene targeting and therapy for hepatic colorectal cancer tumors

    PubMed Central

    Li, Hua-Jung; Everts, Maaike; Yamamoto, Masato; Curiel, David T.; Herschman, Harvey R.

    2009-01-01

    Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors, and other epithelial tumors, often express both cyclooxygenase 2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus vectors infect liver and lack tumor tropism, they cannot be utilized for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional adenovirus hepatic untargeting and tumor retargeting by a bispecific adapter, sCARhMFE, composed of sCAR (the Coxsackie and Adenovirus Receptor ectodomain) and MFE-23 (a single-chain anti-CEA antibody), to untarget liver following intravenous administration of adenovirus vectors expressing firefly luciferase and to retarget virus to hepatic colorectal tumor xenografts and non-small cell lung tumor xenografts. To improve both liver untargeting and tumor retargeting, we developed sCARfMFE, a trimerized sCARhMFE adapter. Trimerization greatly improves both untargeting of CAR-dependent adenovirus infection and CEA-dependent virus retargeting, in culture and in vivo. Combining sCARfMFE bispecific adapter transductional liver untargeting and transductional tumor retargeting with COX-2 transcriptional tumor-restricted transgene expression increases systemically-administered adenovirus therapeutic efficacy for hepatic CRC tumors, using Herpes Virus Type 1 thymidine kinase as a therapeutic gene in conjunction with the prodrug ganciclovir. Both transductional untargeting and COX-2 transcriptional restriction also reduce HSV1-tk/GCV hepatic toxicity. In addition, transductional sCARfMFE untargeting reduces the innate immune response to systemic adenovirus administration. Combined transductional liver Ad untargeting, transductional tumor retargeting and transcriptional transgene restriction suggests a means to engineer practical, effective therapeutic agents for hepatic CRC metastases in particular, as well as hepatic metastases of other epithelial

  15. Combined transductional untargeting/retargeting and transcriptional restriction enhances adenovirus gene targeting and therapy for hepatic colorectal cancer tumors.

    PubMed

    Li, Hua-Jung; Everts, Maaike; Yamamoto, Masato; Curiel, David T; Herschman, Harvey R

    2009-01-15

    Unresectable hepatic colorectal cancer (CRC) metastases are a leading cause of cancer mortality. These tumors and other epithelial tumors often express both cyclooxygenase-2 (COX-2) and carcinoembryonic antigen (CEA). Because adenovirus (Ad) vectors infect the liver and lack tumor tropism, they cannot be used for systemic therapy of hepatic metastases. We used COX-2 transcriptional restriction, in combination with transductional Ad hepatic untargeting and tumor retargeting by a bispecific adapter, sCARhMFE, composed of sCAR [the coxsackie/Ad receptor (CAR) ectodomain] and MFE-23 (a single-chain anti-CEA antibody), to untarget liver after i.v. administration of Ad vectors expressing firefly luciferase and to retarget virus to hepatic colorectal tumor xenografts and non-small cell lung tumor xenografts. To improve both liver untargeting and tumor retargeting, we developed sCARfMFE, a trimerized sCARhMFE adapter. Trimerization greatly improves both untargeting of CAR-dependent Ad infection and CEA-dependent virus retargeting in culture and in vivo. Combining sCARfMFE bispecific adapter transductional liver untargeting and transductional tumor retargeting with COX-2 transcriptional tumor-restricted transgene expression increases systemically administered Ad therapeutic efficacy for hepatic CRC tumors, using herpes virus type 1 thymidine kinase (HSV1-tk) as a therapeutic gene in conjunction with the prodrug ganciclovir (GCV). Both transductional untargeting and COX-2 transcriptional restriction also reduce HSV1-tk/GCV hepatic toxicity. In addition, transductional sCARfMFE untargeting reduces the innate immune response to systemic Ad administration. Combined transductional liver Ad untargeting, transductional tumor retargeting, and transcriptional transgene restriction suggests a means to engineer practical, effective therapeutic agents for hepatic CRC metastases in particular, as well as hepatic metastases of other epithelial cancers.

  16. Calcium bisulfite oxidation rate in the wet limestone-gypsum flue gas desulfurization process

    SciTech Connect

    Lancia, A.; Musmarra, D.

    1999-06-01

    In this paper oxidation of calcium bisulfite in aqueous solutions was studied, in connection with the limestone-gypsum flue gas desulfurization process. Experimental measurements of the oxidation rate were carried out in a laboratory scale stirred reactor with continuous feeding of both gas and liquid phase. A calcium bisulfite clear solution was used as liquid phase, and pure oxygen or mixtures of oxygen and nitrogen were used as gas phase. Experiments were carried out at T = 45 C varying the composition of the liquid phase and the oxygen partial pressure. Manganous sulfate was used as catalyst. The analysis of the experimental results showed that the kinetics of bisulfite oxidation in the presence of MnSO{sub 4} follow a parallel reaction mechanism, in which the overall reaction rate can be calculated as the sum between the uncatalyzed rate (3/2 order in bisulfite ion) and the catalyzed reaction rate (first order in manganous ion).

  17. Dietary restriction in combination with a nutraceutical supplement for the management of equine metabolic syndrome in horses.

    PubMed

    McGowan, C M; Dugdale, A H; Pinchbeck, G L; Argo, C McG

    2013-05-01

    Few studies have examined the effect of dietary restriction in horses with equine metabolic syndrome (EMS). This study aimed to determine improvements in insulin sensitivity following dietary restriction for 6 weeks, and to determine if the improvement would be greater in horses receiving short-chain fructo-oligosaccharides (sc-FOS). Dietary management involved feeding grass hay, restricted to 1.25% of body mass (BM) as daily dry matter intake and soaked in cold water prior to feeding, with the addition of a vitamin and mineral nutraceutical supplement with or without the addition of sc-FOS (10 g/100 kg). Soaking the hay resulted in a significant reduction in non-structural carbohydrates (38%, P = 0.01), digestible energy (6.78%, P = 0.01) and water soluble minerals. Following 6 weeks of dietary restriction with soaked grass hay and nutraceutical supplement, horses lost an average of 6.8% BM and showed reductions in body condition score (BCS) and belly circumference. Sensitivity to insulin improved overall, as determined by the total insulin response during the combined glucose insulin test. The magnitude of improvement in insulin sensitivity was associated with the degree of insulin resistance recorded at outset, and the extent of overall losses in BM and BCS, but was independent of the addition of sc-FOS. The nutraceutical supplement was highly palatable and no adverse effects were noted. From the findings of this study a strict dietary program in combination with a specifically designed vitamin and mineral nutraceutical supplement can be recommended to obtain rapid improvements in BM, BCS and insulin sensitivity of animals presenting with EMS. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

    PubMed Central

    Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

    2013-01-01

    Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

  19. Dechlorination of chloroacetanilide herbicides by plant growth regulator sodium bisulfite.

    PubMed

    Bian, Haitao; Chen, Jingwen; Cai, Xiyun; Liu, Ping; Wang, Ying; Huang, Liping; Qiao, Xianliang; Hao, Ce

    2009-08-01

    Chloroacetanilide herbicides are frequently detected in groundwater and surface waters, and pose high risks to aquatic biota. In this study, sodium bisulfite (NaHSO(3)), a plant growth regulator used in China, was used to remove three chloroacetanilide herbicides including alachlor, acetochlor and S-metolachlor. These herbicides were rapidly dechlorinated by NaHSO(3) in neutral conditions. The dechlorination was accelerated with increasing pH, temperature and NaHSO(3) concentrations. Kinetic analysis and mass spectrum identification revealed that the reaction followed S(N)2 nucleophilic substitution, in which the chlorine was replaced by the reactive specie sulfite. Alachlor and its isomer acetochlor had similar reaction rates, whereas they were more readily transformed than S-metolachlor that had larger steric hindrance and weaker electrophilicity. The transformation products were chloroacetanilide ethane sulfonic acids (ESAs), which were also encountered as major metabolites of these herbicides in natural environment via common metabolic pathways and were less toxic to green algae compared to the parent herbicides. These results indicate that NaHSO(3) can accelerate transformation of chloroacetanilide herbicides to the less toxic transformation products by nucleophilic substitution and dechlorination in aquatic environment. NaHSO(3) can be potentially used for the removal of chloroacetanilide herbicides from wastewater effluent, spill sites and accidental discharge.

  20. Information recovery from low coverage whole-genome bisulfite sequencing

    PubMed Central

    Libertini, Emanuele; Heath, Simon C.; Hamoudi, Rifat A.; Gut, Marta; Ziller, Michael J.; Czyz, Agata; Ruotti, Victor; Stunnenberg, Hendrik G.; Frontini, Mattia; Ouwehand, Willem H.; Meissner, Alexander; Gut, Ivo G.; Beck, Stephan

    2016-01-01

    The cost of whole-genome bisulfite sequencing (WGBS) remains a bottleneck for many studies and it is therefore imperative to extract as much information as possible from a given dataset. This is particularly important because even at the recommend 30X coverage for reference methylomes, up to 50% of high-resolution features such as differentially methylated positions (DMPs) cannot be called with current methods as determined by saturation analysis. To address this limitation, we have developed a tool that dynamically segments WGBS methylomes into blocks of comethylation (COMETs) from which lost information can be recovered in the form of differentially methylated COMETs (DMCs). Using this tool, we demonstrate recovery of ∼30% of the lost DMP information content as DMCs even at very low (5X) coverage. This constitutes twice the amount that can be recovered using an existing method based on differentially methylated regions (DMRs). In addition, we explored the relationship between COMETs and haplotypes in lymphoblastoid cell lines of African and European origin. Using best fit analysis, we show COMETs to be correlated in a population-specific manner, suggesting that this type of dynamic segmentation may be useful for integrated (epi)genome-wide association studies in the future. PMID:27346250

  1. Fluorescence detection of endogenous bisulfite in liver cancer cells using an effective ESIPT enhanced FRET platform.

    PubMed

    Li, Dong-Peng; Wang, Zhao-Yang; Su, Hao; Miao, Jun-Ying; Zhao, Bao-Xiang

    2017-01-03

    Probe L-HF1, which featured large (pseudo) Stokes shifts and high FRET efficiency, was designed on a new ESIPT enhanced FRET platform for the detection of HSO3(-)/SO3(2-). L-HF1 could detect endogenous bisulfite in HepG2 cells but not in L-02 cells, implying the different bisulfite levels in normal and cancer cells of liver.

  2. Exogenous DNA internalisation by sperm cells is improved by combining lipofection and restriction enzyme mediated integration.

    PubMed

    Churchil, R R; Gupta, J; Singh, A; Sharma, D

    2011-06-01

    1. Three types of exogenous DNA inserts, i.e. complete linearised pVIVO2-GFP/LacZ vector (9620 bp), the LacZ gene (5317 bp) and the GFP gene (2152 bp) were used to transfect chicken spermatozoa through simple incubation of sperm cells with insert. 2. PCR assay, Dot Blot hybridisation and Southern hybridisation showed the successful internalisation of exogenous DNA by chicken sperm cells. 3. Lipofection and Restriction Enzyme Mediated Integration (REMI) were used to improve the rate of internalisation of exogenous DNA by sperm cells. 4. Results from dot blot as well as Southern hybridisation were semi-quantified and improved exogenous DNA uptake by sperm cells through lipofection and REMI. Stronger signals were observed from hybridisation of LacZ as well as GFP specific probe with the DNA from lipofected exogenous DNA transfected sperm DNA in comparison with those transfected with nude exogenous DNA.

  3. GPU-BSM: A GPU-Based Tool to Map Bisulfite-Treated Reads

    PubMed Central

    Manconi, Andrea; Orro, Alessandro; Manca, Emanuele; Armano, Giuliano; Milanesi, Luciano

    2014-01-01

    Cytosine DNA methylation is an epigenetic mark implicated in several biological processes. Bisulfite treatment of DNA is acknowledged as the gold standard technique to study methylation. This technique introduces changes in the genomic DNA by converting cytosines to uracils while 5-methylcytosines remain nonreactive. During PCR amplification 5-methylcytosines are amplified as cytosine, whereas uracils and thymines as thymine. To detect the methylation levels, reads treated with the bisulfite must be aligned against a reference genome. Mapping these reads to a reference genome represents a significant computational challenge mainly due to the increased search space and the loss of information introduced by the treatment. To deal with this computational challenge we devised GPU-BSM, a tool based on modern Graphics Processing Units. Graphics Processing Units are hardware accelerators that are increasingly being used successfully to accelerate general-purpose scientific applications. GPU-BSM is a tool able to map bisulfite-treated reads from whole genome bisulfite sequencing and reduced representation bisulfite sequencing, and to estimate methylation levels, with the goal of detecting methylation. Due to the massive parallelization obtained by exploiting graphics cards, GPU-BSM aligns bisulfite-treated reads faster than other cutting-edge solutions, while outperforming most of them in terms of unique mapped reads. PMID:24842718

  4. Shotgun Bisulfite Sequencing of the Betula platyphylla Genome Reveals the Tree’s DNA Methylation Patterning

    PubMed Central

    Su, Chang; Wang, Chao; He, Lin; Yang, Chuanping; Wang, Yucheng

    2014-01-01

    DNA methylation plays a critical role in the regulation of gene expression. Most studies of DNA methylation have been performed in herbaceous plants, and little is known about the methylation patterns in tree genomes. In the present study, we generated a map of methylated cytosines at single base pair resolution for Betula platyphylla (white birch) by bisulfite sequencing combined with transcriptomics to analyze DNA methylation and its effects on gene expression. We obtained a detailed view of the function of DNA methylation sequence composition and distribution in the genome of B. platyphylla. There are 34,460 genes in the whole genome of birch, and 31,297 genes are methylated. Conservatively, we estimated that 14.29% of genomic cytosines are methylcytosines in birch. Among the methylation sites, the CHH context accounts for 48.86%, and is the largest proportion. Combined transcriptome and methylation analysis showed that the genes with moderate methylation levels had higher expression levels than genes with high and low methylation. In addition, methylated genes are highly enriched for the GO subcategories of binding activities, catalytic activities, cellular processes, response to stimulus and cell death, suggesting that methylation mediates these pathways in birch trees. PMID:25514241

  5. Evaluation of preprocessing, mapping and postprocessing algorithms for analyzing whole genome bisulfite sequencing data.

    PubMed

    Tsuji, Junko; Weng, Zhiping

    2016-11-01

    Cytosine methylation regulates many biological processes such as gene expression, chromatin structure and chromosome stability. The whole genome bisulfite sequencing (WGBS) technique measures the methylation level at each cytosine throughout the genome. There are an increasing number of publicly available pipelines for analyzing WGBS data, reflecting many choices of read mapping algorithms as well as preprocessing and postprocessing methods. We simulated single-end and paired-end reads based on three experimental data sets, and comprehensively evaluated 192 combinations of three preprocessing, five postprocessing and five widely used read mapping algorithms. We also compared paired-end data with single-end data at the same sequencing depth for performance of read mapping and methylation level estimation. Bismark and LAST were the most robust mapping algorithms. We found that Mott trimming and quality filtering individually improved the performance of both read mapping and methylation level estimation, but combining them did not lead to further improvement. Furthermore, we confirmed that paired-end sequencing reduced error rate and enhanced sensitivity for both read mapping and methylation level estimation, especially for short reads and in repetitive regions of the human genome. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  6. Novel restricted access materials combined to molecularly imprinted polymers for selective solid-phase extraction of organophosphorus pesticides from honey.

    PubMed

    He, Juan; Song, Lixin; Chen, Si; Li, Yuanyuan; Wei, Hongliang; Zhao, Dongxin; Gu, Keren; Zhang, Shusheng

    2015-11-15

    A novel restricted access materials (RAM) combined to molecularly imprinted polymers (MIPs), using malathion as template molecule and glycidilmethacrylate (GMA) as pro-hydrophilic co-monomer, were prepared for the first time. RAM-MIPs with hydrophilic external layer were characterized by scanning electron microscopy and recognition and selectivity properties were compared with the restricted access materials-non-molecularly imprinted polymers (RAM-NIPs) and unmodified MIPs. RAM-MIPs were used as the adsorbent enclosed in solid phase extraction column and several important extraction parameters were comprehensively optimized to evaluate the extraction performance. Under the optimum extraction conditions, RAM-MIPs exhibited comparable or even higher selectivity with greater extraction capacity toward six kinds of organophosphorus pesticides (including malathion, ethoprophos, phorate, terbufos, dimethoate, and fenamiphos) compared with the MIPs and commercial solid phase extraction columns. The RAM-MIPs solid phase extraction coupled with gas chromatography was successfully applied to simultaneously determine six kinds of organophosphorus pesticides from honey sample. The new established method showed good linearity in the range of 0.01-1.0 μg mL(-1), low limits of detection (0.0005-0.0019 μg mL(-1)), acceptable reproducibility (RSD, 2.26-4.81%, n = 6), and satisfactory relative recoveries (90.9-97.6%). It was demonstrated that RAM-MIPs solid phase extraction with excellent selectivity and restricted access function was a simple, rapid, selective, and effective sample pretreatment method.

  7. Combined effects of exercise and restriction of energy intake on moderately obese women.

    PubMed Central

    Thomas, D P; Belko, A Z; Mulroy, G L; Haas, J D; Roe, D A

    1986-01-01

    The current study was designed to assess the contribution of dietary-induced weight reduction on improvements in functional capacity in moderate obesity. Twelve females (means age = 29 yr, means fat percentage = 37%) served as subjects for the study. Subjects trained on a cycle ergometer 30 min.day-1, six day.wk-1 for three or six weeks at 75 to 85 per cent of maximum heart rate (HR max). Improvements in maximal oxygen uptake (delta VO2 max I.min-1) and functional capacity (delta VO2 max ml.kg-1 min-1) were compared in an attempt to separate out training and dietary effects respectively. Measurements were also taken on both the cycle ergometer and treadmill to test for any specificity of training effects. Changes in body composition were assessed by densitometry. Following three weeks of training, there was an average increase in VO2 max ml.kg-1 min-1 on the cycle ergometer and the treadmill of 14 and 19 per cent respectively. By six weeks, this had increased to 18 and 26 per cent respectively. The contribution of weight reduction to the improvements in functional capacity was calculated to range from 20 to 33 per cent. Thus, both energy restriction and exercise training appear to be effective means of improving functional capacity in moderately obese women. PMID:3730763

  8. Is Patent “Evergreening” Restricting Access to Medicine/Device Combination Products?

    PubMed Central

    Beall, Reed F.; Nickerson, Jason W.; Kaplan, Warren A.; Attaran, Amir

    2016-01-01

    Background Not all new drug products are truly new. Some are the result of marginal innovation and incremental patenting of existing products, but in such a way that confers no major therapeutic improvement. This phenomenon, pejoratively known as “evergreening”, can allow manufacturers to preserve market exclusivity, but without significantly bettering the standard of care. Other studies speculate that evergreening is especially problematic for medicine/device combination products, because patents on the device component may outlast expired patents on the medicine component, and thereby keep competing, possibly less-expensive generic products off the market. Materials and Methods We focused on four common conditions that are often treated by medicine/device product combinations: asthma and chronic obstructive pulmonary disease (COPD), diabetes, and severe allergic reactions. The patent data for a sample of such products (n = 49) for treating these conditions was extracted from the United States Food and Drug Administration’s Orange Book. Additional patent-related data (abstracts, claims, etc) were retrieved using LexisNexis TotalPatent. Comparisons were then made between each product’s device patents and medicine patents. Results Unexpired device patents exist for 90 percent of the 49 medicine/device product combinations studied, and were the only sort of unexpired patent for 14 products. Overall, 55 percent of the 235 patents found by our study were device patents. Comparing the last-to-expire device patent to that of the last-to-expire active ingredient patent, the median additional years of patent protection afforded by device patents was 4.7 years (range: 1.3–15.2 years). Conclusion Incremental, patentable innovation in devices to extend the overall patent protection of medicine/device product combinations is very common. Whether this constitutes “evergreening” depends on whether these incremental innovations and the years of extra patent

  9. Is Patent "Evergreening" Restricting Access to Medicine/Device Combination Products?

    PubMed

    Beall, Reed F; Nickerson, Jason W; Kaplan, Warren A; Attaran, Amir

    2016-01-01

    Not all new drug products are truly new. Some are the result of marginal innovation and incremental patenting of existing products, but in such a way that confers no major therapeutic improvement. This phenomenon, pejoratively known as "evergreening", can allow manufacturers to preserve market exclusivity, but without significantly bettering the standard of care. Other studies speculate that evergreening is especially problematic for medicine/device combination products, because patents on the device component may outlast expired patents on the medicine component, and thereby keep competing, possibly less-expensive generic products off the market. We focused on four common conditions that are often treated by medicine/device product combinations: asthma and chronic obstructive pulmonary disease (COPD), diabetes, and severe allergic reactions. The patent data for a sample of such products (n = 49) for treating these conditions was extracted from the United States Food and Drug Administration's Orange Book. Additional patent-related data (abstracts, claims, etc) were retrieved using LexisNexis TotalPatent. Comparisons were then made between each product's device patents and medicine patents. Unexpired device patents exist for 90 percent of the 49 medicine/device product combinations studied, and were the only sort of unexpired patent for 14 products. Overall, 55 percent of the 235 patents found by our study were device patents. Comparing the last-to-expire device patent to that of the last-to-expire active ingredient patent, the median additional years of patent protection afforded by device patents was 4.7 years (range: 1.3-15.2 years). Incremental, patentable innovation in devices to extend the overall patent protection of medicine/device product combinations is very common. Whether this constitutes "evergreening" depends on whether these incremental innovations and the years of extra patent protection they confer are proportionately matched by therapeutic

  10. Enhanced degradation of organic contaminants in water by peroxydisulfate coupled with bisulfite.

    PubMed

    Qi, Chengdu; Liu, Xitao; Li, Yang; Lin, Chunye; Ma, Jun; Li, Xiaowan; Zhang, Huijuan

    2017-04-15

    In this study, the bisulfite-peroxydisulfate system (S(IV)/PDS) widely used in polymerization was innovatively applied for organic contaminants degradation in water. The addition of S(IV) into PDS system remarkably enhanced the degradation efficiency of bisphenol A (BPA, a frequently detected endocrine disrupting chemical in the environments) from 17.0% to 84.7% within 360 min. The degradation efficiency of BPA in the S(IV)/PDS system followed pseudo-first-order kinetics, with rate constant values ranging from 0.00005min(-1) to 0.02717min(-1) depending on the operating parameters, such as the initial S(IV) and PDS dosage, solution pH, reaction temperature, chloride and water type. Furthermore, nitrogen purging experiment, radical scavenging experiment and electron spin resonance (ESR) analysis were used to elucidate the possible mechanism. The results revealed that sulfate radical was the dominant reactive species in the S(IV)/PDS system. Finally, based on the results of liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), the BPA degradation pathway was proposed to involve β-scission (CC), hydroxylation, dehydration, oxidative skeletal rearrangement, and ring opening. This study helps to characterize the combination of PDS and inorganic S(IV), a common industrial contaminant, to generate reactive species to enhance organic contaminants degradation in water. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Withanone-rich combination of Ashwagandha withanolides restricts metastasis and angiogenesis through hnRNP-K.

    PubMed

    Gao, Ran; Shah, Navjot; Lee, Jung-Sun; Katiyar, Shashank P; Li, Ling; Oh, Eonju; Sundar, Durai; Yun, Chae-Ok; Wadhwa, Renu; Kaul, Sunil C

    2014-12-01

    Ashwagandha is an important herb used in the Indian system of traditional home medicine, Ayurveda. Alcoholic extract (i-Extract) from its leaves and its component, withanone, were previously shown to possess anticancer activity. In the present study, we developed a combination of withanone and withaferin A, major withanolides in the i-Extract, that retained the selective cancer cell killing activity and found that it also has significant antimigratory, -invasive, and -angiogenic activities, in both in vitro and in vivo assays. Using bioinformatics and biochemical approaches, we demonstrate that these phytochemicals caused downregulation of migration-promoting proteins hnRNP-K, VEGF, and metalloproteases and hence are candidate natural drugs for metastatic cancer therapy. ©2014 American Association for Cancer Research.

  12. Escherichia coli Free Radical-Based Killing Mechanism Driven by a Unique Combination of Iron Restriction and Certain Antibiotics

    PubMed Central

    Ma, Li; Gao, Yongjun

    2015-01-01

    ABSTRACT Bacterial resistance to antibiotics is precipitating a medical crisis, and new antibacterial strategies are being sought. Hypothesizing that a growth-restricting strategy could be used to enhance the efficacy of antibiotics, we determined the effect of FDA-approved iron chelators and various antibiotic combinations on invasive and multidrug-resistant extraintestinal pathogenic Escherichia coli (ExPEC), the Gram-negative bacterium most frequently isolated from the bloodstreams of hospitalized patients. We report that certain antibiotics used at sublethal concentrations display enhanced growth inhibition and/or killing when combined with the iron chelator deferiprone (DFP). Inductively coupled plasma optical emission spectrometry reveals abnormally high levels of cell-associated iron under these conditions, a response that correlates with an iron starvation response and supraphysiologic levels of reactive oxygen species (ROS). The high ROS level is reversed upon the addition of antioxidants, which restores bacterial growth, suggesting that the cells are inhibited or killed by excessive free radicals. A model is proposed in which peptidoglycan-targeting antibiotics facilitate the entry of lethal levels of iron-complexed DFP into the bacterial cytoplasm, a process that drives the generation of ROS. This new finding suggests that, in addition to restriction of access to iron as a general growth-restricting strategy, targeting of cellular pathways or networks that selectively disrupt normal iron homeostasis can have potent bactericidal outcomes. IMPORTANCE The prospect that common bacteria will become resistant to all antibiotics is challenging the medical community. In addition to the development of next-generation antibiotics, new bacterial targets that display cytotoxic properties when altered need to be identified. Data presented here demonstrate that combining subinhibitory levels of both iron chelators and certain antibiotics kills pathogenic Escherichia

  13. Ultrasensitive electrochemical biosensing for DNA using quantum dots combined with restriction endonuclease.

    PubMed

    Zhang, Can; Lou, Jing; Tu, Wenwen; Bao, Jianchun; Dai, Zhihui

    2015-01-21

    A universal and sensitive electrochemical biosensing platform for the detection and identification of DNA using CdSe quantum dots (CdSe QDs) as signal markers was designed. The detection mechanism was based on the specific recognition of MspI endonuclease combined with the signal amplification of gold nanoparticles (AuNPs). MspI endonuclease could recognize its specific sequence in the double-strand DNA (dsDNA) and cleave the dsDNA fragments linked with CdSe QDs from the electrode. The remaining attached CdSe QDs can be easily read out by square-wave voltammetry using an electrodeposited bismuth (Bi) film-modified glass carbon electrode. The concentrations of target DNA could be simultaneously detected by the signal of metal markers. Using mycobacterium tuberculosis (Mtb) DNA as a model, under the optimal conditions, the proposed biosensor could detect Mtb DNA down to 8.7 × 10(-15) M with a linear range of 5 orders of magnitude (from 1.0 × 10(-14) to 1.0 × 10(-9) M) and discriminate mismatched DNA with high selectivity. This strategy presented a universal and convenient biosensing platform for DNA assay, and its satisfactory performances make it a potential candidate for the early diagnosis of gene-related diseases.

  14. BS-RNA: An efficient mapping and annotation tool for RNA bisulfite sequencing data.

    PubMed

    Liang, Fang; Hao, Lili; Wang, Jinyue; Shi, Shuo; Xiao, Jingfa; Li, Rujiao

    2016-12-01

    Cytosine methylation is one of the most important RNA epigenetic modifications. With the development of experimental technology, scientists attach more importance to RNA cytosine methylation and find bisulfite sequencing is an effective experimental method for RNA cytosine methylation study. However, there are only a few tools can directly deal with RNA bisulfite sequencing data efficiently. Herein, we developed a specialized tool BS-RNA, which can analyze cytosine methylation of RNA based on bisulfite sequencing data and support both paired-end and single-end sequencing reads from directional bisulfite libraries. For paired-end reads, simply removing the biased positions from the 5' end may result in "dovetailing" reads, where one or both reads seem to extend past the start of the mate read. BS-RNA could map "dovetailing" reads successfully. The annotation result of BS-RNA is exported in BED (.bed) format, including locations, sequence context types (CG/CHG/CHH, H=A,T, or C), reference sequencing depths, cytosine sequencing depths, and methylation levels of covered cytosine sites on both Watson and Crick strands. BS-RNA is an efficient, specialized and highly automated mapping and annotation tool for RNA bisulfite sequencing data. It performs better than the existing program in terms of accuracy and efficiency. BS-RNA is developed by Perl language and the source code of this tool is freely available from the website: http://bs-rna.big.ac.cn. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Methy-Pipe: an integrated bioinformatics pipeline for whole genome bisulfite sequencing data analysis.

    PubMed

    Jiang, Peiyong; Sun, Kun; Lun, Fiona M F; Guo, Andy M; Wang, Huating; Chan, K C Allen; Chiu, Rossa W K; Lo, Y M Dennis; Sun, Hao

    2014-01-01

    DNA methylation, one of the most important epigenetic modifications, plays a crucial role in various biological processes. The level of DNA methylation can be measured using whole-genome bisulfite sequencing at single base resolution. However, until now, there is a paucity of publicly available software for carrying out integrated methylation data analysis. In this study, we implemented Methy-Pipe, which not only fulfills the core data analysis requirements (e.g. sequence alignment, differential methylation analysis, etc.) but also provides useful tools for methylation data annotation and visualization. Specifically, it uses Burrow-Wheeler Transform (BWT) algorithm to directly align bisulfite sequencing reads to a reference genome and implements a novel sliding window based approach with statistical methods for the identification of differentially methylated regions (DMRs). The capability of processing data parallelly allows it to outperform a number of other bisulfite alignment software packages. To demonstrate its utility and performance, we applied it to both real and simulated bisulfite sequencing datasets. The results indicate that Methy-Pipe can accurately estimate methylation densities, identify DMRs and provide a variety of utility programs for downstream methylation data analysis. In summary, Methy-Pipe is a useful pipeline that can process whole genome bisulfite sequencing data in an efficient, accurate, and user-friendly manner. Software and test dataset are available at http://sunlab.lihs.cuhk.edu.hk/methy-pipe/.

  16. Development of a restriction length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses.

    PubMed

    Wan, Chun-He; Chen, Hong-Mei; Fu, Qiu-Ling; Shi, Shao-Hua; Fu, Guang-Hua; Cheng, Long-Fei; Chen, Cui-Teng; Huang, Yu; Hu, Kai-Hui

    2016-06-01

    A restriction fragment length polymorphism combined with direct PCR technique to differentiate goose and Muscovy duck parvoviruses (GPV and MDPV) was developed based on comparison of the NS gene of GPV and MDPV. Both GPV and MDPV genomic DNA can be amplified with 641 bp using the specific PCR primers. The PCR fragments can be cut into 463 bp and 178 bp only in the case of MDPV-derived PCR products, whereas the GPV-derived PCR products cannot. The method established in this study can be used to differentiate GPV and MDPV with high specificity and precision, by using a direct PCR kit and QuickCut enzyme, as quickly as conventional PCR.

  17. Intermittent fasting combined with calorie restriction is effective for weight loss and cardio-protection in obese women

    PubMed Central

    2012-01-01

    Background Intermittent fasting (IF; severe restriction 1 d/week) facilitates weight loss and improves coronary heart disease (CHD) risk indicators. The degree to which weight loss can be enhanced if IF is combined with calorie restriction (CR) and liquid meals, remains unknown. Objective This study examined the effects of IF plus CR (with or without a liquid diet) on body weight, body composition, and CHD risk. Methods Obese women (n = 54) were randomized to either the IFCR-liquid (IFCR-L) or IFCR-food based (IFCR-F) diet. The trial had two phases: 1) 2-week weight maintenance period, and 2) 8-week weight loss period. Results Body weight decreased more (P = 0.04) in the IFCR-L group (3.9 ± 1.4 kg) versus the IFCR-F group (2.5 ± 0.6 kg). Fat mass decreased similarly (P < 0.0001) in the IFCR-L and IFCR-F groups (2.8 ± 1.2 kg and 1.9 ± 0.7 kg, respectively). Visceral fat was reduced (P < 0.001) by IFCR-L (0.7 ± 0.5 kg) and IFCR-F (0.3 ± 0.5 kg) diets. Reductions in total and LDL cholesterol levels were greater (P = 0.04) in the IFCR-L (19 ± 10%; 20 ± 9%, respectively) versus the IFCR-F group (8 ± 3%; 7 ± 4%, respectively). LDL peak particle size increased (P < 0.01), while heart rate, glucose, insulin, and homocysteine decreased (P < 0.05), in the IFCR-L group only. Conclusion These findings suggest that IF combined with CR and liquid meals is an effective strategy to help obese women lose weight and lower CHD risk. PMID:23171320

  18. Intermittent fasting combined with calorie restriction is effective for weight loss and cardio-protection in obese women.

    PubMed

    Klempel, Monica C; Kroeger, Cynthia M; Bhutani, Surabhi; Trepanowski, John F; Varady, Krista A

    2012-11-21

    Intermittent fasting (IF; severe restriction 1 d/week) facilitates weight loss and improves coronary heart disease (CHD) risk indicators. The degree to which weight loss can be enhanced if IF is combined with calorie restriction (CR) and liquid meals, remains unknown. This study examined the effects of IF plus CR (with or without a liquid diet) on body weight, body composition, and CHD risk. Obese women (n = 54) were randomized to either the IFCR-liquid (IFCR-L) or IFCR-food based (IFCR-F) diet. The trial had two phases: 1) 2-week weight maintenance period, and 2) 8-week weight loss period. Body weight decreased more (P = 0.04) in the IFCR-L group (3.9 ± 1.4 kg) versus the IFCR-F group (2.5 ± 0.6 kg). Fat mass decreased similarly (P < 0.0001) in the IFCR-L and IFCR-F groups (2.8 ± 1.2 kg and 1.9 ± 0.7 kg, respectively). Visceral fat was reduced (P < 0.001) by IFCR-L (0.7 ± 0.5 kg) and IFCR-F (0.3 ± 0.5 kg) diets. Reductions in total and LDL cholesterol levels were greater (P = 0.04) in the IFCR-L (19 ± 10%; 20 ± 9%, respectively) versus the IFCR-F group (8 ± 3%; 7 ± 4%, respectively). LDL peak particle size increased (P < 0.01), while heart rate, glucose, insulin, and homocysteine decreased (P < 0.05), in the IFCR-L group only. These findings suggest that IF combined with CR and liquid meals is an effective strategy to help obese women lose weight and lower CHD risk.

  19. Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors.

    PubMed

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z; Prior, Allan M; Alliston, Kevin R; Hua, Duy H; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

  20. A Flexible, Efficient Binomial Mixed Model for Identifying Differential DNA Methylation in Bisulfite Sequencing Data

    PubMed Central

    Lea, Amanda J.

    2015-01-01

    Identifying sources of variation in DNA methylation levels is important for understanding gene regulation. Recently, bisulfite sequencing has become a popular tool for investigating DNA methylation levels. However, modeling bisulfite sequencing data is complicated by dramatic variation in coverage across sites and individual samples, and because of the computational challenges of controlling for genetic covariance in count data. To address these challenges, we present a binomial mixed model and an efficient, sampling-based algorithm (MACAU: Mixed model association for count data via data augmentation) for approximate parameter estimation and p-value computation. This framework allows us to simultaneously account for both the over-dispersed, count-based nature of bisulfite sequencing data, as well as genetic relatedness among individuals. Using simulations and two real data sets (whole genome bisulfite sequencing (WGBS) data from Arabidopsis thaliana and reduced representation bisulfite sequencing (RRBS) data from baboons), we show that our method provides well-calibrated test statistics in the presence of population structure. Further, it improves power to detect differentially methylated sites: in the RRBS data set, MACAU detected 1.6-fold more age-associated CpG sites than a beta-binomial model (the next best approach). Changes in these sites are consistent with known age-related shifts in DNA methylation levels, and are enriched near genes that are differentially expressed with age in the same population. Taken together, our results indicate that MACAU is an efficient, effective tool for analyzing bisulfite sequencing data, with particular salience to analyses of structured populations. MACAU is freely available at www.xzlab.org/software.html. PMID:26599596

  1. Pipeline for large-scale microdroplet bisulfite PCR-based sequencing allows the tracking of hepitype evolution in tumors.

    PubMed

    Herrmann, Alexander; Haake, Andrea; Ammerpohl, Ole; Martin-Guerrero, Idoia; Szafranski, Karol; Stemshorn, Kathryn; Nothnagel, Michael; Kotsopoulos, Steve K; Richter, Julia; Warner, Jason; Olson, Jeff; Link, Darren R; Schreiber, Stefan; Krawczak, Michael; Platzer, Matthias; Nürnberg, Peter; Siebert, Reiner; Hampe, Jochen

    2011-01-01

    Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.

  2. Pipeline for Large-Scale Microdroplet Bisulfite PCR-Based Sequencing Allows the Tracking of Hepitype Evolution in Tumors

    PubMed Central

    Martin-Guerrero, Idoia; Szafranski, Karol; Stemshorn, Kathryn; Nothnagel, Michael; Kotsopoulos, Steve K.; Richter, Julia; Warner, Jason; Olson, Jeff; Link, Darren R.; Schreiber, Stefan; Krawczak, Michael; Platzer, Matthias; Nürnberg, Peter; Siebert, Reiner; Hampe, Jochen

    2011-01-01

    Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into “hepitypes” and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer. PMID:21750708

  3. Direct Quantitative Bisulfite Sequencing Using Tag-modified Primers and Internal Normalization.

    PubMed

    Dietrich, Dimo

    2016-12-01

    For the investigation of DNA methylation patterns, bisulfite conversion of the DNA followed by polymerase chain reaction (PCR) amplification and sequencing of the region of interest is the method of choice when information at single CpG site resolution is desired. In this study, a simple method for direct quantitative bisulfite sequencing based on the Sanger method is shown to be usable for the accurate analysis of single CpG sites. This method is based on the usage of tag-modified primers to obtain an internal normalization signal within the PCR product.

  4. Neonatal parenteral nutrition hypersensitivity: a case report implicating bisulfite sensitivity in a newborn infant.

    PubMed

    Huston, Robert K; Baxter, Louise M; Larrabee, Paige B

    2009-01-01

    This report describes a case of parenteral nutrition hypersensitivity in a 37 weeks' gestation infant with congenital diaphragmatic hernia complicated by bowel necrosis and functional short bowel syndrome. The patient developed a rash with subsequent urticaria beginning on the 50th day of life. The reactions were confirmed with a positive rechallenge. After the amino acid solution was replaced with a non-bisulfite-containing product, the infant was able to continue to receive nutrition support through parenteral nutrition without recurrence of symptoms. It is speculated that the bisulfite additive in the amino acid solution may have interacted with the lipid emulsion to sensitize the patient.

  5. The Effect of Long Term Calorie Restriction on in Vivo Hepatic Proteostatis: A Novel Combination of Dynamic and Quantitative Proteomics

    PubMed Central

    Price, John C.; Khambatta, Cyrus F.; Li, Kelvin W.; Bruss, Matthew D.; Shankaran, Mahalakshmi; Dalidd, Marcy; Floreani, Nicholas A.; Roberts, Lindsay S.; Turner, Scott M.; Holmes, William E.; Hellerstein, Marc K.

    2012-01-01

    Calorie restriction (CR) promotes longevity. A prevalent mechanistic hypothesis explaining this effect suggests that protein degradation, including mitochondrial autophagy, is increased with CR, removing damaged proteins and improving cellular fitness. At steady state, increased catabolism must be balanced by increasing mitochondrial biogenesis and protein synthesis, resulting in faster protein replacement rates. To test this hypothesis, we measured replacement kinetics and relative concentrations of hundreds of proteins in vivo in long-term CR and ad libitum-fed mice using metabolic 2H2O-labeling combined with the Stable Isotope Labeling in Mammals protocol and LC-MS/MS analysis of mass isotopomer abundances in tryptic peptides. CR reduced absolute synthesis and breakdown rates of almost all measured hepatic proteins and prolonged the half-lives of most (∼80%), particularly mitochondrial proteins (but not ribosomal subunits). Proteins with related functions exhibited coordinated changes in relative concentration and replacement rates. In silico expression pathway interrogation allowed the testing of potential regulators of altered network dynamics (e.g. peroxisome proliferator-activated receptor gamma coactivator 1-alpha). In summary, our combination of dynamic and quantitative proteomics suggests that long-term CR reduces mitochondrial biogenesis and mitophagy. Our findings contradict the theory that CR increases mitochondrial protein turnover and provide compelling evidence that cellular fitness is accompanied by reduced global protein synthetic burden. PMID:22984287

  6. BSviewer: a genotype-preserving, nucleotide-level visualizer for bisulfite sequencing data.

    PubMed

    Sun, Kun; Lun, Fiona F M; Jiang, Peiyong; Sun, Hao

    2017-08-08

    The bisulfite sequencing technology has been widely used to study the DNA methylation profile in many species. However, most of the current visualization tools for bisulfite sequencing data only provide high-level views (i.e., overall methylation densities) while miss the methylation dynamics at nucleotide level. Meanwhile, they also focus on CpG sites while omit other information (such as genotypes on SNP sites) which could be helpful for interpreting the methylation pattern of the sequencing data. A bioinformatics tool that visualizes the methylation statuses at nucleotide level and preserves the most essential information of the sequencing data is thus valuable and needed. We have developed BSviewer, a lightweight nucleotide-level visualization tool for bisulfite sequencing data. Using an imprinting gene as an example, we show that BSviewer could be specifically helpful for interpreting the bisulfite sequencing data with allele-specific DNA methylation pattern. BSviewer is implemented in Perl and runs on most GNU/Linux platforms. Source code and testing dataset are freely available at http://sunlab.cpy.cuhk.edu.hk/BSviewer/ . haosun@cuhk.edu.hk.

  7. Toxicity of pH, heavy metals and bisulfite to a freshwater green alga

    SciTech Connect

    Baker, M.D.; Mayfield, C.I.; Inniss, W.E.; Wong, P.T.S.

    1983-01-01

    Low pH markedly reduced the growth and photosynthetic activity of an Ankistrodesmus sp. The alga could not grow at pH 3 and only slight growth occurred at pH 4. The alga grew well above pH 4 with maximum growth occurring at pH 6. The fixation of /sup 14/CO/sub 2/ followed a similar pattern with pH. The algal cells were also sensitive to mercury and bisulfite in acidic conditions. Algal growth and photosynthesis were reduced by mercury and bisulfite more at pH 5 but not at pH 7. Lead was relatively non-toxic to the algal cells at both pH levels. Bisulfite inhibited the membrane transport of ..cap alpha..-amino-/sup 14/ C-isobutyric acid at pH 5 but not at pH7. The results suggest that algal growth and activity may be reduced in acidic lakes by low pH and that the toxicity of mercury and bisulfite is enhanced in acidic conditions.

  8. Resin in bisulfite pulp from Pinus radiata wood and its relationship to pitch troubles

    Treesearch

    P.J. Nelson; Richard W. Hemingway

    1971-01-01

    The resin content of bisulfite pulp from Pinus radiata D. Don was determined at various stages in its manufacture and the changes in the composition of the resin studied. About 50% of the resin present in the wood was removed during cooking, an additional 11% by blowpit washing, and a further 8% by screening. Resin acids and fatty acids were...

  9. Methylation analysis of plasma cell-free DNA for breast cancer early detection using bisulfite next-generation sequencing.

    PubMed

    Li, Zibo; Guo, Xinwu; Tang, Lili; Peng, Limin; Chen, Ming; Luo, Xipeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Xia, Kun; Wang, Jun

    2016-10-01

    Circulating cell-free DNA (cfDNA) has been considered as a potential biomarker for non-invasive cancer detection. To evaluate the methylation levels of six candidate genes (EGFR, GREM1, PDGFRB, PPM1E, SOX17, and WRN) in plasma cfDNA as biomarkers for breast cancer early detection, quantitative analysis of the promoter methylation of these genes from 86 breast cancer patients and 67 healthy controls was performed by using microfluidic-PCR-based target enrichment and next-generation bisulfite sequencing technology. The predictive performance of different logistic models based on methylation status of candidate genes was investigated by means of the area under the ROC curve (AUC) and odds ratio (OR) analysis. Results revealed that EGFR, PPM1E, and 8 gene-specific CpG sites showed significantly hypermethylation in cancer patients' plasma and significantly associated with breast cancer (OR ranging from 2.51 to 9.88). The AUC values for these biomarkers were ranging from 0.66 to 0.75. Combinations of multiple hypermethylated genes or CpG sites substantially improved the predictive performance for breast cancer detection. Our study demonstrated the feasibility of quantitative measurement of candidate gene methylation in cfDNA by using microfluidic-PCR-based target enrichment and bisulfite next-generation sequencing, which is worthy of further validation and potentially benefits a broad range of applications in clinical oncology practice. Quantitative analysis of methylation pattern of plasma cfDNA by next-generation sequencing might be a valuable non-invasive tool for early detection of breast cancer.

  10. Effects of a natural community intervention intensifying alcohol law enforcement combined with a restrictive alcohol policy on adolescent alcohol use.

    PubMed

    Schelleman-Offermans, Karen; Knibbe, Ronald A; Kuntsche, Emmanuel; Casswell, Sally

    2012-12-01

    Determining whether intensified inspections on alcohol retailers, combined with a policy withdrawing liquor licenses if retailers are fined twice per annum, is effective in reducing adolescents' odds to initiate weekly drinking and drunkenness. Causal pathways by which the intervention was assumed to work were tested. A longitudinal (2008, 2009, and 2010) quasi-experimental comparison group design including two Dutch communities, one intervention and one comparison, was used. Outcomes were assessed by following a cohort of 1,327 adolescents (aged 13-15 years at baseline). The intervention resulted in increased retail inspections but only seven sanctions and no repeated sanctions in 1 year. The intervention did not reduce adolescents' odds to initiate weekly drinking. Weekly drinking adolescents in the intervention community were at reduced risk to initiate drunkenness. This effect was not mediated by smaller increases in the frequency of adolescents' alcohol purchases or their perceived ease of purchasing alcohol. Intensified enforcement was effective in preventing adolescent drunkenness. No mediating causal pathways were detected. Effectiveness of enforcement could be increased by adopting enforcement methods with a high likelihood of apprehension, increasing social support for restrictive measures, and mobilizing the community to be more outspoken against adolescent (heavy) drinking. Copyright © 2012 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.

  11. Calorie restriction combined with resveratrol induces autophagy and protects 26-month-old rat hearts from doxorubicin-induced toxicity

    PubMed Central

    Dutta, Debariya; Xu, Jinze; Dirain, Marvin L.S.; Leeuwenburgh, Christiaan

    2014-01-01

    The multiple beneficial effects of calorie restriction (CR) on several organs, including the heart, are widely known. Recently, the plant polyphenol resveratrol has been shown to possess beneficial effects similar to that of CR. Among the host of effects on cardiac muscle, a cellular self-eating process called autophagy has been shown to be induced by both CR and resveratrol. Autophagy is vital in removing dysfunctional organelles and damaged proteins from the cell, thereby maintaining cellular quality control. In this study, we explored whether short-term moderate CR (20%), either alone or in combination with resveratrol, can induce autophagy in the hearts of 26-month old Fischer 344 × Brown Norway (FBN) rats. Autophagy stimulation was investigated by measuring protein expression levels of autophagy proteins Beclin-1, Atg5, p62, and LC3-II/LC3-I ratio. We found that 20% CR or resveratrol alone for 6 weeks could not induce autophagy, but 20% CR in combination with 50 mg/kg/day resveratrol resulted in an induction of autophagy in the hearts of 26 month old rats. Although oxidative stress has been proposed to be an inducer of autophagy, treatment with the chemotherapeutic drug doxorubicin was unable to stimulate autophagy. The enhanced autophagy due to CR + resveratrol was associated with protection from doxorubicin-induced damage, as measured by cardiac apoptotic levels, serum creatine kinase (CK) and lactate dehydrogenase (LDH) activity. We propose that a combinatorial approach of low-dose CR and resveratrol has the potential to be used therapeutically to induce autophagy and provides protection against doxorubicin-mediated toxicity. PMID:24975655

  12. Detection of Cytosine Methylation in Ancient DNA from Five Native American Populations Using Bisulfite Sequencing

    PubMed Central

    Smith, Rick W. A.; Monroe, Cara; Bolnick, Deborah A.

    2015-01-01

    While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches. PMID:26016479

  13. Caloric Restriction

    PubMed Central

    Bales, Connie W.; Kraus, William E.

    2013-01-01

    PURPOSE While the impact of caloric restriction on human health is not fully understood, there is strong evidence to support further studies of its influence on cardiovascular health. The purpose of this review is to update the state of the science by examining the relevant literature regarding calorie restriction effects on aging and cardiovascular health and to discuss the possible role(s) of calorie restriction in preserving cardiovascular function in humans. METHODS For purpose of this review, we have defined calorie restriction as a reduction in energy intake well below the amount of calories that would be consumed ad libitum (≥ 10% in humans, ≥20% in animals). We examined the relevant literature on calorie restriction effects on longevity and cardiovascular health, with an emphasis on the state of the science regarding calorie restriction in humans. We have emphasized the importance of the preliminary and expected findings from the Comprehensive Assessment of the Long-term Effect of Reducing Intake of Energy (CALERIE) trial. RESULTS Evidence from animal studies and a limited number of human trials indicates that calorie restriction has the potential to both delay cardiac aging and help prevent atherosclerotic cardiovascular disease via beneficial effects on blood pressure, lipids, inflammatory processes, and potentially other mechanisms. CONCLUSIONS Based upon its known benefits to cardiometabolic health, including modest calorie restriction in a combined lifestyle program is likely to improve heart health and prevent subsequent cardiovascular events in overweight and obese individuals. Additional study is needed to further illuminate its long-term applicability for older adults and for those with significant comorbidities such as heart failure. PMID:23748374

  14. Effect of thermal stress, restricted feeding and combined stresses (thermal stress and restricted feeding) on growth and plasma reproductive hormone levels of Malpura ewes under semi-arid tropical environment.

    PubMed

    Sejian, V; Maurya, V P; Naqvi, S M K

    2011-04-01

    A study was conducted to assess the effect of thermal, nutritional and combined stresses (thermal and nutritional) on the growth, oestradiol and progesterone levels during oestrus cycles in Malpura ewes. Twenty-eight adult Malpura ewes were used in the present study. The ewes were randomly allocated into four groups, viz., GI (n=7; control), GII (n=7; thermal stress), GIII (n=7; restricted feeding) and GIV (n=7; combined stress). The animals were stall fed with a diet consisting of 60% roughage and 40% concentrate. GI and GII ewes were provided with ad libitum feeding while GIII and GIV ewes were provided with restricted feed (30% intake of GI and GII ewes) to induce nutritional insufficiency. GII and GIV ewes were kept in climatic chamber at 40°C and 55% RH for 6 h a day between 10:00 and 16:00 hours to induce thermal stress for a period of two oestrous cycles. Parameters studied were body weight, oestrus incidences, plasma oestradiol 17-β, plasma progesterone, conception rate, gestation period, lambing rate, and birth weight of lambs. The results indicate that combined stress significantly (p<0.05) reduced body weight, oestrus duration, birth weight of lambs, and oestradiol 17-β whereas significantly (p < 0.05) increased oestrus cycle length and progesterone. Furthermore, the results reveal that on comparative basis, ewes were able to better adapt in terms of growth and reproduction to restricted feeding than thermal stress. However, when restricted feeding was coupled with thermal stress it had significant (p<0.05) influence on body weight, average daily gain, oestradiol 17-β and progesterone concentrations. This showed that combined stress were more detrimental for these reproductive hormones in Malpura ewes under a hot semi-arid environment. © 2010 Blackwell Verlag GmbH.

  15. Restrictive Atrial Septum Defect Becomes a Risk Factor for Norwood Palliation of Hypoplastic Left Heart Syndrome Only When It Is Combined with Mitral or Aortic Atresia.

    PubMed

    Sata, Sojiro; Sinzobahamvya, Nicodème; Arenz, Claudia; Zartner, Peter; Asfour, Boulos; Hraska, Viktor

    2015-08-01

    Restrictive atrial septal defect (ASD) is described as risk factor for Norwood procedure because of elevated pulmonary resistance. We hypothesized that it invariably could not cause pulmonary hypertension, unless it was combined with mitral valve or aortic valve atresia. We investigated how restrictive ASD influenced survival of patients with hypoplastic left heart syndrome (HLHS) who underwent Norwood operation. A total of 118 HLHS patients who underwent surgery between January 2005 and December 2012 were grouped into three groups. Group 1 included 31 patients with restrictive ASD combined with mitral or aortic atresia; Group 2 composed of 12 patients with restrictive ASD and mitral and aortic stenosis; Group 3 (n = 75) had no ASD restriction. Survival was determined for each group. Multivariate analysis was conducted to test risk factors for mortality. Mean follow-up was 26.3 ± 24.1 months. Survival was 78.7% ± 4.2% at 30-month interval and onward after Norwood procedure for the whole cohort; it was 43.8% ± 10.0%, 91.7% ± 8.0%, and 77.3% ± 5.0% for Group 1, 2, and 3, respectively. The difference was significant between Group 1 and Group 2 and 3: p < 0.001. Survival was similar for Group 2 and Group 3: p = 0.45. Combination of restrictive ASD and mitral or aortic atresia was found to be the sole risk factor for early and late mortality (odds ratio: 3.5, 95% confidence interval: 1.8-7.1, p < 0.001). Restrictive ASD only affects survival of HLHS patients following Norwood procedure if it is associated with mitral or aortic atresia. Georg Thieme Verlag KG Stuttgart · New York.

  16. Bisulfite pretreatment changes the structure and properties of oil palm empty fruit bunch to improve enzymatic hydrolysis and bioethanol production.

    PubMed

    Tan, Liping; Sun, Wan; Li, Xuezhi; Zhao, Jian; Qu, Yinbo; Choo, Yuen May; Loh, Soh Kheang

    2015-06-01

    Bisulfite pretreatment is a proven effective method for improving the enzymatic hydrolysis of empty fruit bunch (EFB) from oil palm for bioethanol production. In this study, we set out to determine the changes that occur in the structure and properties of EFB materials and fractions of hemicellulose and lignin during the bisulfite pretreatment process. The results showed that the crystallinity of cellulose in EFB increased after bisulfite pretreatment, whereas the EFB surface was damaged to various degrees. The orderly structure of EFB, which was maintained by hydrogen bonds, was destroyed by bisulfite pretreatment. Bisulfite pretreatment also hydrolyzed the glycosidic bonds of the xylan backbone of hemicellulose, thereby decreasing the molecular weight and shortening the xylan chains. The lignin fractions obtained from EFB and pretreated EFB were typically G-S lignin, and with low content of H units. Meanwhile, de-etherification occurred at the β-O-4 linkage, which was accompanied by polymerization and demethoxylation as a result of bisulfite pretreatment. The adsorption ability of cellulase differed for the various lignin fractions, and the water-soluble lignin fractions had higher adsorption capacity on cellulase than the milled wood lignin. In general, the changes in the structure and properties of EFB provided insight into the benefits of bisulfite pretreatment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Hydrophilic Indolium Cycloruthenated Complex System for Visual Detection of Bisulfite with a Large Red Shift in Absorption.

    PubMed

    Su, Xianlong; Hu, Rongrong; Li, Xianghong; Zhu, Jun; Luo, Facheng; Niu, Xuehu; Li, Mei; Zhao, Qiang

    2016-01-19

    Bisulfite, as an important additive in foodstuffs, is one of the most widely distributed environmental pollutants. The excessive intake of bisulfite may cause asthmatic attacks and allergic reactions. Therefore, the determination and visual detection of bisulfite are very important. Herein, a newly designed hydrophilic indolium cycloruthenated complex, [Ru(mepbi)(bpy)2](+) [1; bpy = 2,2'-bipyridine and Hmepbi = 3,3-dimethyl-1-ethyl-2-[4-(pyridin-2-yl)styryl]benzo[e]indolium iodide (3)], was successfully synthesized and used as a bisulfite probe. The bisulfite underwent a 1,4-addition reaction with complex 1 in PBS buffer (10 mM, pH 7.40), resulting in a dramatic change in absorption spectra with a red shift of over 100 nm and a remarkable change in solution color from yellow to pink. It is worth noting that this obvious bathochromic shift is rarely observed in the detection of bisulfite through an addition reaction. The detection limit was calculated to be as low as 0.12 μM by UV-vis absorption spectroscopy. Moreover, complex 1 was also used to detect bisulfite in sugar samples (granulated and crystal sugar) with good recovery.

  18. Analysis and Visualization Tool for Targeted Amplicon Bisulfite Sequencing on Ion Torrent Sequencers

    PubMed Central

    Pabinger, Stephan; Ernst, Karina; Pulverer, Walter; Kallmeyer, Rainer; Valdes, Ana M.; Metrustry, Sarah; Katic, Denis; Nuzzo, Angelo; Kriegner, Albert; Vierlinger, Klemens; Weinhaeusel, Andreas

    2016-01-01

    Targeted sequencing of PCR amplicons generated from bisulfite deaminated DNA is a flexible, cost-effective way to study methylation of a sample at single CpG resolution and perform subsequent multi-target, multi-sample comparisons. Currently, no platform specific protocol, support, or analysis solution is provided to perform targeted bisulfite sequencing on a Personal Genome Machine (PGM). Here, we present a novel tool, called TABSAT, for analyzing targeted bisulfite sequencing data generated on Ion Torrent sequencers. The workflow starts with raw sequencing data, performs quality assessment, and uses a tailored version of Bismark to map the reads to a reference genome. The pipeline visualizes results as lollipop plots and is able to deduce specific methylation-patterns present in a sample. The obtained profiles are then summarized and compared between samples. In order to assess the performance of the targeted bisulfite sequencing workflow, 48 samples were used to generate 53 different Bisulfite-Sequencing PCR amplicons from each sample, resulting in 2,544 amplicon targets. We obtained a mean coverage of 282X using 1,196,822 aligned reads. Next, we compared the sequencing results of these targets to the methylation level of the corresponding sites on an Illumina 450k methylation chip. The calculated average Pearson correlation coefficient of 0.91 confirms the sequencing results with one of the industry-leading CpG methylation platforms and shows that targeted amplicon bisulfite sequencing provides an accurate and cost-efficient method for DNA methylation studies, e.g., to provide platform-independent confirmation of Illumina Infinium 450k methylation data. TABSAT offers a novel way to analyze data generated by Ion Torrent instruments and can also be used with data from the Illumina MiSeq platform. It can be easily accessed via the Platomics platform, which offers a web-based graphical user interface along with sample and parameter storage. TABSAT is freely

  19. Generating Multiple Base-Resolution DNA Methylomes Using Reduced Representation Bisulfite Sequencing.

    PubMed

    Chatterjee, Aniruddha; Rodger, Euan J; Stockwell, Peter A; Le Mée, Gwenn; Morison, Ian M

    2017-01-01

    Reduced representation bisulfite sequencing (RRBS) is an effective technique for profiling genome-wide DNA methylation patterns in eukaryotes. RRBS couples size selection, bisulfite conversion, and second-generation sequencing to enrich for CpG-dense regions of the genome. The progressive improvement of second-generation sequencing technologies and reduction in cost provided an opportunity to examine the DNA methylation patterns of multiple genomes. Here, we describe a protocol for sequencing multiple RRBS libraries in a single sequencing reaction to generate base-resolution methylomes. Furthermore, we provide a brief guideline for base-calling and data analysis of multiplexed RRBS libraries. These strategies will be useful to perform large-scale, genome-wide DNA methylation analysis.

  20. PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR.

    PubMed

    Lu, Jennifer; Johnston, Andrew; Berichon, Philippe; Ru, Ke-Lin; Korbie, Darren; Trau, Matt

    2017-01-24

    The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com).

  1. PrimerSuite: A High-Throughput Web-Based Primer Design Program for Multiplex Bisulfite PCR

    PubMed Central

    Lu, Jennifer; Johnston, Andrew; Berichon, Philippe; Ru, Ke-lin; Korbie, Darren; Trau, Matt

    2017-01-01

    The analysis of DNA methylation at CpG dinucleotides has become a major research focus due to its regulatory role in numerous biological processes, but the requisite need for assays which amplify bisulfite-converted DNA represents a major bottleneck due to the unique design constraints imposed on bisulfite-PCR primers. Moreover, a review of the literature indicated no available software solutions which accommodated both high-throughput primer design, support for multiplex amplification assays, and primer-dimer prediction. In response, the tri-modular software package PrimerSuite was developed to support bisulfite multiplex PCR applications. This software was constructed to (i) design bisulfite primers against multiple regions simultaneously (PrimerSuite), (ii) screen for primer-primer dimerizing artefacts (PrimerDimer), and (iii) support multiplex PCR assays (PrimerPlex). Moreover, a major focus in the development of this software package was the emphasis on extensive empirical validation, and over 1300 unique primer pairs have been successfully designed and screened, with over 94% of them producing amplicons of the expected size, and an average mapping efficiency of 93% when screened using bisulfite multiplex resequencing. The potential use of the software in other bisulfite-based applications such as methylation-specific PCR is under consideration for future updates. This resource is freely available for use at PrimerSuite website (www.primer-suite.com). PMID:28117430

  2. Genotypic characterization of Indian isolates of infectious bursal disease virus strains by reverse transcription-polymerase chain reaction combined with restriction fragment length polymorphism analysis.

    PubMed

    Priyadharsini, C V; Senthilkumar, T M A; Raja, P; Kumanan, K

    2016-03-01

    The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

  3. Effect of a High-Protein Energy-Restricted Diet Combined with Resistance Training on Metabolic Profile in Older Individuals with Metabolic Impairments.

    PubMed

    Amamou, T; Normandin, E; Pouliot, J; Dionne, I J; Brochu, M; Riesco, E

    2017-01-01

    Adequate protein intake and resistance training are effective strategies to maintain muscle mass, but the effect of their combination on metabolic profile during weight loss remains to be determined in older adults. The main objective of this study was to determine the effect of a 16-week high-protein caloric restriction combined with resistance training on chronic disease risk factors in obese older individuals with metabolic impairments. A total of 26 overweight adults aged between 60 and 75 years (BMI 32.4 ± 3.9 kg/m2) with at least 2 factors of the metabolic syndrome participated in this study and were randomized into two groups: 1) high-protein caloric restriction (HP; n= 12) and 2) high-protein caloric restriction combined with dynamic-resistance training (HP+RT; n=14). Caloric intake was reduced by 500 kcal/d in all participants and protein intake equated 25-30% of total calories (~1.4 g/kg/d). Exercise training consisted of 3 session/week of resistance training on pulley machines. Outcome measures included total and trunk fat mass (FM), total and appendicular lean body mass (LBM), fasting glucose level, lipid profile and blood pressure. Our results showed that total and trunk FM (all p<0.0001) as well as fasting glucose (p<0.0001), triglycerides (p=0.002) and total cholesterol (p=0.03) levels decreased similarly in both groups. However, total (p=0.04) and appendicular (p=0.02) LBM decreased in the HP group only. Our data show that high-protein energy restriction improves health profile of obese elderly at high risk of chronic disease but needs to be combined with resistance training to maintain LBM.

  4. Combining metformin therapy with caloric restriction for the management of type 2 diabetes and nonalcoholic fatty liver disease in obese rats

    PubMed Central

    Linden, Melissa A.; Lopez, Kristi T.; Fletcher, Justin A.; Morris, E. Matthew; Meers, Grace M.; Siddique, Sameer; Laughlin, M. Harold; Sowers, James R.; Thyfault, John P.; Ibdah, Jamal A.; Rector, R. Scott

    2016-01-01

    Weight loss is recommended for patients with nonalcoholic fatty liver disease (NAFLD), while metformin may lower liver enzymes in type 2 diabetics. Yet, the efficacy of the combination of weight loss and metformin in the treatment of NAFLD is unclear. We assessed the effects of metformin, caloric restriction, and their combination on NAFLD in diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Male OLETF rats (age 20 weeks; n = 6–8 per group) were fed ad libitum (AL), given metformin (300 mg·kg−1·day−1; Met), calorically restricted (70% of AL; CR), or calorically restricted and given metformin (CR+Met) for 12 weeks. Met lowered adiposity compared with AL but not to the same magnitude as CR or CR+Met (p < 0.05). Although only CR improved fasting insulin and glucose, the combination of CR+Met was needed to improve post-challenge glucose tolerance. All treatments lowered hepatic triglycerides, but further improvements were observed in the CR groups (p < 0.05, Met vs. CR or CR+Met) and a further reduction in serum alanine aminotransferases was observed in CR+Met rats. CR lowered markers of hepatic de novo lipogenesis (fatty acid synthase, acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase-1 (SCD-1)) and increased hepatic mitochondrial activity (palmitate oxidation and β-hydroxyacyl CoA dehydrogenase (β-HAD) activity). Changes were enhanced in the CR+Met group for ACC, SCD-1, β-HAD, and the mitophagy marker BNIP3. Met decreased total hepatic mTOR content and inhibited mTOR complex 1, which may have contributed to Met-induced reductions in de novo lipogenesis. These findings in the OLETF rat suggest that the combination of caloric restriction and metformin may provide a more optimal approach than either treatment alone in the management of type 2 diabetes and NAFLD. PMID:26394261

  5. Improvement in the high-fat diet-induced dyslipidemia and adiponectin levels by fish oil feeding combined with food restriction in obese KKAy mice.

    PubMed

    Wakutsu, Masaki; Tsunoda, Nobuyo; Mochi, Yasuki; Numajiri, Mitsuki; Shiba, Sachiko; Muraki, Etsuko; Kasono, Keizo

    2012-01-01

    The effect on weight reduction of fish oil combined with food restriction in comparison with that of beef tallow was investigated in high-fat diet-induced obese KKAy mice. Although the reduction of body and white adipose tissue weight was similar in the two groups, fish oil increased adiponectin levels in the plasma, improved dyslipidemia accompanied by suppression of lipid synthesis in the liver when compared with beef tallow.

  6. Combining metformin therapy with caloric restriction for the management of type 2 diabetes and nonalcoholic fatty liver disease in obese rats.

    PubMed

    Linden, Melissa A; Lopez, Kristi T; Fletcher, Justin A; Morris, E Matthew; Meers, Grace M; Siddique, Sameer; Laughlin, M Harold; Sowers, James R; Thyfault, John P; Ibdah, Jamal A; Rector, R Scott

    2015-10-01

    Weight loss is recommended for patients with nonalcoholic fatty liver disease (NAFLD), while metformin may lower liver enzymes in type 2 diabetics. Yet, the efficacy of the combination of weight loss and metformin in the treatment of NAFLD is unclear. We assessed the effects of metformin, caloric restriction, and their combination on NAFLD in diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Male OLETF rats (age 20 weeks; n = 6-8 per group) were fed ad libitum (AL), given metformin (300 mg·kg(-1)·day(-1); Met), calorically restricted (70% of AL; CR), or calorically restricted and given metformin (CR+Met) for 12 weeks. Met lowered adiposity compared with AL but not to the same magnitude as CR or CR+Met (p < 0.05). Although only CR improved fasting insulin and glucose, the combination of CR+Met was needed to improve post-challenge glucose tolerance. All treatments lowered hepatic triglycerides, but further improvements were observed in the CR groups (p < 0.05, Met vs. CR or CR+Met) and a further reduction in serum alanine aminotransferases was observed in CR+Met rats. CR lowered markers of hepatic de novo lipogenesis (fatty acid synthase, acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase-1 (SCD-1)) and increased hepatic mitochondrial activity (palmitate oxidation and β-hydroxyacyl CoA dehydrogenase (β-HAD) activity). Changes were enhanced in the CR+Met group for ACC, SCD-1, β-HAD, and the mitophagy marker BNIP3. Met decreased total hepatic mTOR content and inhibited mTOR complex 1, which may have contributed to Met-induced reductions in de novo lipogenesis. These findings in the OLETF rat suggest that the combination of caloric restriction and metformin may provide a more optimal approach than either treatment alone in the management of type 2 diabetes and NAFLD.

  7. ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

    PubMed

    Scala, Giovanni; Affinito, Ornella; Palumbo, Domenico; Florio, Ermanno; Monticelli, Antonella; Miele, Gennaro; Chiariotti, Lorenzo; Cocozza, Sergio

    2016-11-25

    CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

  8. Structural characterization of reaction products of caftaric acid and bisulfite present in a commercial wine using high resolution mass spectrometric and nuclear magnetic resonance techniques.

    PubMed

    Hayasaka, Yoji; Black, Cory A; Hack, Jeremy; Smith, Paul

    2017-09-01

    Reaction products of bisulfite and caftaric acid were found in wines containing sulfites as a preservative. Acidic compounds were separated from wine and analyzed by HPLC combined with DAD and QTOF mass spectrometer. HPLC chromatograms of the expected [M-H](-) ion and UV absorption revealed the presence of five possible reaction products (a-e). These compounds were isolated then characterized by NMR and confirmed to be the reaction products as follows; 5-sulfo-(E)-caftaric acid (a), 2-sulfo-(Z)-caftaric acid (b), 2-sulfo-(E)-caftaric acid (c), (E)-caftaric acid-4-O-sulfate (d) and (E)-caftaric acid-3-O-sulfate (e). UV spectra and high resolution product ion spectra of the five compounds also supported their identity. The reaction products were confirmed to be commonly present in commercial wines across four vintages and two varieties. Their concentration was found to be as much as that of 2-S-glutathionyl caftaric acid, suggesting that bisulfite consistently competes as a nucleophile with glutathione for the o-quinone of caftaric acid. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Promoter targeted bisulfite sequencing reveals DNA methylation profiles associated with low sperm motility in asthenozoospermia.

    PubMed

    Du, Ye; Li, Meiyan; Chen, Jing; Duan, Yonggang; Wang, Xuebin; Qiu, Yong; Cai, Zhiming; Gui, Yaoting; Jiang, Hui

    2016-01-01

    Is there an association between sperm DNA methylation profiles and asthenozoospermia? DNA methylation, at specific CpGs but not at the global level, was significantly different between low motile sperm cells of asthenozoospermic individuals and high motile sperm cells of normozoospermic controls. Aberrant DNA methylation, both globally and restricted to a specific gene locus, has been associated with male infertility and abnormal semen parameters. This was a case-control study investigating the differences in DNA methylation at CpGs in promoter regions between high and low motile sperm cells from eight normozoospermic controls and seven asthenozoospermic patients. The liquid hybridization capture-based bisulfite sequencing method was used to determine DNA methylation at CpGs in promoter regions. The global inter-individual and intra-individual methylation variability were estimated by evaluating the methylation variance between and within different motile sperm fractions from the same or different individuals. Asthenozoospermia-associated differentially methylated or variable CpGs and differentially methylated regions were identified by comparing the DNA methylation of high motile sperm cells from normozoospermic controls with that of low motile sperm cells from asthenozoospermic patients. In this study, we determined the global DNA methylation level (24.7%), inter-individual variance (14.4%) and intra-individual differences between high and low motile sperm fractions (3.9%). We demonstrated that there were no statistically significant differences in either the global DNA methylation level or global methylation variability between sperm from men with normozoospermia or asthenozoospermia. Between high motile sperm from men with normozoospermia and low motile sperm from men with asthenozoospermia, we identified 134 differentially methylated CpGs, 41 differentially methylated regions and 134 differentially variable CpGs. The genomic distribution patterns of the

  10. Persistent use of against-label statin-fibrate combinations from 2003-2009 despite United States Food and Drug Administration dose restrictions.

    PubMed

    Alford, Julie C; Saseen, Joseph J; Allen, Richard R; Nair, Kavita V

    2012-07-01

    To describe the prevalence of prescribing against-label statin-fibrate combination therapy. Retrospective cohort study. Medstat MarketScan Commercial Claims and Encounter database. Adults (aged 18-89 yrs) who were prescribed statin-fibrate combination therapy between January 1, 2003, and June 30, 2009, had pharmacy claims demonstrating two or more concurrently filled prescriptions for a statin and a fibrate, and had continuous insurance enrollment for at least 12 months. Claims data were used to identify patients with dyslipidemia based on International Classification of Diseases, Ninth Revision, Clinical Modification diagnosis codes. National Drug Codes were used to describe concurrent statin-fibrate combination therapy. The primary outcome was recent use of against-label statin-fibrate combination therapy, defined as use during the last 18 months (January 1, 2008-June 30, 2009) of the study period. Patients were stratified according to statin and dosage to identify against-label combination use (e.g., simvastatin > 10 mg/day with gemfibrozil). Within the recent-use period, 131,394 patients were prescribed concurrent statin-fibrate combination therapy; of these patients, 13,420 (10.2%) had against-label therapy. Simvastatin-gemfibrozil accounted for 8978 (66.9%) of all against-label combinations. Of all 9877 simvastatin-gemfibrozil combinations prescribed in the recent-use period (both on-label and against-label use), 8978 (90.9%) were against label. The secondary outcome was prevalence of against-label statin-fibrate combination therapy on an annual basis: 15.5% in 2003, 18.7% in 2004, 9.1% in 2005, 8.3% in 2006, 9.2% in 2007, and 9.8% in 2008. Against-label statin-fibrate combination therapy continues to be prescribed despite established United States Food and Drug Administration (FDA) dose restrictions. Nearly every time the simvastatin-gemfibrozil combination was prescribed, it was against label because simvastatin exceeded the maximum dose restriction

  11. Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

    PubMed Central

    Laird, Charles D.; Pleasant, Nicole D.; Clark, Aaron D.; Sneeden, Jessica L.; Hassan, K. M. Anwarul; Manley, Nathan C.; Vary, Jay C.; Morgan, Todd; Hansen, R. Scott; Stöger, Reinhard

    2004-01-01

    Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA. PMID:14673087

  12. MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing

    PubMed Central

    2010-01-01

    Background Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. Findings MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. Conclusions The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org. PMID:21159174

  13. MethVisual - visualization and exploratory statistical analysis of DNA methylation profiles from bisulfite sequencing.

    PubMed

    Zackay, Arie; Steinhoff, Christine

    2010-12-15

    Exploration of DNA methylation and its impact on various regulatory mechanisms has become a very active field of research. Simultaneously there is an arising need for tools to process and analyse the data together with statistical investigation and visualisation. MethVisual is a new application that enables exploratory analysis and intuitive visualization of DNA methylation data as is typically generated by bisulfite sequencing. The package allows the import of DNA methylation sequences, aligns them and performs quality control comparison. It comprises basic analysis steps as lollipop visualization, co-occurrence display of methylation of neighbouring and distant CpG sites, summary statistics on methylation status, clustering and correspondence analysis. The package has been developed for methylation data but can be also used for other data types for which binary coding can be inferred. The application of the package, as well as a comparison to existing DNA methylation analysis tools and its workflow based on two datasets is presented in this paper. The R package MethVisual offers various analysis procedures for data that can be binarized, in particular for bisulfite sequenced methylation data. R/Bioconductor has become one of the most important environments for statistical analysis of various types of biological and medical data. Therefore, any data analysis within R that allows the integration of various data types as provided from different technological platforms is convenient. It is the first and so far the only specific package for DNA methylation analysis, in particular for bisulfite sequenced data available in R/Bioconductor enviroment. The package is available for free at http://methvisual.molgen.mpg.de/ and from the Bioconductor Consortium http://www.bioconductor.org.

  14. Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue.

    PubMed

    Arai, Yoshikazu; Fukukawa, Hisho; Atozi, Takanori; Matsumoto, Shoma; Hanazono, Yutaka; Nagashima, Hiroshi; Ohgane, Jun

    2016-01-01

    DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.

  15. Photoinduced reductive dissolution of. cap alpha. -Fe/sub 2/O/sub 3/ by bisulfite

    SciTech Connect

    Faust, B.C.; Hoffmann, M.R.

    1986-09-01

    The kinetics of the photoinduced reductive dissolution of ..cap alpha..-Fe/sub 2/O/sub 3/ by bisulfite in anoxic colloidal suspensions have been investigated. Quantum yields, ..cap alpha../sub Fe(II)/, as a function of wavelength for Fe(II)/sub aq/ production were obtained. Photochemical reactivity has been discussed in terms of the excitation of the O/sup 2 -/ to Fe/sup 3 +/ charge-transfer band of ..cap alpha..-Fe/sub 2/O/sub 3/ and in terms of photoassisted charge-transfer reactions of Fe(III)-S(IV) curficial complexes.

  16. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity.

    PubMed

    Smallwood, Sébastien A; Lee, Heather J; Angermueller, Christof; Krueger, Felix; Saadeh, Heba; Peat, Julian; Andrews, Simon R; Stegle, Oliver; Reik, Wolf; Kelsey, Gavin

    2014-08-01

    We report a single-cell bisulfite sequencing (scBS-seq) method that can be used to accurately measure DNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNA methylome, which makes scBS-seq a versatile tool to explore DNA methylation in rare cells and heterogeneous populations.

  17. Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling.

    PubMed

    Boyle, Patrick; Clement, Kendell; Gu, Hongcang; Smith, Zachary D; Ziller, Michael; Fostel, Jennifer L; Holmes, Laurie; Meldrim, Jim; Kelley, Fontina; Gnirke, Andreas; Meissner, Alexander

    2012-10-03

    Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.

  18. Inhibition of photosystem II precedes thylakoid membrane lipid peroxidation in bisulfite-treated leaves of Phaseolus vulgaris

    SciTech Connect

    Covello, P.S.; Dumbroff, E.B.; Thompson, J.E. Univ. of Guelph, Ontario ); Chang, A. )

    1989-08-01

    Exposure of leaves to SO{sub 2} bisulfite is known to induce peroxidation of thylakoid lipids and to inhibit photosynthetic electron transport. In the present study, we have examined the temporal relationship between bisulfite-induced thylakoid lipid peroxidation and inhibition of electron transport in an attempt to clarify the primary mechanism of SO{sub 2} phytotoxicity. Primary leaves of bean (Phaseolus vulgaris L. cv Kinghorn) were floated on a solution of NaHSO{sub 3}, and the effects of this treatment on photosynthetic electron transport were determined in vivo by measurements of chlorophyll a fluorescence induction and in vitro by biochemical measurements of the light reactions using isolated thylakoids. Lipid peroxidation in treated leaves was followed by monitoring ethane emission from leaf segments and by measuring changes in fatty acid composition and lipid fluidity in isolated thylakoids. A 1 hour treatment with bisulfite inhibited photosystem II (PSII) activity by 70% without modifying Photosystem I, and this inhibitory effect was not light-dependent. By contrast, lipid peroxidation was not detectable until after the inhibition of PSII and was strongly light dependent. This temporal separation of events together with the differential effect of light suggests that bisulfite-induced inhibition of PSII is not a secondary effect of lipid peroxidation, and that bisulfite acts directly on one or more components of PSII.

  19. Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

    PubMed

    Masser, Dustin R; Stanford, David R; Hadad, Niran; Giles, Cory B; Wren, Jonathan D; Sonntag, William E; Richardson, Arlan; Freeman, Willard M

    2016-06-01

    Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

  20. Identification of spermatozoa by tissue-specific differential DNA methylation using bisulfite modification and pyrosequencing.

    PubMed

    Balamurugan, Kuppareddi; Bombardi, Robin; Duncan, George; McCord, Bruce

    2014-11-01

    The focus of this study is to evaluate the application of epigenetic markers as a forensic tool for the determination of semen present in sexual assault cases. A series of genetic loci were screened in order to identify certain epigenetic markers displaying differential methylation that can allow semen to be differentiated from blood, buccal cells, skin epidermis, and vaginal epithelial cells. Of the different loci tested, a panel of six markers, DACT1, USP49, DDX4, Hs_INSL6_03, Hs_ZC3H12D_05, and B_SPTB_03 were identified to contain tissue-specific differential methylation. Samples ranging from 9-21 for each tissue type were collected and subjected to bisulfite modification. The bisulfite modified DNA was amplified by PCR, and analyzed by pyrosequencing to quantitate the level of methylation at each marker. All six markers successfully differentiated semen samples from the other four tissue types analyzed. Sperm DNA was hypomethylated in all but one marker, B_SPTB_03, where this marker showed hypermethylation. Mean methylation percentages for semen samples were statistically significant from mean methylation percentages of the other four tissues studied (p < 0.01). The results of this study demonstrate the applicability of epigenetic markers as a novel tool for determination of spermatozoa and to identify the tissue source of origin of a DNA sample.

  1. P3BSseq: parallel processing pipeline software for automatic analysis of bisulfite sequencing data.

    PubMed

    Luu, Phuc-Loi; Gerovska, Daniela; Arrospide-Elgarresta, Mikel; Retegi-Carrión, Sugoi; Schöler, Hans R; Araúzo-Bravo, Marcos J

    2017-02-01

    Bisulfite sequencing (BSseq) processing is among the most cumbersome next generation sequencing (NGS) applications. Though some BSseq processing tools are available, they are scattered, require puzzling parameters and are running-time and memory-usage demanding. We developed P3BSseq, a parallel processing pipeline for fast, accurate and automatic analysis of BSseq reads that trims, aligns, annotates, records the intermediate results, performs bisulfite conversion quality assessment, generates BED methylome and report files following the NIH standards. P3BSseq outperforms the known BSseq mappers regarding running time, computer hardware requirements (processing power and memory use) and is optimized to process the upcoming, extended BSseq reads. We optimized the P3BSseq parameters for directional and non-directional libraries, and for single-end and paired-end reads of Whole Genome and Reduced Representation BSseq. P3BSseq is a user-friendly streamlined solution for BSseq upstream analysis, requiring only basic computer and NGS knowledge. P3BSseq binaries and documentation are available at: http://sourceforge.net/p/p3bsseq/wiki/Home/ mararabra@yahoo.co.uk Supplementary data are available at Bioinformatics online.

  2. Enhancement of high-solids enzymatic hydrolysis of corncob residues by bisulfite pretreatment for biorefinery.

    PubMed

    Xing, Yang; Bu, Lingxi; Zheng, Tianran; Liu, Shijie; Jiang, Jianxin

    2016-12-01

    Co-production of glucose, furfural and other green materials based on a lignocellulosic biorefinery is a promising way to realize the commercial application of corncob residues. An effective process was developed for glucose production using low temperature bisulfite pretreatment and high-solids enzymatic hydrolysis. Corncob residues from furfural production (FRs) were pretreated with 0.1g NaHSO3/g dry substrate at 100°C for 3h. Lignin was sulfonated and sulfonic groups were produced during pretreatment, which resulted in decreasing the zeta potential of the samples. Compared with raw material, bisulfite pretreatment of FRs increased the glucose yield from 18.6 to 99.45% after 72h hydrolysis at a solids loading of 12.5%. The hydrolysis residues showed a relatively high thermal stability and concentrated high derivatives. Direct pretreatment followed by enzymatic hydrolysis is an environmentally-friendly and economically-feasible method for the production of glucose and high-purity lignin, which could be further converted into high-value products. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

    PubMed Central

    Meller, Sebastian; Leisse, Annette; Sailer, Verena; Zech, Julie; Mengdehl, Martina; Garbe, Leif-Alexander; Uhl, Barbara; Dietrich, Dimo

    2014-01-01

    DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of

  4. Effects of Moderate Aerobic Exercise Combined with Caloric Restriction on Circulating Estrogens and IGF-I in Premenopausal Women

    DTIC Science & Technology

    2002-10-01

    239 kcals; High BMI =2125 + 287 to 1450 + 264 kcals; P< 0.05 pre vs post in both groups). The combination of moderate exercise and diet produced...combination of moderate exercise and diet produced significant weight loss in both groups (Low BMI -X%; High BMI -X% P< 0.05). Significant changes in body...of Exercise for Older Adults 50 Kines496C Independent Study 3 1 Spring 1998 Kines456 Fitness Appraisal 4 96 Kines496C Independent Study 3 5 Kines

  5. Effects of the dietary approaches to stop hypertension diet alone and in combination with exercise and caloric restriction on insulin sensitivity and lipids.

    PubMed

    Blumenthal, James A; Babyak, Michael A; Sherwood, Andrew; Craighead, Linda; Lin, Pao-Hwa; Johnson, Julie; Watkins, Lana L; Wang, Jenny T; Kuhn, Cynthia; Feinglos, Mark; Hinderliter, Alan

    2010-05-01

    This study examined the effects of the Dietary Approaches to Stop Hypertension (DASH) diet on insulin sensitivity and lipids. In a randomized control trial, 144 overweight (body mass index: 25 to 40) men (n=47) and women (n=97) with high blood pressure (130 to 159/85 to 99 mm Hg) were randomly assigned to one of the following groups: (1) DASH diet alone; (2) DASH diet with aerobic exercise and caloric restriction; or (3) usual diet controls (UC). Body composition, fitness, insulin sensitivity, and fasting lipids were measured before and after 4 months of treatment. Insulin sensitivity was estimated on the basis of glucose and insulin levels in the fasting state and after an oral glucose load. Participants in the DASH diet with aerobic exercise and caloric restriction condition lost weight (-8.7 kg [95% CI: -2.0 to -9.7 kg]) and exhibited a significant increase in aerobic capacity, whereas the DASH diet alone and UC participants maintained their weight (-0.3 kg [95% CI: -1.2 to 0.5 kg] and +0.9 kg [95% CI: 0.0 to 1.7 kg], respectively) and had no improvement in exercise capacity. DASH diet with aerobic exercise and caloric restriction demonstrated lower glucose levels after the oral glucose load, improved insulin sensitivity, and lower total cholesterol and triglycerides compared with both DASH diet alone and UC, as well as lower fasting glucose and low-density lipoprotein cholesterol compared with UC. DASH diet alone participants generally did not differ from UC in these measures. Combining the DASH diet with exercise and weight loss resulted in significant improvements in insulin sensitivity and lipids. Despite clinically significant reductions in blood pressure, the DASH diet alone, without caloric restriction or exercise, resulted in minimal improvements in insulin sensitivity or lipids.

  6. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-10-25

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides.

  7. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA.

    PubMed Central

    Hayatsu, H; Yamashita, Y; Yui, S; Yamagata, Y; Tomita, K; Negishi, K

    1982-01-01

    When guanine-, adenine- and cytosine-nucleosides and nucleotides were treated with formaldehyde and then with bisulfite, stable N-sulfomethyl compounds were formed. N2-Sulfomethylguanine, N6-sulfomethyladenine, N4-sulfomthylcytosine and N6-sulfomethyl-9-beta-D-arabinofuranosyladenine were isolated as crystals and characterized. A guanine-specific sulfomethylation was brought about by treatment and denatured single-stranded DNA with formaldehyde and then with bisulfite at pH 7 and 4 degrees C. Since native double-stranded DNA was not modified by this treatment, this new method of modification is expected to be useful as a conformational probe for polynucleotides. PMID:7177848

  8. A combination of epitope prediction and molecular docking allows for good identification of MHC class I restricted T-cell epitopes.

    PubMed

    Zhang, Xue Wu

    2013-08-01

    In silico identification of T-cell epitopes is emerging as a new methodology for the study of epitope-based vaccines against viruses and cancer. In order to improve accuracy of prediction, we designed a novel approach, using epitope prediction methods in combination with molecular docking techniques, to identify MHC class I restricted T-cell epitopes. Analysis of the HIV-1 p24 protein and influenza virus matrix protein revealed that the present approach is effective, yielding prediction accuracy of over 80% with respect to experimental data. Subsequently, we applied such a method for prediction of T-cell epitopes in SARS coronavirus (SARS-CoV) S, N and M proteins. Based on available experimental data, the prediction accuracy is up to 90% for S protein. We suggest the use of epitope prediction methods in combination with 3D structural modelling of peptide-MHC-TCR complex to identify MHC class I restricted T-cell epitopes for use in epitope based vaccines like HIV and human cancers, which should provide a valuable step forward for the design of better vaccines and may provide in depth understanding about activation of T-cell epitopes by MHC binding peptides.

  9. A combined EPR and MD simulation study of a nitroxyl spin label with restricted internal mobility sensitive to protein dynamics

    NASA Astrophysics Data System (ADS)

    Oganesyan, Vasily S.; Chami, Fatima; White, Gaye F.; Thomson, Andrew J.

    2017-01-01

    EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions.

  10. Software updates in the Illumina HiSeq platform affect whole-genome bisulfite sequencing.

    PubMed

    Toh, Hidehiro; Shirane, Kenjiro; Miura, Fumihito; Kubo, Naoki; Ichiyanagi, Kenji; Hayashi, Katsuhiko; Saitou, Mitinori; Suyama, Mikita; Ito, Takashi; Sasaki, Hiroyuki

    2017-01-05

    Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be

  11. Mechanisms for independent and combined effects of calorie restriction and acute exercise on insulin-stimulated glucose uptake by skeletal muscle of old rats.

    PubMed

    Sharma, Naveen; Wang, Haiyan; Arias, Edward B; Castorena, Carlos M; Cartee, Gregory D

    2015-04-01

    Either calorie restriction [CR; consuming 60-65% of ad libitum (AL) intake] or acute exercise can independently improve insulin sensitivity in old age, but their combined effects on muscle insulin signaling and glucose uptake have previously been unknown. Accordingly, we assessed the independent and combined effects of CR (beginning at 14 wk old) and acute exercise (3-4 h postexercise) on insulin signaling and glucose uptake in insulin-stimulated epitrochlearis muscles from 30-mo-old rats. Either CR alone or exercise alone vs. AL sedentary controls induced greater insulin-stimulated glucose uptake. Combined CR and exercise vs. either treatment alone caused an additional increase in insulin-stimulated glucose uptake. Either CR or exercise alone vs. AL sedentary controls increased Akt Ser(473) and Akt Thr(308) phosphorylation. Combined CR and exercise further elevated Akt phosphorylation on both sites. CR alone, but not exercise alone, vs. AL sedentary controls significantly increased Akt substrate of 160 kDa (AS160) Ser(588) and Thr(642) phosphorylation. Combined CR and exercise did not further enhance AS160 phosphorylation. Exercise alone, but not CR alone, modestly increased GLUT4 abundance. Combined CR and exercise did not further elevate GLUT4 content. These results suggest that CR or acute exercise independently increases insulin-stimulated glucose uptake via overlapping (greater Akt phosphorylation) and distinct (greater AS160 phosphorylation for CR, greater GLUT4 for exercise) mechanisms. Our working hypothesis is that greater insulin-stimulated glucose uptake in the combined CR and exercise group vs. CR or exercise alone relies on greater Akt activation, leading to greater phosphorylation of one or more Akt substrates other than AS160.

  12. Restrictive cardiomyopathy

    MedlinePlus

    Cardiomyopathy - restrictive; Infiltrative cardiomyopathy; Idiopathic myocardial fibrosis ... In a case of restrictive cardiomyopathy, the heart muscle is of normal size or slightly enlarged. Most of the time, it also pumps normally. However, it does ...

  13. Bisulfite and sulfite as derivatives of sulfur dioxide alters biomechanical behaviors of airway smooth muscle cells in culture.

    PubMed

    Song, Aijing; Lin, Feng; Li, Jianming; Liao, Qingfeng; Liu, Enmei; Jiang, Xuemei; Deng, Linhong

    2014-02-01

    Sulfur dioxide (SO2) is a common air pollutant that triggers asthmatic symptoms, but its toxicological mechanisms are not fully understood. Specifically, it is unclear how SO2 in vivo affects airway smooth muscle (ASM) cells of which the mechanics is known to ultimately mediate airway hyperresponsiveness (AHR) - a hallmark feature of asthma. To this end, we investigated the effects of bisulfite/sulfite (1:3 M/M in neutral fluid to simulate the in vivo derivatives of inhaled SO2 in the airways), on the viability, migration, stiffness and contractility of ASM cells cultured in vitro. The results showed that bisulfite/sulfite consistently increased viability, migration, F-actin intensity and stiffness of ASM cells in similar fashion as concentration increasing from 10(-4) to 10(-1) mmol/L. However, bisulfite/sulfite increased the ASM cell contractility induced by KCl only at the concentration between 10(-4) and 10(-3) mmol/L (p < 0.05), while having no consistent effect on that induced by histamine. At the concentration of 10(0) mmol/L, bisulfite/sulfite became acutely toxic to the ASM cells. Taken together, the data suggest that SO2 derivatives at low levels in vivo may directly increase the mass, stiffness and contractility of ASM cells, which may help understand the mechanism in which specific air pollutants contribute in vivo to the pathogenesis of asthma.

  14. Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

    PubMed Central

    Lu, Jennifer; Ru, Kelin; Candiloro, Ida; Dobrovic, Alexander; Korbie, Darren; Trau, Matt

    2017-01-01

    Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG’s contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates. PMID:28327639

  15. Kinetics of oxygen exchange between bisulfite ion and water as studied by oxygen-17 nuclear magnetic resonance spectroscopy

    SciTech Connect

    Horner, D.A.

    1984-08-01

    The nuclear magnetic relaxation times of oxygen-17 have been measured in aqueous sodium bisulfite solutions in the pH range from 2.5 to 5 as a function of temperature, pH, and S(IV) concentration, at an ionic strength of 1.0 m. The rate law for oxygen exchange between bisulfite ion and water was obtained from an analysis of the data, and is consistent with oxygen exchange occurring via the reaction SO/sub 2/ + H/sub 2/O right reversible H/sup +/ + SHO/sub 3//sup -/. The value of k/sub -1/ is in agreement with relaxation measurements. Direct spectroscopic evidence was found for the existence of two isomers of bisulfite ion: one with the proton bonded to the sulfur (HSO/sub 3//sup -/) and the other with the proton bonded to an oxygen (SO/sub 3/H/sup -/). (The symbol SHO/sub 3//sup -/ in the above chemical equation refers to both isomeric forms of bisulfite ion.) The relative amounts of the two isomers were determined as a function of temperature, and the rate and mechanism of oxygen exchange between the two was investigated. One of the two isomers, presumably SO/sub 3/H/sup -/, exchanges oxygens with water much more rapidly than does the other. A two-pulse sequence was developed which greatly diminished the solvent peak in the NMR spectrum.

  16. DNA methylation profiling using bisulfite-based epityping of pooled genomic DNA.

    PubMed

    Docherty, Sophia J; Davis, Oliver S P; Haworth, Claire M A; Plomin, Robert; Mill, Jonathan

    2010-11-01

    DNA methylation plays a vital role in normal cellular function, with aberrant methylation signatures being implicated in a growing number of human pathologies and complex human traits. Methods based on the modification of genomic DNA with sodium bisulfite are considered the 'gold-standard' for DNA methylation profiling on genomic DNA; however they require large amounts of DNA and may be prohibitively expensive when used on the large sample sizes necessary to detect small effects. DNA pooling approaches are already widely used in large-scale studies of DNA sequence and gene expression. In this paper, we describe the application of this economical DNA pooling technique to the study of DNA methylation profiles. This method generates accurate quantitative assessments of group DNA methylation averages, reducing the time, cost and amount of DNA starting material required for large-scale epigenetic investigation of disease phenotypes.

  17. Bisulfite-free and Base-resolution Analysis of 5-formylcytosine at Whole-genome Scale

    PubMed Central

    Xia, Bo; Han, Dali; Lu, Xingyu; Sun, Zhaozhu; Zhou, Ankun; Yin, Qiangzong; Zeng, Hu; Liu, Menghao; Jiang, Xiang; Xie, Wei; He, Chuan; Yi, Chengqi

    2015-01-01

    Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for whole-genome analysis of 5fC, based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones. PMID:26344045

  18. Study on the electrochemiluminescent behavior of menadione sodium bisulfite in presence of luminol.

    PubMed

    Lin, Zhenyu; Chen, Jinhua; Chen, Guonan

    2007-07-31

    Menadione sodium bisulfite (MSB) is a stable water-soluble derivative of Vitamin K(3), which is found to be able to enhance the ECL of luminol at potential of 0.88 V in phosphate buffer solution. The conditions for the enhanced ECL, such as the selection of the type of buffer solution, applied potential mode, scanning rate, the effect of pH and concentration of luminol have been investigated in detail in this paper. Under the optimum conditions, the enhanced ECL intensity is linear with the concentration of MSB over a wide range, the detection limit for MSB is 3.0x10(-7)mol/L. The proposed method has been applied to determine the MSB in the commercial injection samples. A possible mechanism for the enhanced ECL of luminol by MSB has also been proposed.

  19. Effect of sodium bisulfite modification on the arginine acceptance of E. coli tRNA Arg.

    PubMed Central

    Chakraburtty, K

    1975-01-01

    Escherichia coli tRNA Arg was treated with sodium bisulfite to convert exposed cytosine residues to uracil. This treatment resulted in the loss of amino acid acceptance of the tRNA Arg with pseudo first-order reaction kinetics. The active and inactive molecules were separated after about 60e active and inactive molecules were separated after about 60 percent inactivation and analyzed for U in various positions by finger-printing of the oligonucleotides produced by nucleases. The results show that C to U base transitions in the dihydrouridine loop and in the CCA terminus have no effect on the aminoacylation of this tRNA. Deamination of a cytosine residue at the second position of the anticodon resulted in the loss of amino acid acceptor activity of arginine transfer RNA. Images PMID:1103086

  20. Nanostructured alkyl carboxylic acid-based restricted access solvents: Application to the combined microextraction and cleanup of polycyclic aromatic hydrocarbons in mosses.

    PubMed

    Caballero-Casero, N; Çabuk, H; Martínez-Sagarra, G; Devesa, J A; Rubio, S

    2015-08-26

    Alkyl carboxylic acid-based nanostructured solvents, synthesized in mixtures of tetrahydrofuran (THF) and water through self-assembly and coacervation, were proved to behave as restricted access liquids. Both physical and chemical mechanisms were found responsible for exclusion of macromolecules such as proteins and polysaccharides. The potential of these solvents for extracting small molecules from complex solid samples, without interference from large biomolecules, was here evaluated. For this purpose, they were applied to the extraction of 14 priority polycyclic aromatic hydrocarbons (PAHs) from mosses prior to their separation by liquid chromatography and fluorescence detection (LC-FLD). Sample treatment involved the vortex shaking of 200 mg of moss with 200 μL of decanoic acid-based solvent for 5 min, subsequent centrifugation for 8 min and analysis of the extract by LC-FLD using external calibration. Proteins precipitated during extraction because of both the decrease of the dielectric constant of the solution caused by THF and the formation of macromolecular complexes with decanoic acid. Polysaccharides were not solubilized in the aqueous cavities of the solvent because of their size exclusion. In-house method validation was performed according to the recommendations of the European Commission Decision 202/657/EC. Method detection and quantification limits for the different PAHs were in the ranges 0.04-0.24 and 0.14-0.80 μg kg(-1), respectively. The method was applied to the determination of different moss species collected in both polluted and unpolluted sites in the South of Spain. Recoveries were within the range 71-110%. The results obtained show that solvents with restricted access properties have the potential to expand the scope of application of restricted access materials to areas other than biological fluids because of their suitability to combine analyte isolation and sample cleanup of solid samples in a single step. Copyright © 2015 Elsevier B

  1. Impact of restrictive intravenous fluid replacement and combined epidural analgesia on perioperative volume balance and renal function within a Fast Track program.

    PubMed

    Hübner, Martin; Schäfer, Markus; Demartines, Nicolas; Müller, Sven; Maurer, Konrad; Baulig, Werner; Clavien, Pierre A; Zalunardo, Marco P

    2012-03-01

    Key factors of Fast Track (FT) programs are fluid restriction and epidural analgesia (EDA). We aimed to challenge the preconception that the combination of fluid restriction and EDA might induce hypotension and renal dysfunction. A recent randomized trial (NCT00556790) showed reduced complications after colectomy in FT patients compared with standard care (SC). Patients with an effective EDA were compared with regard to hemodynamics and renal function. 61/76 FT patients and 59/75 patients in the SC group had an effective EDA. Both groups were comparable regarding demographics and surgery-related characteristics. FT patients received significantly less i.v. fluids intraoperatively (1900 mL [range 1100-4100] versus 2900 mL [1600-5900], P < 0.0001) and postoperatively (700 mL [400-1500] versus 2300 mL [1800-3800], P < 0.0001). Intraoperatively, 30 FT compared with 19 SC patients needed colloids or vasopressors, but this was statistically not significant (P = 0.066). Postoperative requirements were low in both groups (3 versus 5 patients; P = 0.487). Pre- and postoperative values for creatinine, hematocrit, sodium, and potassium were similar, and no patient developed renal dysfunction in either group. Only one of 82 patients having an EDA without a bladder catheter had urinary retention. Overall, FT patients had fewer postoperative complications (6 versus 20 patients; P = 0.002) and a shorter median hospital stay (5 [2-30] versus 9 d [6-30]; P< 0.0001) compared with the SC group. Fluid restriction and EDA in FT programs are not associated with clinically relevant hemodynamic instability or renal dysfunction. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. An evaluation of methods to test predefined genomic regions for differential methylation in bisulfite sequencing data.

    PubMed

    Klein, Hans-Ulrich; Hebestreit, Katja

    2016-09-01

    In the biology of tissue development and diseases, DNA methylation plays an important role. For a deeper understanding, it is crucial to accurately compare DNA methylation patterns between groups of samples representing different conditions. A widely used method to investigate DNA methylation in the CpG context is bisulfite sequencing, which produces data on the single-nucleotide scale. While there are benefits to analyzing CpG sites on a basepair level, there are both biological and statistical reasons to test entire genomic regions for differential methylation. However, the analysis of DNA methylation is hampered by the lack of best practice standards. Here, we compared multiple approaches for testing predefined genomic regions for differential DNA methylation in bisulfite sequencing data. Nine methods were evaluated: BiSeq, COHCAP, Goeman's Global Test, Limma, methylKit/eDMR, RADMeth and three log-linear regression approaches with different distribution assumptions. We applied these methods to simulated data and determined their sensitivity and specificity. This revealed performance differences, which were also seen when applied to real data. Methods that first test single CpG sites and then test regions based on transformed CpG-wise P-values performed better than methods that summarize methylation levels or raw reads. Interestingly, smoothing of methylation levels had a negligible impact. In particular, Global Test, BiSeq and RADMeth/z-test outperformed the other methods we evaluated, providing valuable guidance for more accurate analysis of DNA methylation. © The Author 2015. Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

  3. Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite.

    PubMed

    An, Dongshan; Dong, Xiaoli; An, Annie; Park, Hyung S; Strous, Marc; Voordouw, Gerrit

    2016-01-01

    Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S(0)) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe(0)). Overall this accelerates reaction of of S(0) and Fe(0) to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function.

  4. Metagenomic Analysis Indicates Epsilonproteobacteria as a Potential Cause of Microbial Corrosion in Pipelines Injected with Bisulfite

    PubMed Central

    An, Dongshan; Dong, Xiaoli; An, Annie; Park, Hyung S.; Strous, Marc; Voordouw, Gerrit

    2016-01-01

    Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S0) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe0). Overall this accelerates reaction of of S0 and Fe0 to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function. PMID:26858705

  5. Metabolic Effects of a 24-Week Energy-Restricted Intervention Combined with Low or High Dairy Intake in Overweight Women: An NMR-Based Metabolomics Investigation

    PubMed Central

    Zheng, Hong; Lorenzen, Janne K.; Astrup, Arne; Larsen, Lesli H.; Yde, Christian C.; Clausen, Morten R.; Bertram, Hanne Christine

    2016-01-01

    We investigated the effect of a 24-week energy-restricted intervention with low or high dairy intake (LD or HD) on the metabolic profiles of urine, blood and feces in overweight/obese women by NMR spectroscopy combined with ANOVA-simultaneous component analysis (ASCA). A significant effect of dairy intake was found on the urine metabolome. HD intake increased urinary citrate, creatinine and urea excretion, and decreased urinary excretion of trimethylamine-N-oxide (TMAO) and hippurate relative to the LD intake, suggesting that HD intake was associated with alterations in protein catabolism, energy metabolism and gut microbial activity. In addition, a significant time effect on the blood metabolome was attributed to a decrease in blood lipid and lipoprotein levels due to the energy restriction. For the fecal metabolome, a trend for a diet effect was found and a series of metabolites, such as acetate, butyrate, propionate, malonate, cholesterol and glycerol tended to be affected. Overall, even though these effects were not accompanied by a higher weight loss, the present metabolomics data reveal that a high dairy intake is associated with endogenous metabolic effects and effects on gut microbial activity that potentially impact body weight regulation and health. Moreover, ASCA has a great potential for exploring the effect of intervention factors and identifying altered metabolites in a multi-factorial metabolomic study. PMID:26907339

  6. Metabolic Effects of a 24-Week Energy-Restricted Intervention Combined with Low or High Dairy Intake in Overweight Women: An NMR-Based Metabolomics Investigation.

    PubMed

    Zheng, Hong; Lorenzen, Janne K; Astrup, Arne; Larsen, Lesli H; Yde, Christian C; Clausen, Morten R; Bertram, Hanne Christine

    2016-02-23

    We investigated the effect of a 24-week energy-restricted intervention with low or high dairy intake (LD or HD) on the metabolic profiles of urine, blood and feces in overweight/obese women by NMR spectroscopy combined with ANOVA-simultaneous component analysis (ASCA). A significant effect of dairy intake was found on the urine metabolome. HD intake increased urinary citrate, creatinine and urea excretion, and decreased urinary excretion of trimethylamine-N-oxide (TMAO) and hippurate relative to the LD intake, suggesting that HD intake was associated with alterations in protein catabolism, energy metabolism and gut microbial activity. In addition, a significant time effect on the blood metabolome was attributed to a decrease in blood lipid and lipoprotein levels due to the energy restriction. For the fecal metabolome, a trend for a diet effect was found and a series of metabolites, such as acetate, butyrate, propionate, malonate, cholesterol and glycerol tended to be affected. Overall, even though these effects were not accompanied by a higher weight loss, the present metabolomics data reveal that a high dairy intake is associated with endogenous metabolic effects and effects on gut microbial activity that potentially impact body weight regulation and health. Moreover, ASCA has a great potential for exploring the effect of intervention factors and identifying altered metabolites in a multi-factorial metabolomic study.

  7. The effects of water-based exercise in combination with blood flow restriction on strength and functional capacity in post-menopausal women.

    PubMed

    Araújo, Joamira P; Neto, Gabriel R; Loenneke, Jeremy P; Bemben, Michael G; Laurentino, Gilberto C; Batista, Gilmário; Silva, Júlio C G; Freitas, Eduardo D S; Sousa, Maria S C

    2015-12-01

    Water-based exercise and low-intensity exercise in combination with blood flow restriction (BFR) are two methods that have independently been shown to improve muscle strength in those of advancing age. The objective of this study was to assess the long-term effect of water-based exercise in combination with BFR on maximum dynamic strength and functional capacity in post-menopausal women. Twenty-eight women underwent an 8-week water-based exercise program. The participants were randomly allocated to one of the three groups: (a) water exercise only, (b) water exercise + BFR, or (c) a non-exercise control group. Functional capacity (chair stand test, timed up and go test, gait speed, and dynamic balance) and strength testing were tested before and after the 8-week aquatic exercise program. The main findings were as follows: (1) water-based exercise in combination with BFR significantly increased the lower limb maximum strength which was not observed with water-based exercise alone and (2) water-based exercise, regardless of the application of BFR, increased functional performance measured by the timed up and go test over a control group. Although we used a healthy population in the current study, these findings may have important implications for those who may be contraindicated to using traditional resistance exercise. Future research should explore this promising modality in these clinical populations.

  8. Intrauterine growth restriction combined with a maternal high-fat diet increases hepatic cholesterol and low-density lipoprotein receptor activity in rats.

    PubMed

    Zinkhan, Erin K; Zalla, Jennifer M; Carpenter, Jeanette R; Yu, Baifeng; Yu, Xing; Chan, Gary; Joss-Moore, Lisa; Lane, Robert H

    2016-07-01

    Intrauterine growth restriction (IUGR) and maternal consumption of a high-saturated-fat diet (HFD) increase the risk of hypercholesterolemia, a leading cause of morbidity and mortality. Many pregnant women eat a HFD, thus exposing the fetus to a HFD in utero. The cumulative effect of in utero exposure to IUGR and a HFD on offspring cholesterol levels remains unknown. Furthermore, little is known about the mechanism through which IUGR and maternal HFD consumption increase cholesterol. We hypothesize that IUGR combined with a maternal HFD would increase offspring serum and hepatic cholesterol accumulation via alteration in levels of key proteins involved in cholesterol metabolism. To test our hypothesis we used a rat model of surgically induced IUGR and fed the dams a regular diet or a HFD HFD-fed dams consumed the same kilocalories as regular diet-fed dams, with no difference between surgical intervention groups. In the offspring, IUGR combined with a maternal HFD increased hepatic cholesterol levels, low-density lipoprotein (LDL) receptor protein levels, and Ldlr activity in female rat offspring at birth and both sexes at postnatal day 14 relative to non-IUGR offspring both from regular diet- and HFD-fed dams. These findings suggest that IUGR combined with a maternal HFD increases hepatic cholesterol accumulation via increased LDL cholesterol uptake into the liver with resulting persistent increases in hepatic cholesterol accumulation.

  9. Effectiveness of the sulfur(IV) compound, sodium bisulfite, in reducing chlorine, chlorine dioxide, and chlorite toxicity to Daphnia magna in well water and pond water.

    PubMed

    Yonkos, L T; Fisher, D J; Burton, D T; Whitekettle, W K; Peterille, J C

    2001-03-01

    Flow-through toxicity tests were conducted with Daphnia magna to determine the residual toxicity of chlorine, chlorine dioxide, and chlorite after treatment with the sulfur(IV) compound sodium bisulfite. Daphnids were exposed separately to 0.5-mg/L concentrations of each of the three compounds without the addition of sodium bisulfite, with a low stoichiometric dose of sodium bisulfite, and with a high stoichiometric dose of sodium bisulfite. Tests were performed in well water with a low total organic carbon (TOC) content and pond water with a high TOC content. Analysis of results indicated that sodium bisulfite did not eliminate the toxicity of chlorine dioxide or chlorite to D. magna. Total residual oxidant (TRO) concentrations were reduced and survival times were extended, but acute toxicity persisted even with a S(IV) concentration 10.0 times the stoichiometric ratio of oxidant. Mortality occurred in chlorine dioxide treatments in which no TRO was detected, indicating that standard analytical (amperometric) techniques may be inadequate to detect toxicity. Sodium bisulfite did succeed in eliminating chlorine toxicity except in pond water receiving a low (3.0x) sodium bisulfite dose. Oxidant reactions with organic substrates may have produced chlorinated residuals that were resistant to S(IV) dechlorination.

  10. Impact of heat stress, nutritional restriction and combined stresses (heat and nutritional) on growth and reproductive performance of Malpura rams under semi-arid tropical environment.

    PubMed

    Maurya, V P; Sejian, V; Kumar, D; Naqvi, S M K

    2016-10-01

    A study was conducted to assess the combined effect of heat stress and nutritional restriction on growth and reproductive performances in Malpura rams. Twenty-eight adult Malpura rams (average body weight (BW) 66.0 kg) were used in this study. The rams were divided into four groups: CON (n = 7; control), HES (n = 7; heat stress), NUS (n = 7; nutritional stress) and COS (n = 7; combined stress). The study was conducted for a period of 2 months. CON and HES rams had ad libitum access to their feed while NUS and COS rams were under restricted feed (30% intake of CON rams) to induce nutritional stress. The HES and COS rams were kept in climatic chamber at 42 °C and 55% relative humidity for 6 h a day between 10 : 00 h and 16 : 00 h to induce heat stress. Body weight increased significantly (p < 0.05) in CON as compared to NUS and COS. When compared within groups, scrotal width morning, scrotal width afternoon, scrotal circumference morning and scrotal circumference afternoon were significantly (p < 0.05) larger in CON while smaller in COS rams. The higher testicular length was recorded both during morning (p < 0.05) and afternoon (p < 0.01) in COS rams while the lowest in NUS rams. The highest plasma testosterone concentration was recorded in CON and lowest in COS rams. Semen volume and mass motility also differed significantly (p < 0.05) between the groups. The highest semen volume and mass motility was recorded in CON and NUS while lowest in both HES and COS rams. It can be concluded from this study that when two stressors occur simultaneously, they may have severe impact on reproductive performance of rams. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

  11. Inhibition of superoxide dismutase, Vitamin C and glutathione on chemiluminescence produced by luminol and the mixture of sulfite and bisulfite

    NASA Astrophysics Data System (ADS)

    Geng, Hong; Meng, Ziqiang

    2006-05-01

    In a system which consisted of luminol (3-aminophthalhydrazide), cobalt sulfate (CoSO 4), alkaline buffer and the mixture of NaSO 3 and sodium bisulfite (NaHSO 3) (sulfite and bisulfite = 3:1, m/m), a strong chemiluminescence (CL) was observed using a BPCL ultra-weak luminometer. The CL signals resulted from 3-aminophthalate (the product of oxidized luminol), and were affected by the buffer pH, buffer medium and the concentrations of luminol, CoSO 4 and the NaSO 3-NaHSO 3 mixture. The observation that the CL intensities were inhibited by superoxide dismutase (SOD), Vitamin C (Vc) and glutathione (GSH) in a dose-dependent manner suggested that superoxide radical (O 2rad -) was involved in the CL reaction and responsible for oxidation of luminol.

  12. (Bi)sulfite Oxidation by Copper,Zinc-Superoxide Dismutase: Sulfite-Derived, Radical-Initiated Protein Radical Formation

    PubMed Central

    Ranguelova, Kalina; Bonini, Marcelo G.; Mason, Ronald P.

    2010-01-01

    Background Sulfur dioxide, formed during the combustion of fossil fuels, is a major air pollutant near large cities. Its two ionized forms in aqueous solution, sulfite and (bi)sulfite, are widely used as preservatives and antioxidants to prevent food and beverage spoilage. (Bi)sulfite can be oxidized by peroxidases to form the very reactive sulfur trioxide anion radical (•SO3−). This free radical further reacts with oxygen to form the peroxymonosulfate anion radical (−O3SOO•) and sulfate anion radical (SO4• −). Objective To explore the critical role of these radical intermediates in further oxidizing biomolecules, we examined the ability of copper,zinc-superoxide dismutase (Cu,Zn-SOD) to initiate this radical chain reaction, using human serum albumin (HSA) as a model target. Methods We used electron paramagnetic resonance, optical spectroscopy, oxygen uptake, and immuno-spin trapping to study the protein oxidations driven by sulfite-derived radicals. Results We found that when Cu,Zn-SOD reacted with (bi)sulfite, •SO3− was produced, with the concomitant reduction of SOD-Cu(II) to SOD-Cu(I). Further, we demonstrated that sulfite oxidation mediated by Cu,Zn-SOD induced the formation of radical-derived 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin-trapped HSA radicals. Conclusions The present study suggests that protein oxidative damage resulting from (bi)sulfite oxidation promoted by Cu,Zn-SOD could be involved in oxidative damage and tissue injury in (bi)sulfite-exacerbated allergic reactions. PMID:20348042

  13. Investigation into length heteroplasmy in the mitochondrial DNA control region after treatment with bisulfite.

    PubMed

    Lee, James Chun-I; Tsai, Li-Chin; Yu, Yu-Jen; Lin, Chun-Yen; Linacre, Adrian; Hsieh, Hsing-Mei

    2016-04-01

    We report on a method to analyze length heteroplasmy within the human mitochondrial genome in which there are polycytosine [poly(C)] stretches. These poly(C) tracts induce heteroplasmy with the resultant inherent problems of accurate sequence designations. In this study, 20 samples that exhibited length heteroplasmy due to variation in the C-tracts within hypervariable region I (HVI) were treated with bisulfite, and one or more cytosine bases in these C-tracts were converted randomly to uracil. This resulted in an accurate sequence designation for nearly all samples. The only exceptions in which the DNA sequence could still not be determined occurred when there was total conversion, or a lack of conversion, of the cytosine bases. Replicate tests on the same samples showed that individual cytosine bases were randomly converted to uracil. This simple method was useful for investigating length heteroplasmy due to 16189C and 310C transitions in the mitochondrial-DNA control region. It is valuable for medical and forensic investigations.

  14. Measuring δ(13)C values of atmospheric acetaldehyde via sodium bisulfite adsorption and cysteamine derivatisation.

    PubMed

    Guo, Songjun; Chen, Mei; Wen, Sheng; Sheng, Guoying; Fu, Jiamo

    2012-01-01

    δ(13)C values of gaseous acetaldehyde were measured by gas chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS) via sodium bisulfite (NaHSO(3)) adsorption and cysteamine derivatisation. Gaseous acetaldehyde was collected via NaHSO(3)-coated Sep-Pak(®) silica gel cartridge, then derivatised with cysteamine, and then the δ(13)C value of the acetaldehyde-cysteamine derivative was measured by GC-C-IRMS. Using two acetaldehydes with different δ(13)C values, derivatisation experiments were carried out to cover concentrations between 0.009×10(-3) and 1.96×10(-3) mg·l(-1)) of atmospheric acetaldehyde, and then δ(13)C fractionation was evaluated in the derivatisation of acetaldehyde based on stoichiometric mass balance after measuring the δ(13)C values of acetaldehyde, cysteamine and the acetaldehyde-cysteamine derivative. δ(13)C measurements in the derivertisation process showed good reproducibility (<0.5 ‰) for gaseous acetaldehyde. The differences between predicted and measured δ(13)C values were 0.04-0.31 ‰ for acetaldehyde-cysteamine derivative, indicating that the derivatisation introduces no isotope fractionation for gaseous acetaldehyde, and obtained δ(13)C values of acetaldehyde in ambient air at the two sites were distinct (-34.00 ‰ at an urban site versus-31.00 ‰ at a forest site), implying potential application of the method to study atmospheric acetaldehyde.

  15. Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations.

    PubMed

    Mattox, Austin K; Wang, Yuxuan; Springer, Simeon; Cohen, Joshua D; Yegnasubramanian, Srinivasan; Nelson, William G; Kinzler, Kenneth W; Vogelstein, Bert; Papadopoulos, Nickolas

    2017-05-02

    The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

  16. Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing.

    PubMed

    Yuan, Xiao-Long; Gao, Ning; Xing, Yan; Zhang, Hai-Bin; Zhang, Ai-Ling; Liu, Jing; He, Jin-Long; Xu, Yuan; Lin, Wen-Mian; Chen, Zan-Mou; Zhang, Hao; Zhang, Zhe; Li, Jia-Qi

    2016-02-25

    Substantial evidence has shown that DNA methylation regulates the initiation of ovarian and sexual maturation. Here, we investigated the genome-wide profile of DNA methylation in porcine ovaries at single-base resolution using reduced representation bisulfite sequencing. The biological variation was minimal among the three ovarian replicates. We found hypermethylation frequently occurred in regions with low gene abundance, while hypomethylation in regions with high gene abundance. The DNA methylation around transcriptional start sites was negatively correlated with their own CpG content. Additionally, the methylation level in the bodies of genes was higher than that in their 5' and 3' flanking regions. The DNA methylation pattern of the low CpG content promoter genes differed obviously from that of the high CpG content promoter genes. The DNA methylation level of the porcine ovary was higher than that of the porcine intestine. Analyses of the genome-wide DNA methylation in porcine ovaries would advance the knowledge and understanding of the porcine ovarian methylome.

  17. Effects of sodium bisulfite with or without procaine derivatives on axons of cultured mouse dorsal root ganglion neurons.

    PubMed

    Takenami, Tamie; Hiruma, Hiromi; Kaneko, Haruka; Okamoto, Hirotsugu; Kawakami, Tadashi

    2015-01-01

    Sodium bisulfite (NaHSO3) was clinically used as a preservative agent for local anesthetics but was later suspected to be neurotoxic. However, recent studies reported that NaHSO3 reduces the neurotoxicity of local anesthetics. The purpose of this study was to examine the effects of NaHSO3 with and without procaine on axonal transport in cultured mouse dorsal root ganglion (DRG) neurons. Experiment 1 served to determine the dose-dependent effects of NaHSO3 on axonal transport (DRG neurons were treated with 0.01, 0.1, 1, 10, or 20 mM of NaHSO3), whereas experiment 2 investigated the effect of 0.1 mM NaHSO3 on the action of local anesthetics on axonal transport (DRG neurons were treated with 1 mM procaine alone, or with 0.1 mM NaHSO3 plus 1 mM procaine). As an additional experiment, DRG neurons were also treated with 1 mM chloroprocaine alone, or with 0.1 mM NaHSO3 plus 1 mM chloroprocaine. In these experiments, we analyzed the percent change in the number of anterogradely and retrogradely transported organelles and recorded changes in neurite morphology using video-enhanced microscopy. In experiment 1, NaHSO3 at more than 1 mM caused cell membrane damage and complete inhibition of axonal transport, whereas 0.1 mM NaHSO3 maintained axonal transport at 40% to 60% of control with intact cell membrane. In experiment 2, 1 mM procaine alone maintained axonal transport at 90% to 100%. However, application of 1 mM procaine-0.1 mM NaHSO3 solution resulted in deformation of neurites and with complete cessation of axonal transport. Likewise, although 1 mM chloroprocaine maintain axonal transport at 80% to 100%, 1 mM chloroprocaine-0.1 mM NaHSO3 arrested axonal transport. NaHSO3 resulted in a dose-dependent damage to the cell membrane and axonal transport, especially when used in combination with procaine or chloroprocaine.

  18. Identification of quantitative trait loci for flowering time by a combination of restriction site–associated DNA sequencing and bulked segregant analysis in soybean

    PubMed Central

    Watanabe, Satoshi; Tsukamoto, Chikaharu; Oshita, Tatsuki; Yamada, Tetsuya; Anai, Toyoaki; Kaga, Akito

    2017-01-01

    Soybean (Glycine max) has a paleopolyploid genome, and many re-sequencing experiments to characterize soybean genotypes have been conducted using next-generation sequencing platforms. The accumulation of information about single nucleotide polymorphisms (SNPs) throughout the soybean genome has accelerated identification of genomic regions related to agronomically important traits through association studies. However, although many efficient mapping techniques that use next-generation sequencing are available, the number of practical approaches to identify genes/loci is still limited. In this study, we used a combination of restriction site–associated DNA sequencing (RAD-seq) and bulk segregant analysis (BSA) to identify quantitative trait locus (QTLs) for flowering time in a segregating population derived from a cross between Japanese soybean cultivars. Despite the homogeneous genetic background of the parents, over 7000 SNPs were identified and can be used to detect QTLs by RAD-seq BSA analysis. By comparing genotype frequency between early and late-flowering bulks from the F3 segregating population, we identified a QTL on Gm10, which corresponds to the previously identified E2 locus, and a QTL on Gm04, which is close to the E8 locus. Out of these SNPs, more than 2000 were easily converted to conventional DNA markers. Our approach would improve the efficiency of genetic mapping. PMID:28744181

  19. Effects of low-intensity concentric and eccentric exercise combined with blood flow restriction on indices of exercise-induced muscle damage

    PubMed Central

    Yasuda, Tomohiro; Loenneke, Jeremy P.; Abe, Takashi

    2013-01-01

    Low-intensity blood-flow restriction (BFR) resistance training significantly increases strength and muscle size, but some studies report it produces exercise-induced muscle damage (EIMD) in the lower body after exercise to failure. Purpose: To investigate the effects of a pre-set number of repetitions of upper body concentric and eccentric exercise when combined with BFR on changes in EIMD. Methods: Ten young men had arms randomly assigned to either concentric BFR (CON-BFR) or eccentric BFR (ECC-BFR) dumbbell curl exercise (30% one-repetition maximum (1-RM), 1 set of 30 repetitions followed by 3 sets of 15 repetitions). Maximal isometric voluntary contraction force (MVC), muscle thickness (MTH), circumference, range of motion (ROM), ratings of perceived exertion (RPE), and muscle soreness were measured before, immediately after, and daily for 4 days post-exercise. Results: MVC decreased by 36% for CON-BFR and 12% for ECC-BFR immediately after exercise but was not changed 1–4 days post-exercise (p > 0.05). Only CON-BFR had significant changes in MTH and circumference immediately after exercise (p < 0.05). Muscle soreness was observed in the ECC-BFR arm at 1 and 2 days after exercise. Conclusions: Low-intensity ECC-BFR produces significant muscle soreness at 24 h but neither ECC-BFR nor CON-BFR exercise produces significant changes in multiple indices of EIMD. PMID:24265891

  20. Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus.

    PubMed

    Raue, R; Hess, M

    1998-08-01

    Three different polymerase chain reactions (PCRs), two of them combined with restriction enzyme analysis (REA), were developed for detection and differentiation of all 12 fowl adenovirus (FAV) serotypes and the egg drop syndrome (EDS) virus. For primer construction FAV1, FAV10 and EDS virus hexon proteins were aligned and conserved and variable regions were determined. Two primer sets (H1/H2 and H3/H4) for single use were constructed which hybridize in three conserved regions of hexon genes. Each primer pair amplifies approximately half of the hexon gene including two loop regions. An amplification product was detected with both primer sets using purified DNA from all FAV1-12 reference strains. Viral EDS DNA was negative using the H1/H2 or H3/H4 primer pair. HaeII digestion of the H1/H2 amplification products differentiates between all viruses except FAV4 and FAV5. In comparison, much more clustering among genomic closely related FAV serotypes was seen after HpaII digestion of the H3/H4 PCR products. Oligonucleotides H5/H6 located in the variable regions of EDS virus hexon gene do not detect any of the FAV serotypes. The PCRs and REA described are suitable to detect all avian adenoviruses infecting chickens, to distinguish all 12 FAV reference strains and to differentiate FAVs from the EDS virus.

  1. Effects of low-load resistance training combined with blood flow restriction or hypoxia on muscle function and performance in netball athletes.

    PubMed

    Manimmanakorn, Apiwan; Hamlin, Michael J; Ross, Jenny J; Taylor, Robert; Manimmanakorn, Nuttaset

    2013-07-01

    To investigate the effect of blood flow restriction or normobaric hypoxic exposure combined with low-load resistant exercise (LRE), on muscular strength and endurance. A randomised controlled trial. Well-trained netball players (n=30) took part in a 5 weeks training of knee flexor and extensor muscles in which LRE (20% of one repetition maximum) was combined with (1) an occlusion pressure of approximately 230mmHg around the upper thigh (KT, n=10), (2) hypoxic air to generate blood oxyhaemoglobin levels of approximately 80% (HT, n=10) or (3) with no additional stimulus (CT, n=10). The training was of the same intensity and amount in all groups. One to five days before and after training, participants performed a series of strength and endurance tests of the lower limbs (3-s maximal voluntary contraction [MVC3], area under 30-s force curve [MVC30], number of repetitions at 20% 1RM [Reps201RM]). In addition, the cross-sectional area (CSA) of the quadriceps and hamstrings were measured. Relative to CT, KT and HT increased MVC3 (11.0±11.9% and 15.0±13.1%), MVC30 (10.2±9.0% and 18.3±17.4%) and Reps201RM (28.9±23.7% and 23.3±24.0%, mean±90% confidence interval) after training. CSA increased by 6.6±4.5%, 6.1±5.1% and 2.9±2.7% in the KT, HT and CT groups respectively. LRE in conjunction with KT or HT can provide substantial improvements in muscle strength and endurance and may be useful alternatives to traditional training practices. Copyright © 2012 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

  2. A single nucleotide polymorphism chip-based method for combined genetic and epigenetic profiling: validation in decitabine therapy and tumor/normal comparisons.

    PubMed

    Yuan, Eric; Haghighi, Fatemeh; White, Susan; Costa, Ramiro; McMinn, Julie; Chun, Kathy; Minden, Mark; Tycko, Benjamin

    2006-04-01

    Progress on several unresolved issues in cancer epigenetics will benefit from rapid and standardized methods for profiling DNA methylation genome-wide. In the area of epigenetic therapy, the demethylating drug decitabine (5-aza-2'-deoxycytidine) is increasingly used to treat acute myelogenous leukemia and myelodysplastic syndrome, but the mechanisms of its anticancer activity have remained unclear. Given the clinical efficacy of decitabine and the uncertainties about its mode of action, it will be useful to optimize methods for following DNA methylation as a biochemical response in individual patients. Here, we describe a single nucleotide polymorphism (SNP) chip-based method (MSNP) for profiling DNA methylation. Using this procedure, the extent of demethylation in bone marrow aspirates from patients with leukemia receiving decitabine can be assessed genome-wide using commercially available (Affymetrix) SNP chips. We validated the accuracy of MSNP by comparing the results with combined bisulfite restriction analysis and by sequencing cloned PCR products from bisulfite-converted DNA. We further validated MSNP in a Wilms' tumor/normal kidney comparison, comparing the results with methylation-sensitive Southern blotting. MSNP simultaneously detects aberrations in DNA copy number and loss of heterozygosity, making it a generally useful approach for combined genetic and epigenetic profiling in tissue samples from cancer patients.

  3. Prediction of fetal growth restriction using estimated fetal weight versus a combined screening model at 32-36 weeks of gestation.

    PubMed

    Miranda, Jezid; Rodriguez-Lopez, Merida; Triunfo, Stefania; Sairanen, Mikko; Kouru, Heikki; Parra-Saavedra, Miguel; Crovetto, Francesca; Figueras, Francesc; Crispi, Fatima; Gratacos, Eduard

    2016-12-22

    To compare the performance of a third trimester screening based on estimated fetal weight (EFW) versus a combined model composed by maternal baseline characteristics, mean arterial pressure (MAP), ultrasound and biochemical markers for the prediction of smallness for gestational age (SGA) and late-onset fetal growth restriction (FGR). Nested case-control study within a prospective cohort of 1,590 singleton gestations referred for third-trimester evaluation (32-36 weeks of gestation). Maternal baseline characteristics, MAP, feto-placental ultrasound and circulating biochemical markers [placental growth factor (PlGF), lipocalin-2, unconjugated estriol, and inhibin A] were assessed in all women who subsequently delivered a SGA neonate defined as a birth weight (BW) <10(th) centile by customized standards (n = 175) and in a control group (n = 875). Among SGA cases, those with a BW <3rd centile and/or abnormal uterine artery (UtA) Doppler and/or abnormal cerebroplacental ratio (CPR) were classified as FGR. Logistic regression predictive models were developed for SGA and FGR, and their performance was compared to that obtained using EFWc alone. EFW, CPR and maternal serum concentrations of unconjugated estriol and PlGF were significantly lower in SGA cases than in controls, while mean UtA-PI values and lipocalin-2 concentrations were significantly higher. Using EFWc alone, 52% (AUC 0.82, 95% CI 0.77-0.85) and 64% (AUC 0.86, 95% CI 0.81-0.91) of SGA and FGR cases were predicted at a 10% false positive rate, respectively. A combined screening model including a priori risk (maternal characteristics), EFW, UtA Doppler, PlGF, and unconjugated estriol, achieves a detection rate of 61% (AUC 0.86, 95% CI 0.83-0.89) for SGA cases and 77% (AUC 0.92, 95% CI 0.88-0.95) for FGR. The detection rate of a combined model for the prediction of SGA and FGR performs significantly better than that obtained using EFW alone (p < 0.01 and p = 0.002, respectively). A

  4. Gastroprotective effect of andrographolide sodium bisulfite against indomethacin-induced gastric ulceration in rats.

    PubMed

    Liu, Yu-Hong; Zhang, Zhen-Biao; Zheng, Yi-Feng; Chen, Hai-Ming; Yu, Xiu-Ting; Chen, Xiao-Ying; Zhang, Xie; Xie, Jian-Hui; Su, Zu-Qing; Feng, Xue-Xuan; Zeng, Hui-Fang; Su, Zi-Ren

    2015-06-01

    Andrographolide sodium bisulfite (ASB), a water-soluble sulfonate of andrographolide has been shown to possess anti-inflammatory, antipyretic and analgesic activities. However, there is no report on the gastroprotective effect of ASB against indomethacin-induced gastric ulcer. Here we investigated the possible anti-ulcerogenic potential of ASB and the underlying mechanism against indomethacin-induced gastric ulcer in rats. The ulcer area, histopathological assessment, contents of gastric mucosal glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malonaldehyde (MDA) and prostaglandin E2 (PGE2) were examined. In addition, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) mRNA expression and immunohistochemical evaluation of HSP70, Bcl-2 and Bax proteins were also investigated. Results indicated that ASB pre-treatment significantly reduced the ulcer areas induced by indomethacin compared with the vehicle group. The gastric levels of GSH, CAT and SOD were markedly increased by ASB while the level of MDA was decreased. In addition, ASB pretreatment significantly promoted the gastric PGE2 levels and up-regulated the COX-1 and COX-2 mRNA expression in comparison with the vehicle group. Immunohistochemistry analysis showed obvious up-regulation of HSP70 and Bcl-2 protein expression while suppression of Bax protein in the gastric tissue of ASB-pretreated group. Taken together, these findings indicated that the gastroprotective effect of ASB might be associated with the improvement of antioxidative status, activation of COX-mediated PGE2 synthesis, down-regulation of Bax proteins and up-regulation of Bcl-2 and HSP70 proteins. ASB might have the potential for further development as a promising alternative for antiulcer treatment.

  5. Comparative analysis of 12 different kits for bisulfite conversion of circulating cell-free DNA.

    PubMed

    Worm Ørntoft, Mai-Britt; Jensen, Sarah Østrup; Hansen, Thomas Birkballe; Bramsen, Jesper Bertram; Andersen, Claus Lindbjerg

    2017-05-30

    Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. To compare cfDNA recovery of different BSC methods, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only 3 kits recovered DNA fragments of <100 bp well. In contrast, DNA input amount did not seem to affect DNA recovery: for quantities spanning between 820 and ∼25,000 genome equivalents per BSC, a linear relation was found between input and recovery amount. Overall, mean recovery ranged between 9 and 32%, with BSC efficiency of 97-99.9%. When plasma cfDNA was used as input for BSC, recovery varied from 22% for the poorest and 66% for the best performing kits, while conversion efficiency ranged from 96 to 100% among different kits. In conclusion, clear performance differences exist between commercially available BSC kits, both in terms of DNA recovery and conversion efficiency. The choice of BSC kit can substantially impact the amount of converted cfDNA available for downstream analysis, which is critical in a cfDNA methylation marker setting.

  6. Restrictive cardiomyopathies.

    PubMed

    Nihoyannopoulos, Petros; Dawson, David

    2009-12-01

    Restrictive cardiomyopathies constitute a heterogenous group of heart muscle conditions that all have, in common, the symptoms of heart failure. Diastolic dysfunction with preserved systolic function is often the only echocardiographic abnormality that may be noted, although systolic dysfunction may also be an integral part of some specific pathologies, particularly in the most advanced cases such as amyloid infiltration of the heart. By far, the majority of restrictive cardiomyopathies are secondary to a systemic disorder such as amyloidosis, sarcoidosis, scleroderma, haemochromatosis, eosinophilic heart disease, or as a result of radiation treatment. The much more rare diagnosis of idiopathic restrictive cardiomyopathy is supported only by the absence of specific pathology on either endomyocardial biopsies or at post-mortem. Restrictive cardiomyopathy is diagnosed based on medical history, physical examination, and tests: such as blood tests, electrocardiogram, chest X-ray, echocardiography, and magnetic resonance imaging. With its wide availability, echocardiography is probably the most important investigation to identify the left ventricular dysfunction and should be performed early and by groups that are familiar with the wide variety of aetiologies. Finally, on rare occasions, the differential diagnosis from constrictive pericarditis may be necessary.

  7. Effect of sodium bisulfite injection on the microbial community composition in a brackish-water-transporting pipeline.

    PubMed

    Park, Hyung Soo; Chatterjee, Indranil; Dong, Xiaoli; Wang, Sheng-Hung; Sensen, Christoph W; Caffrey, Sean M; Jack, Thomas R; Boivin, Joe; Voordouw, Gerrit

    2011-10-01

    Pipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) of Pseudomonas not found in sessile pipe samples. The sessile population of PAS-616P had methanogens (Methanobacteriaceae) as the main (56%) community component, whereas Deltaproteobacteria of the genera Desulfomicrobium and Desulfocapsa were not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) of Methanobacteriaceae archaea but increased fractions of sulfate-reducing Desulfomicrobium (18% and 48%) and of bisulfite-disproportionating Desulfocapsa (35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters.

  8. Effect of Sodium Bisulfite Injection on the Microbial Community Composition in a Brackish-Water-Transporting Pipeline▿†

    PubMed Central

    Park, Hyung Soo; Chatterjee, Indranil; Dong, Xiaoli; Wang, Sheng-Hung; Sensen, Christoph W.; Caffrey, Sean M.; Jack, Thomas R.; Boivin, Joe; Voordouw, Gerrit

    2011-01-01

    Pipelines transporting brackish subsurface water, used in the production of bitumen by steam-assisted gravity drainage, are subject to frequent corrosion failures despite the addition of the oxygen scavenger sodium bisulfite (SBS). Pyrosequencing of 16S rRNA genes was used to determine the microbial community composition for planktonic samples of transported water and for sessile samples of pipe-associated solids (PAS) scraped from pipeline cutouts representing corrosion failures. These were obtained from upstream (PAS-616P) and downstream (PAS-821TP and PAS-821LP, collected under rapid-flow and stagnant conditions, respectively) of the SBS injection point. Most transported water samples had a large fraction (1.8% to 97% of pyrosequencing reads) of Pseudomonas not found in sessile pipe samples. The sessile population of PAS-616P had methanogens (Methanobacteriaceae) as the main (56%) community component, whereas Deltaproteobacteria of the genera Desulfomicrobium and Desulfocapsa were not detected. In contrast, PAS-821TP and PAS-821LP had lower fractions (41% and 0.6%) of Methanobacteriaceae archaea but increased fractions of sulfate-reducing Desulfomicrobium (18% and 48%) and of bisulfite-disproportionating Desulfocapsa (35% and 22%) bacteria. Hence, SBS injection strongly changed the sessile microbial community populations. X-ray diffraction analysis of pipeline scale indicated that iron carbonate was present both upstream and downstream, whereas iron sulfide and sulfur were found only downstream of the SBS injection point, suggesting a contribution of the bisulfite-disproportionating and sulfate-reducing bacteria in the scale to iron corrosion. Incubation of iron coupons with pipeline waters indicated iron corrosion coupled to the formation of methane. Hence, both methanogenic and sulfidogenic microbial communities contributed to corrosion of pipelines transporting these brackish waters. PMID:21856836

  9. Detection of differentially methylated regions in whole genome bisulfite sequencing data using local Getis-Ord statistics.

    PubMed

    Wen, Yalu; Chen, Fushun; Zhang, Qingzheng; Zhuang, Yan; Li, Zhiguang

    2016-11-15

    DNA methylation is an important epigenetic modification that has essential role in gene regulation, cell differentiation and cancer development. Bisulfite sequencing is a widely used technique to obtain genome-wide DNA methylation profiles, and one of the key tasks of analyzing bisulfite sequencing data is to detect differentially methylated regions (DMRs) among samples under different treatment conditions. Although numerous tools have been proposed to detect differentially methylated single CpG site (DMC) between samples, methods for direct DMR detection, especially for complex study designs, are largely limited. We present a new software, GetisDMR, for direct DMR detection. We use beta-binomial regression to model the whole-genome bisulfite sequencing data, where variations in methylation levels and confounding effects have been accounted for. We employ a region-wise test statistic, which is derived from local Getis-Ord statistics and considers the spatial correlation between nearby CpG sites, to detect DMRs. Unlike existing methods, that attempt to infer DMRs from DMCs based on empirical criteria, we provide statistical inference for direct DMR detection. Through extensive simulations and an application to two mouse datasets, we demonstrate that GetisDMR achieves better sensitivities, positive predictive values, more exact locations and better agreement of DMRs with current biological knowledge. It is available at https://github.com/DMU-lilab/GetisDMR CONTACTS: y.wen@auckland.ac.nz or zhiguangli@dlmedu.edu.cnSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. epiG: statistical inference and profiling of DNA methylation from whole-genome bisulfite sequencing data.

    PubMed

    Vincent, Martin; Mundbjerg, Kamilla; Skou Pedersen, Jakob; Liang, Gangning; Jones, Peter A; Ørntoft, Torben Falck; Dalsgaard Sørensen, Karina; Wiuf, Carsten

    2017-02-21

    The study of epigenetic heterogeneity at the level of individual cells and in whole populations is the key to understanding cellular differentiation, organismal development, and the evolution of cancer. We develop a statistical method, epiG, to infer and differentiate between different epi-allelic haplotypes, annotated with CpG methylation status and DNA polymorphisms, from whole-genome bisulfite sequencing data, and nucleosome occupancy from NOMe-seq data. We demonstrate the capabilities of the method by inferring allele-specific methylation and nucleosome occupancy in cell lines, and colon and tumor samples, and by benchmarking the method against independent experimental data.

  11. A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene for the detection of bisulfite anions and its practical application

    NASA Astrophysics Data System (ADS)

    Chao, Jianbin; Liu, Yuhong; Zhang, Yan; Zhang, Yongbin; Huo, Fangjun; Yin, Caixia; Wang, Yu; Qin, Liping

    2015-07-01

    A new fluorescent enhanced probe based on (E)-9-(2-nitrovinyl)-anthracene is developed, which shows high selectivity and sensitivity for the detection of bisulfite anions at Na2HPO4 citric acid buffer solutions (pH 5.0). When addition of HSO3-, the fluorescence intensity is significantly enhanced and the probe displays apparent fluorescence color changes from non-fluorescence to blue under a UV lamp illumination, the solution color also changes from yellow to colorless. The detection limit is determined to be as low as 6.30 μM. This offers another specific colorimetric and fluorescent probe for bisulfite anions detection, furthermore it is applied in detecting the level of bisulfite in sugar samples.

  12. Bisulfite sequencing of chromatin immunoprecipitated DNA (BisChIP-seq) directly informs methylation status of histone-modified DNA

    PubMed Central

    Statham, Aaron L.; Robinson, Mark D.; Song, Jenny Z.; Coolen, Marcel W.; Stirzaker, Clare; Clark, Susan J.

    2012-01-01

    The complex relationship between DNA methylation, chromatin modification, and underlying DNA sequence is often difficult to unravel with existing technologies. Here, we describe a novel technique based on high-throughput sequencing of bisulfite-treated chromatin immunoprecipitated DNA (BisChIP-seq), which can directly interrogate genetic and epigenetic processes that occur in normal and diseased cells. Unlike most previous reports based on correlative techniques, we found using direct bisulfite sequencing of Polycomb H3K27me3-enriched DNA from normal and prostate cancer cells that DNA methylation and H3K27me3-marked histones are not always mutually exclusive, but can co-occur in a genomic region-dependent manner. Notably, in cancer, the co-dependency of marks is largely redistributed with an increase of the dual repressive marks at CpG islands and transcription start sites of silent genes. In contrast, there is a loss of DNA methylation in intergenic H3K27me3-marked regions. Allele-specific methylation status derived from the BisChIP-seq data clearly showed that both methylated and unmethylated alleles can simultaneously be associated with H3K27me3 histones, highlighting that DNA methylation status in these regions is not dependent on Polycomb chromatin status. BisChIP-seq is a novel approach that can be widely applied to directly interrogate the genomic relationship between allele-specific DNA methylation, histone modification, or other important epigenetic regulators. PMID:22466171

  13. A Combination of Activation and Repression by a Colinear Hox Code Controls Forelimb-Restricted Expression of Tbx5 and Reveals Hox Protein Specificity

    PubMed Central

    Nishimoto, Satoko; Minguillon, Carolina; Wood, Sophie; Logan, Malcolm P. O.

    2014-01-01

    Tight control over gene expression is essential for precision in embryonic development and acquisition of the regulatory elements responsible is the predominant driver for evolution of new structures. Tbx5 and Tbx4, two genes expressed in forelimb and hindlimb-forming regions respectively, play crucial roles in the initiation of limb outgrowth. Evolution of regulatory elements that activate Tbx5 in rostral LPM was essential for the acquisition of forelimbs in vertebrates. We identified such a regulatory element for Tbx5 and demonstrated Hox genes are essential, direct regulators. While the importance of Hox genes in regulating embryonic development is clear, Hox targets and the ways in which each protein executes its specific function are not known. We reveal how nested Hox expression along the rostro-caudal axis restricts Tbx5 expression to forelimb. We demonstrate that Hoxc9, which is expressed in caudal LPM where Tbx5 is not expressed, can form a repressive complex on the Tbx5 forelimb regulatory element. This repressive capacity is limited to Hox proteins expressed in caudal LPM and carried out by two separate protein domains in Hoxc9. Forelimb-restricted expression of Tbx5 and ultimately forelimb formation is therefore achieved through co-option of two characteristics of Hox genes; their colinear expression along the body axis and the functional specificity of different paralogs. Active complexes can be formed by Hox PG proteins present throughout the rostral-caudal LPM while restriction of Tbx5 expression is achieved by superimposing a dominant repressive (Hoxc9) complex that determines the caudal boundary of Tbx5 expression. Our results reveal the regulatory mechanism that ensures emergence of the forelimbs at the correct position along the body. Acquisition of this regulatory element would have been critical for the evolution of limbs in vertebrates and modulation of the factors we have identified can be molecular drivers of the diversity in limb morphology

  14. Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.

    PubMed

    Hajj, Nady El; Kuhtz, Juliane; Haaf, Thomas

    2015-01-01

    Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to circumvent these problems. The bisulfite-treated DNA of a single or a few cells is diluted to an extent, that only a single DNA target molecule is present in the reaction. Then each individual DNA molecule in the starting sample is separately amplified and analyzed by Pyrosequencing. This allows the detection of rare alleles that are easily masked when pools of DNA target molecules are analyzed. Amplicons containing a heterozygous single nucleotide polymorphism (SNP) allow one to delineate the parental origin of the recovered molecules in addition to their methylation status. The number of cells (DNA target molecules) in the starting sample determines the dilution level and the number of reactions that have to be performed. LD-BSP allows methylation analysis of small cell pools (i.e., 5-10 microdissected cells) and even individual cells. The primers and PCR conditions described here have been successfully employed to analyze the methylation status of up to eight target genes in individual 2-16 cell embryos, germinal vesicle (GV) oocytes, and haploid sperms.

  15. Validation of a Stability-Indicating Hydrophilic Interaction Liquid Chromatographic Method for the Quantitative Determination of Vitamin K3 (Menadione Sodium Bisulfite) in Injectable Solution Formulation

    PubMed Central

    Ghanem, Mashhour M.; Abu-Lafi, Saleh A.; Hallak, Hussein O.

    2013-01-01

    A simple, specific, accurate, and stability-indicating method was developed and validated for the quantitative determination of menadione sodium bisulfite in the injectable solution formulation. The method is based on zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled with a photodiode array detector. The desired separation was achieved on the ZIC-HILIC column (250 mm × 4.6 mm, 5 μm) at 25°C temperature. The optimized mobile phase consisted of an isocratic solvent mixture of 200mM ammonium acetate (NH4AC) solution and acetonitrile (ACN) (20:80; v/v) pH-adjusted to 5.7 by glacial acetic acid. The mobile phase was fixed at 0.5 ml/min and the analytes were monitored at 261 nm using a photodiode array detector. The effects of the chromatographic conditions on the peak retention, peak USP tailing factor, and column efficiency were systematically optimized. Forced degradation experiments were carried out by exposing menadione sodium bisulfite standard and the injectable solution formulation to thermal, photolytic, oxidative, and acid-base hydrolytic stress conditions. The degradation products were well-resolved from the main peak and the excipients, thus proving that the method is a reliable, stability-indicating tool. The method was validated as per ICH and USP guidelines (USP34/NF29) and found to be adequate for the routine quantitative estimation of menadione sodium bisulfite in commercially available menadione sodium bisulfite injectable solution dosage forms. PMID:24106670

  16. Modeling the production of sugar and byproducts from acid bisulfite pretreatment and enzymatic hydrolysis of Douglas-fir.

    PubMed

    Liu, Yalan; Wang, Jinwu; Wolcott, Michael

    2017-01-01

    The aim of this work was to investigate the kinetics of multiple chemicals in acid bisulfite pretreatment and the relationship between total sugar yields and pretreatment factors (temperature and time). The results showed Saeman model accurately fitted the pretreatment process. According to this kinetic model, a maximum hemicellulose hydrolysis yield was achieved at a treatment time of 75min with a temperature of 145°C. Meantime, the concentrations of acetic acid, hydroxymethylfurfural (HMF), and furfural were 1.54, 0.60, and 1.15gL(-1), respectively. Also, a Lorentzian function described the relationship between total sugar yield and pretreatment factors: temperature and time. The regression parameters from this mathematical fitting have accurately reflected the maximum total sugar yield and the optimal treatment conditions were determined to be 145°C and 110min.

  17. Sensitive Detection of Polyalanine Expansions in PHOX2B by Polymerase Chain Reaction Using Bisulfite-Converted DNA

    PubMed Central

    Horiuchi, Hidekazu; Sasaki, Ayako; Osawa, Motoki; Kijima, Kazuki; Ino, Yukiko; Matoba, Ryoji; Hayasaka, Kiyoshi

    2005-01-01

    Congenital central hypoventilation syndrome, also known as Ondine’s curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts. PMID:16258163

  18. Effects of sodium meta bisulfite on diffusion fermentation of fodder beets for fuel ethanol production. [Saccharomyces cerevisiae

    SciTech Connect

    Gibbons, W.R.; Westby, C.A.

    1987-01-01

    The authors designed and tested a new process for converting fodder beets to ethanol: continuous diffusion-fermentation. This process utilizes the simultaneous diffusion-fermentation concept of the EX-FERM design; however, it overcomes the material handling problems inherent in that system by utilizing a counterflow tubular auger system. This process also eliminates the need for roller mills or presses and dryers which are required for alcohol recovery from solid phase fermentation. The latter is the only other currently feasible procedure for producing distillably worthwhile amounts of ethanol from fodder beets, sweet sorghum, and other similar feedstocks. Results on the use of sodium meta bisulfite (SMB) for contamination control with fermenting fodder beet cubes are reported.

  19. Clustering of Cancer Cell Lines Using A Promoter- Targeted Liquid Hybridization Capture-Based Bisulfite Sequencing Approach.

    PubMed

    Gao, Fei; Wang, Junwen; Ji, Guanyu; Liu, Siyang; Yao, Yu; Wang, Tong; Wu, Honglong; Xia, Yudong; Gong, Desheng; Jiang, Hui; Yang, Huanming; Zhang, Xiuqing

    2015-08-01

    DNA methylation plays a significant role in assuring cell identity, thus potentiating its application in molecular classification of cancers in respect to tissue-origins or clinically and etiologically distinct subtypes. In this study, we optimized our liquid hybridization capture-based bisulfite sequencing (LHC-BS) approach on the gene promoter regions of 11 cell lines. Our results indicated that promoter methylomes could not only cluster cancer cell lines with respect to tissue origins but also differentiate cancer subtypes based on CpG island methylator phenotype (CIMP). Promoter-targeted LHC-BS as means for comprehensive screening and classifying cancer cells with promoter methylomes provided a powerful strategy for further complex clinical studies. © The Author(s) 2014.

  20. Combination of Recreational Soccer and Caloric Restricted Diet Reduces Markers of Protein Catabolism and Cardiovascular Risk in Patients with Type 2 Diabetes.

    PubMed

    Vieira de Sousa, M; Fukui, R; Krustrup, P; Dagogo-Jack, S; Rossi da Silva, M E

    2017-01-01

    Moderate calorie-restricted diets and exercise training prevent loss of lean mass and cardiovascular risk. Because adherence to routine exercise recommendation is generally poor, we utilized recreational soccer training as a novel therapeutic exercise intervention in type 2 diabetes (T2D) patients. We compared the effects of acute and chronic soccer training plus calorie-restricted diet on protein catabolism and cardiovascular risk markers in T2D. Fifty-one T2D patients (61.1±6.4 years, 29 females: 22 males) were randomly allocated to the soccer+diet-group (SDG) or to the diet-group (DG). The 40-min soccer sessions were held 3 times per week for 12 weeks. Nineteen participants attended 100% of scheduled soccer sessions, and none suffered any injuries. The SDG group showed higher levels of growth hormone (GH), free fatty acids and ammonia compared with DG. After 12 weeks, insulin-like growth factor binding protein (IGFPB)-3 and glucose levels were lower in SDG, whereas insulin-like growth factor (IGF)-1/ IGFBP-3 ratio increased in both groups. After the last training session, an increase in IGF-1/IGFBP-3 and attenuation in ammonia levels were suggestive of lower muscle protein catabolism. Recreational soccer training was popular and safe, and was associated with decreased plasma glucose and IGFBP-3 levels, decreased ammoniagenesis, and increased lipolytic activity and IGF-1/IGFBP-3 ratio, all indicative of attenuated catabolism.

  1. Population whole-genome bisulfite sequencing across two tissues highlights the environment as the principal source of human methylome variation.

    PubMed

    Busche, Stephan; Shao, Xiaojian; Caron, Maxime; Kwan, Tony; Allum, Fiona; Cheung, Warren A; Ge, Bing; Westfall, Susan; Simon, Marie-Michelle; Barrett, Amy; Bell, Jordana T; McCarthy, Mark I; Deloukas, Panos; Blanchette, Mathieu; Bourque, Guillaume; Spector, Timothy D; Lathrop, Mark; Pastinen, Tomi; Grundberg, Elin

    2015-12-23

    CpG methylation variation is involved in human trait formation and disease susceptibility. Analyses within populations have been biased towards CpG-dense regions through the application of targeted arrays. We generate whole-genome bisulfite sequencing data for approximately 30 adipose and blood samples from monozygotic and dizygotic twins for the characterization of non-genetic and genetic effects at single-site resolution. Purely invariable CpGs display a bimodal distribution with enrichment of unmethylated CpGs and depletion of fully methylated CpGs in promoter and enhancer regions. Population-variable CpGs account for approximately 15-20 % of total CpGs per tissue, are enriched in enhancer-associated regions and depleted in promoters, and single nucleotide polymorphisms at CpGs are a frequent confounder of extreme methylation variation. Differential methylation is primarily non-genetic in origin, with non-shared environment accounting for most of the variance. These non-genetic effects are mainly tissue-specific. Tobacco smoking is associated with differential methylation in blood with no evidence of this exposure impacting cell counts. Opposite to non-genetic effects, genetic effects of CpG methylation are shared across tissues and thus limit inter-tissue epigenetic drift. CpH methylation is rare, and shows similar characteristics of variation patterns as CpGs. Our study highlights the utility of low pass whole-genome bisulfite sequencing in identifying methylome variation beyond promoter regions, and suggests that targeting the population dynamic methylome of tissues requires assessment of understudied intergenic CpGs distal to gene promoters to reveal the full extent of inter-individual variation.

  2. The North Wind and the Sun: Pediatric Antimicrobial Stewardship Program Combining Restrictive and Persuasive Approaches in Hematology-Oncology Ward and Hematopoietic Stem Cell Transplant Unit.

    PubMed

    Horikoshi, Yuho; Kaneko, Tetsuji; Morikawa, Yoshihiko; Isogai, Mihoko; Suwa, Junichi; Higuchi, Hiroshi; Yuza, Yuki; Shoji, Takayo; Ito, Kenta

    2017-08-17

    The Japanese government's goal for the reduction of antimicrobial consumption is two-thirds of the 2013 rate by 2020. While the antimicrobial stewardship programs (ASP) are essential in health care facilities, ASP data on pediatric hematology-oncology and hematopoietic stem cell transplant (HSCT) patients are limited. Our aim was to evaluate the impact of restrictive and persuasive ASP on immunocompromised children. The ASP for hematology-oncology and HSCT patients at Tokyo Metropolitan Children's Medical Center was assessed. Phase 1 was a post-prescriptive review of carbapenem conducted between April, 2010 and September, 2011. Phase 2 consisted of the preauthorization of carbapenem, prospective audit with feedback, a weekly luncheon meeting among physicians, consensus on febrile neutropenia management, and implementation of viral molecular diagnostics between October, 2011 and September, 2015. Both phases were compared for day-of-therapy (DOT) per 1,000 patient-days, cost of intravenous antimicrobial agents, average hospitalization duration, all-cause mortality, infection-related mortality at 30 days, and appropriateness of empirical treatment of bacteremia. The ASP did not differ from phase 1 to phase 2 in terms of average hospitalization length, mortality rate, or appropriateness of empirical treatment for bacteremia. DOTs of cefepime, piperacillin/tazobactam, meropenem, vancomycin, liposomal amphotericin B, and fosfluconazole decreased by 20%, 45%, 57%, 38%, 85% and 44%, respectively (p<0.05). The total cost of antibiotic and antifungal agents decreased by 27%, for a savings of $59,905 USD annually. Restrictive and persuasive ASP in the hematology-oncology ward and the HSCT unit safely decreased the use of antibacterial and antifungal agents.

  3. A combined DFT and restricted open-shell configuration interaction method including spin-orbit coupling: Application to transition metal L-edge X-ray absorption spectroscopy

    NASA Astrophysics Data System (ADS)

    Roemelt, Michael; Maganas, Dimitrios; DeBeer, Serena; Neese, Frank

    2013-05-01

    A novel restricted-open-shell configuration interaction with singles (ROCIS) approach for the calculation of transition metal L-edge X-ray absorption spectra is introduced. In this method, one first calculates the ground state and a number of excited states of the non-relativistic Hamiltonian. By construction, the total spin is a good quantum number in each of these states. For a ground state with total spin S excited states with spin S' = S, S - 1, and S + 1 are constructed. Using Wigner-Eckart algebra, all magnetic sublevels with MS = S, …, -S for each multiplet of spin S are obtained. The spin-orbit operator is represented by a mean-field approximation to the full Breit-Pauli spin-orbit operator and is diagonalized over this N-particle basis. This is equivalent to a quasi-degenerate treatment of the spin-orbit interaction to all orders. Importantly, the excitation space spans all of the molecular multiplets that arise from the atomic Russell-Saunders terms. Hence, the method represents a rigorous first-principles approach to the complicated low-symmetry molecular multiplet problem met in L-edge X-ray absorption spectroscopy. In order to gain computational efficiency, as well as additional accuracy, the excitation space is restricted to single excitations and the configuration interaction matrix is slightly parameterized in order to account for dynamic correlation effects in an average way. To this end, it is advantageous to employ Kohn-Sham rather than Hartree-Fock orbitals thus defining the density functional theory/ROCIS method. However, the method can also be used in an entirely non-empirical fashion. Only three global empirical parameters are introduced and have been determined here for future application of the method to any system containing any transition metal. The three parameters were carefully calibrated using the L-edge X-ray absorption spectroscopy spectra of a test set of coordination complexes containing first row transition metals. These

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

    PubMed

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

    2006-02-09

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

  5. Combination of exercise training and diet restriction normalizes limited exercise capacity and impaired skeletal muscle function in diet-induced diabetic mice.

    PubMed

    Suga, Tadashi; Kinugawa, Shintaro; Takada, Shingo; Kadoguchi, Tomoyasu; Fukushima, Arata; Homma, Tsuneaki; Masaki, Yoshihiro; Furihata, Takaaki; Takahashi, Masashige; Sobirin, Mochamad A; Ono, Taisuke; Hirabayashi, Kagami; Yokota, Takashi; Tanaka, Shinya; Okita, Koichi; Tsutsui, Hiroyuki

    2014-01-01

    Exercise training (EX) and diet restriction (DR) are essential for effective management of obesity and insulin resistance in diabetes mellitus. However, whether these interventions ameliorate the limited exercise capacity and impaired skeletal muscle function in diabetes patients remains unexplored. Therefore, we investigated the effects of EX and/or DR on exercise capacity and skeletal muscle function in diet-induced diabetic mice. Male C57BL/6J mice that were fed a high-fat diet (HFD) for 8 weeks were randomly assigned for an additional 4 weeks to 4 groups: control, EX, DR, and EX+DR. A lean group fed with a normal diet was also studied. Obesity and insulin resistance induced by a HFD were significantly but partially improved by EX or DR and completely reversed by EX+DR. Although exercise capacity decreased significantly with HFD compared with normal diet, it partially improved with EX and DR and completely reversed with EX+DR. In parallel, the impaired mitochondrial function and enhanced oxidative stress in the skeletal muscle caused by the HFD were normalized only by EX+DR. Although obesity and insulin resistance were completely reversed by DR with an insulin-sensitizing drug or a long-term intervention, the exercise capacity and skeletal muscle function could not be normalized. Therefore, improvement in impaired skeletal muscle function, rather than obesity and insulin resistance, may be an important therapeutic target for normalization of the limited exercise capacity in diabetes. In conclusion, a comprehensive lifestyle therapy of exercise and diet normalizes the limited exercise capacity and impaired muscle function in diabetes mellitus.

  6. Selective molecularly imprinted polymer combined with restricted access material for in-tube SPME/UHPLC-MS/MS of parabens in breast milk samples.

    PubMed

    Souza, Israel D; Melo, Lidervan P; Jardim, Isabel C S F; Monteiro, Juliana C S; Nakano, Ana Marcia S; Queiroz, Maria Eugênia C

    2016-08-17

    A new molecularly imprinted polymer modified with restricted access material (a hydrophilic external layer), (MIP-RAM) was synthesized via polymerization in situ in an open fused silica capillary. This stationary phase was used as sorbent for in-tube solid phase microextraction (in-tube SPME) to determine parabens in breast milk samples by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Scanning electron micrographs (SEM) illustrate MIP surface modification after glycerol dimethacrylate (hydrophilic monomer) incorporation. The interaction between parabens and MIP-RAM was investigated by Fourier-transform infrared (FTIR) spectroscopy. The Scatchard plot for MIP-RAM presented two linear parts with different slopes, illustrating binding sites with high- and low-affinity. Endogenous compounds exclusion from the MIP-RAM capillary was demonstrated by in-tube SPME/LC-UV assays carried out with blank milk samples. The in-tube SPME/UHPLC-MS/MS method presented linear range from 10 ng mL(-1) (LLOQ) to 400 ng mL(-1) with coefficients of determination higher than 0.99, inter-assay precision with coefficient of variation (CV) values ranging from 2 to 15%, and inter-assay accuracy with relative standard deviation (RSD) values ranging from -1% to 19%. Analytical validation parameters attested that in-tube SPME/UHPLC-MS/MS is an appropriate method to determine parabens in human milk samples to assess human exposure to these compounds. Analysis of breast milk samples from lactating women demonstrated that the proposed method is effective. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Atypical hematological response to combined calorie restriction and chronic hypoxia in Biosphere 2 crew: a possible link to latent features of hibernation capacity.

    PubMed

    Paglia, Donald E; Walford, Roy L

    2005-01-01

    Eight humans were isolated for 2 years in Biosphere 2, a sealed airtight habitat with recycled air, food, water, and wastes. A combination of conditions led to selective decline of oxygen (O2) in the internal atmosphere from 21% to 14%, inducing symptoms of high-altitude sickness but with little or no compensatory increase in red cell production. All crew members exhibited significant decreases in both erythrocyte 2,3-bisphosphoglycerate (2,3-BPG) concentrations and P50 [partial pressure of O2 for 50% hemoglobin (Hb) saturation] values, changes opposite those expected in adaptation to high-altitude hypoxia. Lower P50 with increased Hb-O2 affinity induced by low 2,3-BPG is a characteristic of hibernating species and could be advantageous in O2-impoverished environments. The mechanisms underlying these changes in the Biosphere 2 crew remain obscure but could be related to low-calorie diet (1750-2100 kcal/day). Because the combination of hypoxia and limited caloric intake is also characteristic of hibernation, this unusual response may represent a cross-adaptation phenomenon in which certain features of hibernation capability are expressed in humans.

  8. Bisprimer--a program for the design of primers for bisulfite-based genomic sequencing of both plant and Mammalian DNA samples.

    PubMed

    Kovacova, Viera; Janousek, Bohuslav

    2012-01-01

    Plants and animals differ in the sequence context of the methylated sites in DNA. Plants exhibit cytosine methylation in CG, CHG, and CHH sites, whereas CG methylation is the only form present in mammals (with an exception of the early embryonic development). This fact must be taken into account in the design of primers for bisulfite-based genomic sequencing because CHG and CHH sites can remain unmodified. Surprisingly, no user-friendly primer design program is publicly available that could be used to design primers in plants and to simultaneously check the properties of primers such as the potential for primer-dimer formation. For studies concentrating on particular DNA loci, the correct design of primers is crucial. The program, called BisPrimer, includes 2 different subprograms for the primer design, the first one for mammals and the second one for angiosperm plants. Each subprogram is divided into 2 variants. The first variant serves to design primers that preferentially bind to the bisulfite-modified primer-binding sites (C to U conversion). This type of primer preferentially amplifies the bisulfite-converted DNA strands. This feature can help to avoid problems connected with an incomplete bisulfite modification that can sometimes occur for technical reasons. The second variant is intended for the analysis of samples that are supposed to consist of a mixture of DNA molecules that have different levels of cytosine methylation (e.g., pollen DNA). In this case, the aim is to minimize the selection in favor of either less methylated or more methylated molecules.

  9. LoMA-B: a simple and versatile lab-on-a-chip system based on single-channel bisulfite conversion for DNA methylation analysis.

    PubMed

    Yoon, Jaeyun; Park, Mi Kyoung; Lee, Tae Yoon; Yoon, Yong-Jin; Shin, Yong

    2015-09-07

    Miniaturized lab-on-a-chip (LOC) systems have been developed for genetic and epigenetic analyses in clinical applications because of advantages such as reduced sample size and reagent consumption, rapid processing speed, simplicity, and enhanced sensitivity. Despite tremendous efforts made towards developing LOC systems for use in the clinical setting, the development of LOC systems to analyze DNA methylation, which is an emerging epigenetic marker causing the abnormal silencing of genes including tumor suppressor genes, is still challenging because of the gold standard methods involving a bisulfite conversion step. Existing bisulfite conversion-based techniques are not suitable for clinical use due to their long processing time, labor intensiveness, and the purification steps involved. Here, we present a lab-on-a-chip system for DNA methylation analysis based on bisulfite conversion (LoMA-B), which couples a sample pre-processing module for on-chip bisulfite conversion and a label-free, real-time detection module for rapid analysis of DNA methylation status using an isothermal DNA amplification/detection technique. The methylation status of the RARβ gene in human genomic DNA extracted from MCF-7 cells was analyzed by the LoMA-B system within 80 min (except 16 h for sensor preparation) compared to conventional MS-PCR within 24 h. Furthermore, the LoMA-B system is highly sensitive and can detect as little as 1% methylated DNA in a methylated/unmethylated cell mixture. Therefore, the LoMA-B system is an efficient diagnostic tool for the simple, versatile, and quantitative evaluation of DNA methylation patterns for clinical applications.

  10. swDMR: A Sliding Window Approach to Identify Differentially Methylated Regions Based on Whole Genome Bisulfite Sequencing

    PubMed Central

    Shao, Qianzhi; Liu, Qi; Chen, BingYu; Huang, Dongsheng

    2015-01-01

    DNA methylation is a widespread epigenetic modification that plays an essential role in gene expression through transcriptional regulation and chromatin remodeling. The emergence of whole genome bisulfite sequencing (WGBS) represents an important milestone in the detection of DNA methylation. Characterization of differential methylated regions (DMRs) is fundamental as well for further functional analysis. In this study, we present swDMR (http://sourceforge.net/projects/swdmr/) for the comprehensive analysis of DMRs from whole genome methylation profiles by a sliding window approach. It is an integrated tool designed for WGBS data, which not only implements accessible statistical methods to perform hypothesis test adapted to two or more samples without replicates, but false discovery rate was also controlled by multiple test correction. Downstream analysis tools were also provided, including cluster, annotation and visualization modules. In summary, based on WGBS data, swDMR can produce abundant information of differential methylated regions. As a convenient and flexible tool, we believe swDMR will bring us closer to unveil the potential functional regions involved in epigenetic regulation. PMID:26176536

  11. Activation of Manganese Oxidants with Bisulfite for Enhanced Oxidation of Organic Contaminants: The Involvement of Mn(III).

    PubMed

    Sun, Bo; Guan, Xiaohong; Fang, Jingyun; Tratnyek, Paul G

    2015-10-20

    MnO4(-) was activated by HSO3(-), resulting in a process that oxidizes organic contaminants at extraordinarily high rates. The permanganate/bisulfite (PM/BS) process oxidized phenol, ciprofloxacin, and methyl blue at pHini 5.0 with rates (kobs ≈ 60-150 s(-1)) that were 5-6 orders of magnitude faster than those measured for permanganate alone, and ∼5 to 7 orders of magnitude faster than conventional advanced oxidation processes for water treatment. Oxidation of phenol was fastest at pH 4.0, but still effective at pH 7.0, and only slightly slower when performed in tap water. A smaller, but still considerable (∼3 orders of magnitude) increase in oxidation rates of methyl blue was observed with MnO2 activated by HSO3(-) (MO/BS). The above results, time-resolved spectroscopy of manganese species under various conditions, stoichiometric analysis of pH changes, and the effect of pyrophosphate on UV absorbance spectra suggest that the reactive intermediate(s) responsible for the extremely rapid oxidation of organic contaminants in the PM/BS process involve manganese(III) species with minimal stabilization by complexation. The PM/BS process may lead to a new category of advanced oxidation technologies based on contaminant oxidation by reactive manganese(III) species, rather than hydroxyl and sulfate radicals.

  12. Genome-wide DNA methylation profiles changes associated with constant heat stress in pigs as measured by bisulfite sequencing

    PubMed Central

    Hao, Yue; Cui, Yanjun; Gu, Xianhong

    2016-01-01

    Heat stress affects muscle development and meat quality in food animals; however, little is known regarding its regulatory mechanisms at the epigenetic level, such as via DNA methylation. In this study, we aimed to compare the DNA methylation profiles between control and heat-stressed pigs to identify candidate genes for skeletal muscle development and meat quality. Whole-genome bisulfite sequencing was used to investigate the genome-wide DNA methylation patterns in the longissimus dorsi muscles of the pigs. Both groups showed similar proportions of methylation at CpG sites but exhibited different proportions at non-CpG sites. A total of 57,147 differentially methylated regions were identified between the two groups, which corresponded to 1,422 differentially methylated genes. Gene ontogeny and KEGG pathway analyses indicated that these were mainly involved in energy and lipid metabolism, cellular defense and stress responses, and calcium signaling pathways. This study revealed the global DNA methylation pattern of pig muscle between normal and heat stress conditions. The result of this study might contribute to a better understanding of epigenetic regulation in pig muscle development and meat quality. PMID:27264107

  13. A systematic study of normalization methods for Infinium 450K methylation data using whole-genome bisulfite sequencing data

    PubMed Central

    Wang, Ting; Guan, Weihua; Lin, Jerome; Boutaoui, Nadia; Canino, Glorisa; Luo, Jianhua; Celedón, Juan Carlos; Chen, Wei

    2015-01-01

    DNA methylation plays an important role in disease etiology. The Illumina Infinium HumanMethylation450 (450K) BeadChip is a widely used platform in large-scale epidemiologic studies. This platform can efficiently and simultaneously measure methylation levels at ∼480,000 CpG sites in the human genome in multiple study samples. Due to the intrinsic chip design of 2 types of chemistry probes, data normalization or preprocessing is a critical step to consider before data analysis. To date, numerous methods and pipelines have been developed for this purpose, and some studies have been conducted to evaluate different methods. However, validation studies have often been limited to a small number of CpG sites to reduce the variability in technical replicates. In this study, we measured methylation on a set of samples using both whole-genome bisulfite sequencing (WGBS) and 450K chips. We used WGBS data as a gold standard of true methylation states in cells to compare the performances of 8 normalization methods for 450K data on a genome-wide scale. Analyses on our dataset indicate that the most effective methods are peak-based correction (PBC) and quantile normalization plus β-mixture quantile normalization (QN.BMIQ). To our knowledge, this is the first study to systematically compare existing normalization methods for Illumina 450K data using novel WGBS data. Our results provide a benchmark reference for the analysis of DNA methylation chip data, particularly in white blood cells. PMID:26036609

  14. Determination of menadione sodium bisulfite in pharmaceutical preparations by flow-injection on-line photochemical spectrofluorometry.

    PubMed

    He, Q; Wang, Z; Cao, X; Chen, H; Ke, Y

    2001-10-01

    A flow-injection on-line photochemical spectrofluorometry (FI-PF) was developed for the determination of menadione sodium bisulfite (MSB) using acetone and sodium sulfite as sensitizing reagents. An injected sample band carried by a water stream was on-line merged with a mixed NaOH, Na2SO3 and acetone solution in a "T" connector. It was then driven to pass a knotted PTFE photochemical reactor (0.5 mm i.d. x 200 cm, KR) that was freely coiled around a 6-W low-pressure mercury lamp. While passing the KR, MSB was derived into an intensively fluorescent compound that was on-line delivered into a flow-through cell and detected therein at an emission wavelength of 459 nm and an excitation wavelength of 336 nm. Under optimized conditions a detection limit of 0.38 microg l(-1) was achieved at a sampling rate of 90 h(-1). Eleven determinations of 0.5 mg l(-1) and 0.05 mg l(-1) MSB standard solution gave RSDs of 0.75% and 1.3%, respectively. The calibration curve was linear in the MSB concentration range 0.005-1.5 mg l(-1). The proposed method was successfully applied to assay the MSB content in MSB injection.

  15. Single-cell methylome landscapes of mouse embryonic stem cells and early embryos analyzed using reduced representation bisulfite sequencing

    PubMed Central

    Guo, Hongshan; Zhu, Ping; Wu, Xinglong; Li, Xianlong; Wen, Lu; Tang, Fuchou

    2013-01-01

    DNA methylation is crucial for a wide variety of biological processes, yet no technique suitable for the methylome analysis of DNA methylation at single-cell resolution is available. Here, we describe a methylome analysis technique that enables single-cell and single-base resolution DNA methylation analysis based on reduced representation bisulfite sequencing (scRRBS). The technique is highly sensitive and can detect the methylation status of up to 1.5 million CpG sites within the genome of an individual mouse embryonic stem cell (mESC). Moreover, we show that the technique can detect the methylation status of individual CpG sites in a haploid sperm cell in a digitized manner as either unmethylated or fully methylated. Furthermore, we show that the demethylation dynamics of maternal and paternal genomes after fertilization can be traced within the individual pronuclei of mouse zygotes. The demethylation process of the genic regions is faster than that of the intergenic regions in both male and female pronuclei. Our method paves the way for the exploration of the dynamic methylome landscapes of individual cells at single-base resolution during physiological processes such as embryonic development, or during pathological processes such as tumorigenesis. PMID:24179143

  16. Genome-wide Bisulfite Sequencing in Zygotes Identifies Demethylation Targets and Maps the Contribution of TET3 Oxidation

    PubMed Central

    Peat, Julian R.; Dean, Wendy; Clark, Stephen J.; Krueger, Felix; Smallwood, Sébastien A.; Ficz, Gabriella; Kim, Jong Kyoung; Marioni, John C.; Hore, Timothy A.; Reik, Wolf

    2014-01-01

    Summary Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup. PMID:25497087

  17. Combining positional scanning peptide libraries, HLA-DR transfectants and bioinformatics to dissect the epitope spectrum of HLA class II cross-restricted CD4+ T cell clones.

    PubMed

    Sospedra, Mireia; Zhao, Yingdong; Giulianotti, Marc; Simon, Richard; Pinilla, Clemencia; Martin, Roland

    2010-02-28

    The use of positional scanning peptide libraries in combination with biometrical analysis is one of the few approaches, which allows the identification of stimulatory peptides for T cells of unknown specificity. Despite the successful application of this strategy in different studies, not every T cell is suited for analysis. For as yet unknown reasons some T cells do not recognize these highly complex libraries, and even more importantly the predictive capacity of the current approach shows high variability among individual T cell clones and their TCRs. A number of factors probably contribute to differences in T cell recognition and have to be taken into account in order to overcome these difficulties. Our results suggest that the ability of some T cells to recognize peptides in the context of more than one HLA class II molecule expressed by autologous APCs could diminish the predictive capacity of the approach. In contrast, the use of B cell lines expressing single HLA class II molecules as APCs instead of autologous peripheral blood mononuclear cells markedly improves the capacity to identify target peptides for this type of T cells.

  18. metilene: fast and sensitive calling of differentially methylated regions from bisulfite sequencing data

    PubMed Central

    Jühling, Frank; Kretzmer, Helene; Bernhart, Stephan H.; Otto, Christian; Stadler, Peter F.; Hoffmann, Steve

    2016-01-01

    The detection of differentially methylated regions (DMRs) is a necessary prerequisite for characterizing different epigenetic states. We present a novel program, metilene, to identify DMRs within whole-genome and targeted data with unrivaled specificity and sensitivity. A binary segmentation algorithm combined with a two-dimensional statistical test allows the detection of DMRs in large methylation experiments with multiple groups of samples in minutes rather than days using off-the-shelf hardware. metilene outperforms other state-of-the-art tools for low coverage data and can estimate missing data. Hence, metilene is a versatile tool to study the effect of epigenetic modifications in differentiation/development, tumorigenesis, and systems biology on a global, genome-wide level. Whether in the framework of international consortia with dozens of samples per group, or even without biological replicates, it produces highly significant and reliable results. PMID:26631489

  19. Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation

    PubMed Central

    2011-01-01

    Heterogeneous DNA methylation leads to difficulties in accurate detection and quantification of methylation. Methylation-sensitive high resolution melting (MS-HRM) is unique among regularly used methods for DNA methylation analysis in that heterogeneous methylation can be readily identified, although not quantified, by inspection of the melting curves. Bisulfite pyrosequencing has been used to estimate the level of heterogeneous methylation by quantifying methylation levels present at individual CpG dinucleotides. Sequentially combining the two methodologies using MS-HRM to screen the amplification products prior to bisulfite pyrosequencing would be advantageous. This would not only replace the quality control step using agarose gel analysis prior to the pyrosequencing step but would also provide important qualitative information in its own right. We chose to analyze DAPK1 as it is an important tumor suppressor gene frequently heterogeneously methylated in a number of malignancies, including chronic lymphocytic leukemia (CLL). A region of the DAPK1 promoter was analyzed in ten CLL samples by MS-HRM. By using a biotinylated primer, bisulfite pyrosequencing could be used to directly analyze the samples. MS-HRM revealed the presence of various extents of heterogeneous DAPK1 methylation in all CLL samples. Further analysis of the biotinylated MS-HRM products by bisulfite pyrosequencing provided quantitative information for each CpG dinucleotide analyzed, and confirmed the presence of heterogeneous DNA methylation. Whereas each method could be used individually, MS-HRM and bisulfite pyrosequencing provided complementary information for the assessment of heterogeneous methylation. PMID:21364322

  20. Identification of differential genomic DNA Methylation in the hypothalamus of pubertal rat using reduced representation Bisulfite sequencing.

    PubMed

    Luo, Lei; Yao, Zhiqiu; Ye, Jing; Tian, Yuan; Yang, Chen; Gao, Xiaoxiao; Song, Min; Liu, Ya; Zhang, Yunhai; Li, Yunsheng; Zhang, Xiaorong; Fang, Fugui

    2017-10-06

    There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome. We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis. The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway. Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.

  1. Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains.

    PubMed

    Jensen, Taylor J; Kim, Sung K; Zhu, Zhanyang; Chin, Christine; Gebhard, Claudia; Lu, Tim; Deciu, Cosmin; van den Boom, Dirk; Ehrich, Mathias

    2015-04-15

    Circulating cell-free fetal DNA has enabled non-invasive prenatal fetal aneuploidy testing without direct discrimination of the maternal and fetal DNA. Testing may be improved by specifically enriching the sample material for fetal DNA. DNA methylation may allow for such a separation of DNA; however, this depends on knowledge of the methylomes of circulating cell-free DNA and its cellular contributors. We perform whole genome bisulfite sequencing on a set of unmatched samples including circulating cell-free DNA from non-pregnant and pregnant female donors and genomic DNA from maternal buffy coat and placenta samples. We find CpG cytosines within longer fragments are more likely to be methylated. Comparison of the methylomes of placenta and non-pregnant circulating cell-free DNA reveal many of the 51,259 identified differentially methylated regions are located in domains exhibiting consistent placenta hypomethylation across millions of consecutive bases. We find these placenta hypomethylated domains are consistently located within regions exhibiting low CpG and gene density. Differentially methylated regions identified when comparing placenta to non-pregnant circulating cell-free DNA are recapitulated in pregnant circulating cell-free DNA, confirming the ability to detect differential methylation in circulating cell-free DNA mixtures. We generate methylome maps for four sample types at single-base resolution, identify a link between DNA methylation and fragment length in circulating cell-free DNA, identify differentially methylated regions between sample groups, and uncover the presence of megabase-size placenta hypomethylated domains.

  2. A Bayesian Approach for Analysis of Whole-Genome Bisulfite Sequencing Data Identifies Disease-Associated Changes in DNA Methylation

    PubMed Central

    Rackham, Owen J. L.; Langley, Sarah R.; Oates, Thomas; Vradi, Eleni; Harmston, Nathan; Srivastava, Prashant K.; Behmoaras, Jacques; Dellaportas, Petros; Bottolo, Leonardo; Petretto, Enrico

    2017-01-01

    DNA methylation is a key epigenetic modification involved in gene regulation whose contribution to disease susceptibility remains to be fully understood. Here, we present a novel Bayesian smoothing approach (called ABBA) to detect differentially methylated regions (DMRs) from whole-genome bisulfite sequencing (WGBS). We also show how this approach can be leveraged to identify disease-associated changes in DNA methylation, suggesting mechanisms through which these alterations might affect disease. From a data modeling perspective, ABBA has the distinctive feature of automatically adapting to different correlation structures in CpG methylation levels across the genome while taking into account the distance between CpG sites as a covariate. Our simulation study shows that ABBA has greater power to detect DMRs than existing methods, providing an accurate identification of DMRs in the large majority of simulated cases. To empirically demonstrate the method’s efficacy in generating biological hypotheses, we performed WGBS of primary macrophages derived from an experimental rat system of glomerulonephritis and used ABBA to identify >1000 disease-associated DMRs. Investigation of these DMRs revealed differential DNA methylation localized to a 600 bp region in the promoter of the Ifitm3 gene. This was confirmed by ChIP-seq and RNA-seq analyses, showing differential transcription factor binding at the Ifitm3 promoter by JunD (an established determinant of glomerulonephritis), and a consistent change in Ifitm3 expression. Our ABBA analysis allowed us to propose a new role for Ifitm3 in the pathogenesis of glomerulonephritis via a mechanism involving promoter hypermethylation that is associated with Ifitm3 repression in the rat strain susceptible to glomerulonephritis. PMID:28213474

  3. Formation of the bisulfite anion (HSO(3) (-) , m/z 81) upon collision-induced dissociation of anions derived from organic sulfonic acids.

    PubMed

    Jariwala, Freneil B; Wood, Ryan E; Nishshanka, Upul; Attygalle, Athula B

    2012-04-01

    In the negative-ion collision-induced dissociation mass spectra of most organic sulfonates, the base peak is observed at m/z 80 for the sulfur trioxide radical anion (SO(3) (-·) ). In contrast, the product-ion spectra of a few sulfonates, such as cysteic acid, aminomethanesulfonate, and 2-phenylethanesulfonate, show the base peak at m/z 81 for the bisulfite anion (HSO(3) (-) ). An investigation with an extensive variety of sulfonates revealed that the presence of a hydrogen atom at the β-position relative to the sulfur atom is a prerequisite for the formation of the bisulfite anion. The formation of HSO(3) (-) is highly favored when the atom at the β-position is nitrogen, or the leaving neutral species is a highly conjugated molecule such as styrene or acrylic acid. Deuterium-exchange experiments with aminomethanesulfonate demonstrated that the hydrogen for HSO(3) (-) formation is transferred from the β-position. The presence of a peak at m/z 80 in the spectrum of 2-sulfoacetic acid, in contrast to a peak at m/z 81 in that of 3-sulfopropanoic acid, corroborated the proposed hydrogen transfer mechanism. For diacidic compounds, such as 4-sulfobutanoic acid and cysteic acid, the m/z 81 ion can be formed by an alternative mechanism, in which the negative charge of the carboxylate moiety attacks the α-carbon relative to the sulfur atom. Experiments conducted with deuterium-exchanged and deuterium-labeled analogs of sulfocarboxylic acids demonstrated that the formation of the bisulfite anion resulted either from a hydrogen transfer from the β-carbon, or from a direct attack by the carboxylate moiety on the α-carbon.

  4. Whole-Genome Bisulfite Sequencing of Human Pancreatic Islets Reveals Novel Differentially Methylated Regions in Type 2 Diabetes Pathogenesis.

    PubMed

    Volkov, Petr; Bacos, Karl; Ofori, Jones K; Esguerra, Jonathan Lou S; Eliasson, Lena; Rönn, Tina; Ling, Charlotte

    2017-04-01

    Current knowledge about the role of epigenetics in type 2 diabetes (T2D) remains limited. Only a few studies have investigated DNA methylation of selected candidate genes or a very small fraction of genomic CpG sites in human pancreatic islets, the tissue of primary pathogenic importance for diabetes. Our aim was to characterize the whole-genome DNA methylation landscape in human pancreatic islets, to identify differentially methylated regions (DMRs) in diabetic islets, and to investigate the function of DMRs in islet biology. Here, we performed whole-genome bisulfite sequencing, which is a comprehensive and unbiased method to study DNA methylation throughout the genome at a single nucleotide resolution, in pancreatic islets from donors with T2D and control subjects without diabetes. We identified 25,820 DMRs in islets from individuals with T2D. These DMRs cover loci with known islet function, e.g., PDX1, TCF7L2, and ADCY5 Importantly, binding sites previously identified by ChIP-seq for islet-specific transcription factors, enhancer regions, and different histone marks were enriched in the T2D-associated DMRs. We also identified 457 genes, including NR4A3, PARK2, PID1, SLC2A2, and SOCS2, that had both DMRs and significant expression changes in T2D islets. To mimic the situation in T2D islets, candidate genes were overexpressed or silenced in cultured β-cells. This resulted in impaired insulin secretion, thereby connecting differential methylation to islet dysfunction. We further explored the islet methylome and found a strong link between methylation levels and histone marks. Additionally, DNA methylation in different genomic regions and of different transcript types (i.e., protein coding, noncoding, and pseudogenes) was associated with islet expression levels. Our study provides a comprehensive picture of the islet DNA methylome in individuals with and without diabetes and highlights the importance of epigenetic dysregulation in pancreatic islets and T2D

  5. Biology of DNA restriction.

    PubMed Central

    Bickle, T A; Krüger, D H

    1993-01-01

    Our understanding of the evolution of DNA restriction and modification systems, the control of the expression of the structural genes for the enzymes, and the importance of DNA restriction in the cellular economy has advanced by leaps and bounds in recent years. This review documents these advances for the three major classes of classical restriction and modification systems, describes the discovery of a new class of restriction systems that specifically cut DNA carrying the modification signature of foreign cells, and deals with the mechanisms developed by phages to avoid the restriction systems of their hosts. PMID:8336674

  6. Genetics of restrictive cardiomyopathy.

    PubMed

    Sen-Chowdhry, Srijita; Syrris, Petros; McKenna, William J

    2010-04-01

    Restrictive physiology, a severe form of diastolic dysfunction, is characteristically observed in the setting of constrictive pericarditis and myocardial restriction. The latter is commonly due to systemic diseases, some of which are inherited as mendelian traits (eg, hereditary amyloidosis), while others are multifactorial (eg, sarcoidosis). When restrictive physiology occurs as an early and dominant feature of a primary myocardial disorder, it may be termed restrictive cardiomyopathy. In the past decade, clinical and genetic studies have demonstrated that restrictive cardiomyopathy as such is part of the spectrum of sarcomeric disease and frequently coexists with hypertrophic cardiomyopathy in affected families.

  7. The mTORC1-Signaling Pathway and Hepatic Polyribosome Profile Are Enhanced after the Recovery of a Protein Restricted Diet by a Combination of Soy or Black Bean with Corn Protein.

    PubMed

    Márquez-Mota, Claudia C; Rodriguez-Gaytan, Cinthya; Adjibade, Pauline; Mazroui, Rachid; Gálvez, Amanda; Granados, Omar; Tovar, Armando R; Torres, Nimbe

    2016-09-20

    Between 6% and 11% of the world's population suffers from malnutrition or undernutrition associated with poverty, aging or long-term hospitalization. The present work examined the effect of different types of proteins on the mechanistic target of rapamycin (mTORC1)-signaling pathway in: (1) healthy; and (2) protein restricted rats. (1) In total, 200 rats were divided into eight groups and fed one of the following diets: 20% casein (C), soy (S), black bean (B), B + Corn (BCr), Pea (P), spirulina (Sp), sesame (Se) or Corn (Cr). Rats fed C or BCr had the highest body weight gain; rats fed BCr had the highest pS6K1/S6K1 ratio; rats fed B, BCr or P had the highest eIF4G expression; (2) In total, 84 rats were fed 0.5% C for 21 day and protein rehabilitated with different proteins. The S, soy + Corn (SCr) and BCr groups had the highest body weight gain. Rats fed SCr and BCr had the highest eIF4G expression and liver polysome formation. These findings suggest that the quality of the dietary proteins modulate the mTORC1-signaling pathway. In conclusion, the combination of BCr or SCr are the best proteins for dietary protein rehabilitation due to the significant increase in body weight, activation of the mTORC1-signaling pathway in liver and muscle, and liver polysome formation.

  8. The mTORC1-Signaling Pathway and Hepatic Polyribosome Profile Are Enhanced after the Recovery of a Protein Restricted Diet by a Combination of Soy or Black Bean with Corn Protein

    PubMed Central

    Márquez-Mota, Claudia C.; Rodriguez-Gaytan, Cinthya; Adjibade, Pauline; Mazroui, Rachid; Gálvez, Amanda; Granados, Omar; Tovar, Armando R.; Torres, Nimbe

    2016-01-01

    Between 6% and 11% of the world’s population suffers from malnutrition or undernutrition associated with poverty, aging or long-term hospitalization. The present work examined the effect of different types of proteins on the mechanistic target of rapamycin (mTORC1)-signaling pathway in: (1) healthy; and (2) protein restricted rats. (1) In total, 200 rats were divided into eight groups and fed one of the following diets: 20% casein (C), soy (S), black bean (B), B + Corn (BCr), Pea (P), spirulina (Sp), sesame (Se) or Corn (Cr). Rats fed C or BCr had the highest body weight gain; rats fed BCr had the highest pS6K1/S6K1 ratio; rats fed B, BCr or P had the highest eIF4G expression; (2) In total, 84 rats were fed 0.5% C for 21 day and protein rehabilitated with different proteins. The S, soy + Corn (SCr) and BCr groups had the highest body weight gain. Rats fed SCr and BCr had the highest eIF4G expression and liver polysome formation. These findings suggest that the quality of the dietary proteins modulate the mTORC1-signaling pathway. In conclusion, the combination of BCr or SCr are the best proteins for dietary protein rehabilitation due to the significant increase in body weight, activation of the mTORC1-signaling pathway in liver and muscle, and liver polysome formation. PMID:27657118

  9. Online restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry for the simultaneous determination of vanillin and its vanillic acid metabolite in human plasma.

    PubMed

    Li, De-Qiang; Zhang, Zhi-Qing; Yang, Xiu-Ling; Zhou, Chun-Hua; Qi, Jin-Long

    2016-09-01

    An automated online solid-phase extraction with restricted-access material combined with high-performance liquid chromatography and tandem mass spectrometry was developed and validated for the simultaneous quantification of vanillin and its vanillic acid metabolite in human plasma. After protein precipitation by methanol, which contained the internal standards, the supernatant of plasma samples was injected to the system, the endogenous large molecules were flushed out, and target analytes were trapped and enriched on the adsorbent, resulting in a minimization of sample complexity and ion suppression effects. Calibration curves were linear over the concentrations of 5-1000 ng/mL for vanillin and 10-5000 ng/mL for vanillic acid with a coefficient of determination >0.999 for the determined compounds. The lower limits of quantification of vanillin and vanillic acid were 5.0 and 10.0 ng/mL, respectively. The intra- and inter-run precisions expressed as the relative standard deviation were 2.6-8.6 and 3.2-10.2%, respectively, and the accuracies expressed as the relative error were in the range of -6.1 to 7.3%. Extraction recoveries of analytes were between 89.5 and 97.4%. There was no notable matrix effect for any analyte concentration. The developed method was proved to be sensitive, repeatable, and accurate for the quantification of vanillin and its vanillic acid metabolite in human plasma.

  10. Restricted accessed material-copper(II) ion imprinted polymer solid phase extraction combined with inductively coupled plasma-optical emission spectrometry for the determination of free Cu(II) in urine and serum samples.

    PubMed

    Cui, Chao; He, Man; Chen, Beibei; Hu, Bin

    2013-11-15

    A novel restricted accessed material (RAM)-Cu(II) ion imprinted polymer (IIP) was synthesized by the surface imprinted-emulsion method, and possessed a high selectivity to Cu(II) and good macromolecules exclusion property. And a novel method of RAM-IIP packed microcolumn solid phase extraction (SPE) combined with inductively coupled plasma-optical emission spectrometry (ICP-OES) was developed for the determination of trace free Cu(II) in human body fluids. Under the optimized conditions, the adsorption capacity of RAM-IIP for Cu(II) was 15.9 mg g(-1). With a preconcentration factor of 30, the limit of detection was 0.17 µg L(-1), and the relative standard deviation was 2.2% (n=7, c=1 µg L(-1)). The developed method was validated by the analysis of two Certified Reference Materials, and the determined values were in good agreement with the certified values. This method was also successfully applied for the direct analysis of free Cu(II) in human urine and serum samples. While the total Cu can be determined by the proposed method after microwave digestion. The concentrations of free Cu(II) were much lower than that of total Cu, indicating that Cu is mainly coordinated with macromolecules in these biological samples. From this point of view, the developed method exhibits application potential in speciation of free metal ions and metallic complex molecules in biological samples. © 2013 Elsevier B.V. All rights reserved.

  11. Effects of acute feed restriction combined with targeted use of increasing luteinizing hormone content of follicle-stimulating hormone preparations on ovarian superstimulation, fertilization, and embryo quality in lactating dairy cows.

    PubMed

    Bender, R W; Hackbart, K S; Dresch, A R; Carvalho, P D; Vieira, L M; Crump, P M; Guenther, J N; Fricke, P M; Shaver, R D; Combs, D K; Wiltbank, M C

    2014-02-01

    Multiple metabolic and hormonal factors can affect the success of protocols for ovarian superstimulation. In this study, the effect of acute feed restriction and increased LH content in the superstimulatory FSH preparation on numbers of ovulations, fertilization, and embryo quality in lactating dairy cows was evaluated. Two experiments were performed using a Latin square design with treatments arranged as a 2 × 2 factorial: feed restriction (FR; 25% reduction in dry matter intake) compared with ad libitum (AL) feeding, combined with high (H) versus low (L) LH in the last 4 injections of the superstimulatory protocol. As expected, FR decreased circulating insulin concentrations (26.7 vs. 46.0 μU/mL). Two analyses were performed: one that evaluated the complete Latin square in experiment 2 and a second that evaluated only the first periods of experiments 1 and 2. For both analyses, follicle numbers, ovulation rates, and corpora lutea on d 7 were not different. In the first period analysis of experiments 1 and 2, we observed an interaction between feed allowance and amount of LH on fertilization rates, percentage of embryos or oocytes that were quality 1 and 2 embryos, and number of embryos or oocytes that were degenerate. Fertilization rates were greater for the AL-L (89.4%) and FR-H (80.1%) treatments compared with the AL-H (47.9%) and FR-L (59.9%) treatments. Similarly, the proportion of total embryos or oocytes designated as quality 1 and 2 embryos was greater for AL-L (76.7%) and FR-H (73.4%) treatments compared with AL-H (35.6%) and FR-L (47.3%) treatments. In addition, the number of degenerate embryos was decreased for AL-L (1.3) and FR-H (0.4) treatments compared with the AL-H (2.6) and FR-L (2.3) treatments. Thus, cows with either too low (FR-L) or too high (AL-H) insulin and LH stimulation had lesser embryo production after superstimulation because of reduced fertilization rate and increased percentage of degenerate embryos. Therefore, interaction of the

  12. Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing (scBS-seq).

    PubMed

    Clark, Stephen J; Smallwood, Sébastien A; Lee, Heather J; Krueger, Felix; Reik, Wolf; Kelsey, Gavin

    2017-03-01

    DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome. Here we present a detailed protocol for scBS-seq that includes our most recent developments to optimize recovery of CpGs, mapping efficiency and success rate; reduce hands-on time; and increase sample throughput with the option of using an automated liquid handler. We provide step-by-step instructions for each stage of the method, comprising cell lysis and bisulfite (BS) conversion, preamplification and adaptor tagging, library amplification, sequencing and, lastly, alignment and methylation calling. An individual with relevant molecular biology expertise can complete library preparation within 3 d. Subsequent computational steps require 1-3 d for someone with bioinformatics expertise.

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

    PubMed

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

    2014-01-01

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

  14. Identification of S-3-(hexanal)-glutathione and its bisulfite adduct in grape juice from Vitis vinifera L. cv. Sauvignon blanc as new potential precursors of 3SH.

    PubMed

    Thibon, Cécile; Böcker, Caroline; Shinkaruk, Svitlana; Moine, Virginie; Darriet, Philippe; Dubourdieu, Denis

    2016-05-15

    Two main precursors (S-3-(hexan-1-ol)-l-cysteine and S-3-(hexan-1-ol)-l-glutathione) of 3-sulfanylhexanol (3SH, formerly named 3-mercaptohexanol) have been identified so far in grape juice but a correlation between precursor concentrations in grape juices and 3SH concentrations in wines is not always observed. This suggests that there may be other compounds associated with the aromatic potential. In this work, S-3-(hexanal)-glutathione (Glut-3SH-Al) and its bisulfite (Glut-3SH-SO3) adduct were identified in Sauvignon blanc grape juice by liquid chromatography coupled to Fourier transform mass spectrometry experiments. A partial purification of the compounds was carried out by Medium Pressure Liquid Chromatography (MPLC) on the reverse phase using 5L of grape juice. The addition of synthetized Glut-3SH-Al and Glut-3SH-SO3 in the synthetic medium induced a significant release of 3SH after fermentation. Moreover, we demonstrate that Glut-3SH-Al and its bisulfite adduct are present in grape juice and could be considered as new direct 3SH precursors with molar conversion yields close to 0.4%. Copyright © 2016. Published by Elsevier Ltd.

  15. Incremental value of combining systolic mitral annular velocity and time difference between mitral inflow and diastolic mitral annular velocity to early diastolic annular velocity for differentiating constrictive pericarditis from restrictive cardiomyopathy.

    PubMed

    Choi, Eui-Young; Ha, Jong-Won; Kim, Jin-Mi; Ahn, Jeong-Ah; Seo, Hye-Sun; Lee, Jee-Hyun; Rim, Se-Joong; Chung, Namsik

    2007-06-01

    Although normal or exaggerated early diastolic mitral annular velocity (E') provides an excellent specificity for differentiating constrictive pericarditis (CP) from restrictive cardiomyopathy (RCM), its sensitivity has been shown to be lower, especially in patients with CP who had underlying myocardial abnormality. This study sought to evaluate the incremental value of systolic mitral annular velocity (S') and time difference between onset of mitral inflow and onset of E' (T(E'-E)) for differentiation between CP and RCM. This study included 44 participants (28 male, 16 female; mean age 47 years, range 10-76): 17 patients with CP, 12 with RCM, and 15 control subjects. Standard mitral inflow Doppler and tissue Doppler echocardiography were performed. E' (9.5 +/- 1.7 vs 4.7 +/- 1.6 cm/s, P < .001) and S' (7.7 +/- 1.3 vs 4.6 +/- 1.9 cm/s, P < .001) were significantly higher, whereas T(E'-E) (21.0 +/- 32.0 vs 53.1 +/- 30.4 milliseconds, P = .02) was significantly shorter in patients with CP than with RCM. Diagnostic accuracy of E' for differentiation of CP from RCM was higher than S' or T(E'-E) (area under curve 0.99 vs 0.87 vs 0.74, respectively). E' of 8 cm/s had excellent specificity (100%) for differentiation of CP from RCM but sensitivity (70%) was relatively low. However, when combining E' with S' and T(E'-E), the sensitivity could be increased when compared with E' alone (70% with E', 88% with E' + S', and 94% with E' + S' + T(E'-E)), P = .001). In conclusion, the measurement of S' and T(E'-E) can be helpful for differentiating between CP and RCM by providing incremental diagnostic information to E'.

  16. Combined moderate and high intensity exercise with dietary restriction improves cardiac autonomic function associated with a reduction in central and systemic arterial stiffness in obese adults: a clinical trial.

    PubMed

    Hu, Min; Wang, Shen; Wang, Dan; Lai, Qinhao; Chen, Xiaoying; Duan, Shiwei; Zhao, Mengke; Huang, Junhao

    2017-01-01

    The present study aimed to assess the effects of exercise with dietary restriction on cardiac autonomic activity, arterial stiffness, and cardiovascular biomarkers in obese individuals. Seventeen obese adults completed an 8-week exercise and dietary program. Anthropometry, body composition, and multiple biochemical markers were measured. We used carotid-femoral pulse wave velocity (cfPWV), brachial-ankle pulse wave velocity (baPWV), central blood pressure, and augmentation index (AIx) to assess arterial stiffness. To determine cardiac autonomic activity, heart rate variability (HRV) was analyzed by standard deviation of normal-to-normal intervals (SDNN), square root of the mean squared differences of successive normal-to-normal intervals (RMSSD), total power (TF), low-frequency power in normalized units (LFnu), high-frequency power in normalized units (HFnu), and low-frequency power/high-frequency power (LF/HF). Following the exercise and diet intervention, obese subjects had significant reductions in body weight, body mass index, body fat percentage, brachial systolic blood pressure, and resting heart rate, and they had shown improvements in blood chemistry markers such as lipid profiles, insulin, and high-sensitivity C-reactive protein. There was a significant reduction in both cfPWV and baPWV following the intervention when compared to baseline levels. Moreover, the AIx and aortic systolic blood pressure were significantly reduced after the intervention. The diet and exercise intervention significantly increased cardiac autonomic modulation (determined by improved SDNN, RMSSD, TP LF, HF, and LF/HF), which was partly due to changes in heart rate, insulin resistance, and the inflammatory pattern. Furthermore, we observed a correlation between enhanced cardiac autonomic modulation (LF/HF) and decreased arterial stiffness, as measured by central cfPWV and systemic baPWV. An 8-week combined intervention of diet and exercise is effective in improving cardiac autonomic

  17. A Conformational Restriction in the Influenza A Virus Neuraminidase Binding Site by R152 Results in a Combinational Effect of I222T and H274Y on Oseltamivir Resistance

    PubMed Central

    Huang, Lan; Cao, Yang; Zhou, Jianfang; Qin, Kun; Zhu, Wenfei; Zhu, Yun; Yang, Lei; Wang, Dayan

    2014-01-01

    The I222K, I222R, and I222T substitutions in neuraminidase (NA) have been found in clinically derived 2009 pandemic influenza A/H1N1 viruses with altered susceptibilities to NA inhibitors (NAIs). The effects of these substitutions, together with the most frequently observed resistance-related substitution, H274Y, on viral fitness and resistance mechanisms were further investigated in this study. Reduced sensitivities to oseltamivir were observed in all three mutants (I222K, I222R, and I222T). Furthermore, the I222K and I222T substitutions had a combinational effect of further increasing resistance in the presence of H274Y, which might result from a conformational restriction in the NA binding site. Of note, by using molecular dynamics simulations, R152, the neighbor of T222, was observed to translate to a position closer to T222, resulting in the narrowing of the binding pocket, which otherwise only subtends the residue substitution of H274Y. Moreover, significantly attenuated NA function and viral growth abilities were found in the I222K+H274Y double mutant, while the I222T+H274Y double mutant exhibited slightly delayed growth but had a peak viral titer similar to that of the wild-type virus in MDCK cells. The relative growth advantage of the I222T mutant versus the I222K mutant and the higher frequency of I222T emerging in N1 subtype influenza viruses raise concerns necessitating close monitoring of the dual substitutions I222T and H274Y. PMID:24366752

  18. Short-term caloric restriction, resveratrol, or combined treatment regimens initiated in late-life alter mitochondrial protein expression profiles in a fiber-type specific manner in aged animals.

    PubMed

    Joseph, Anna-Maria; Malamo, Angelina G; Silvestre, Jason; Wawrzyniak, Nick; Carey-Love, Sean; Nguyen, Linda M-D; Dutta, Debapriya; Xu, Jinze; Leeuwenburgh, Christiaan; Adhihetty, Peter J

    2013-09-01

    Aging is associated with a loss in muscle known as sarcopenia that is partially attributed to apoptosis. In aging rodents, caloric restriction (CR) increases health and longevity by improving mitochondrial function and the polyphenol resveratrol (RSV) has been reported to have similar benefits. In the present study, we investigated the potential efficacy of using short-term (6 weeks) CR (20%), RSV (50 mg/kg/day), or combined CR+ RSV (20% CR and 50 mg/kg/day RSV), initiated at late-life (27 months) to protect muscle against sarcopenia by altering mitochondrial function, biogenesis, content, and apoptotic signaling in both glycolytic white and oxidative red gastrocnemius muscle (WG and RG, respectively) of male Fischer 344 × Brown Norway rats. CR but not RSV attenuated the age-associated loss of muscle mass in both mixed gastrocnemius and soleus muscle, while combined treatment (CR + RSV) paradigms showed a protective effect in the soleus and plantaris muscle (P < 0.05). Sirt1 protein content was increased by 2.6-fold (P < 0.05) in WG but not RG muscle with RSV treatment, while CR or CR + RSV had no effect. PGC-1α levels were higher (2-fold) in the WG from CR-treated animals (P < 0.05) when compared to ad-libitum (AL) animals but no differences were observed in the RG with any treatment. Levels of the anti-apoptotic protein Bcl-2 were significantly higher (1.6-fold) in the WG muscle of RSV and CR + RSV groups compared to AL (P < 0.05) but tended to occur coincident with elevations in the pro-apoptotic protein Bax so that the apoptotic susceptibility as indicated by the Bax to Bcl-2 ratio was unchanged. There were no alterations in DNA fragmentation with any treatment in muscle from older animals. Additionally, mitochondrial respiration measured in permeabilized muscle fibers was unchanged in any treatment group and this paralleled the lack of change in cytochrome c oxidase (COX) activity. These data suggest that short-term moderate CR, RSV, or CR + RSV tended to

  19. Evaluating the effect of wood ultrastructural changes from mechanical treatment on kinetics of monomeric sugars and chemicals production in acid bisulfite treatment.

    PubMed

    Liu, Yalan; Wang, Jinwu; Wolcott, Michael P

    2017-02-01

    Currently, various chemical-mechanical treatments were widely used in biofuel production to achieve high total sugar yields. However, the interaction between two treatments was scarcely investigated. In this study, we employed a ball milling process to create ultrastructural changes for Douglas-fir (Pseudotsuga menziesii) micronized wood powders. The 0, 30, and 60min ball milled wood powders resulted in a crystallinity index of 0.41, 0.21, and 0.10 respectively. It was found that the ultrastructural changes accelerate monomeric sugars production without influencing the yield of sugar degradation products. The optimal acid bisulfite treatment time was substantially decreased from 120min to 40min as the cellulose crystallinity decreased. Meanwhile, total sugar yield increased from 65% to 92% and had a linear relation with a decrease of the cellulose crystallinity.

  20. New Methods To Estimate Lattice Energies: Application to the Relative Stabilities of Bisulfite (HSO3 ) and Metabisulfite (S2O52 ) Salts

    NASA Astrophysics Data System (ADS)

    Jenkins, H. Donald B.; Tudela, David

    2003-12-01

    Thermodynamic information is critical for the assessment of the reactivity and stability of materials. New procedures have recently been developed that provide straightforward predictive methods for estimating lattice energy and standard entropy of conventional salts. These procedures also open up new possibilities for estimating the thermochemistry involving salts for which there is little or no structural information available, as well as for conventional, well-characterized, ionic materials. These approaches are based on volume data, obtained from crystal structure determinations or from additive single-ion volume data (or estimates thereof), or on experimental densities. A detailed example explains the observed trend in increasing stability of bisulfites as the size of the cation increases and shows how quantitative free energy information can be obtained. Errors are also discussed.

  1. Locus-specific DNA methylation analysis of retrotransposons in ES, somatic and cancer cells using High-Throughput Targeted Repeat Element Bisulfite Sequencing.

    PubMed

    Bakshi, Arundhati; Ekram, Muhammad B; Kim, Joomyeong

    2015-03-01

    DNA methylation is a major epigenetic mark associated with multiple aspects of retrotransposons within the mammalian genome. In order to study DNA methylation of a large number of retrotransposons on an individual-locus basis, we have developed a new protocol termed High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS) (Ekram and Kim, 2014 [1]). We have used this technique to characterize the locus-specific patterns of DNA methylation of 4799 members of the mouse IAP LTR (Intracisternal A Particle Long Terminal Repeat) retrotransposon family in embryonic stem, somatic and Neuro2A cells (Bakshi and Kim, 2014 [2]). Here we describe in detail the sample preparation and bioinformatics analyses used for these studies. The somatic cell data may be accessed under GEO accession number GSE49222. The ES and Neuro2A data are deposited under GEO accession number GSE60007.

  2. Energy restriction and potential energy restriction mimetics.

    PubMed

    Nikolai, Sibylle; Pallauf, Kathrin; Huebbe, Patricia; Rimbach, Gerald

    2015-12-01

    Energy restriction (ER; also known as caloric restriction) is the only nutritional intervention that has repeatedly been shown to increase lifespan in model organisms and may delay ageing in humans. In the present review we discuss current scientific literature on ER and its molecular, metabolic and hormonal effects. Moreover, criteria for the classification of substances that might induce positive ER-like changes without having to reduce energy intake are summarised. Additionally, the putative ER mimetics (ERM) 2-deoxy-d-glucose, metformin, rapamycin, resveratrol, spermidine and lipoic acid and their suggested molecular targets are discussed. While there are reports on these ERM candidates that describe lifespan extension in model organisms, data on longevity-inducing effects in higher organisms such as mice remain controversial or are missing. Furthermore, some of these candidates produce detrimental side effects such as immunosuppression or lactic acidosis, or have not been tested for safety in long-term studies. Up to now, there are no known ERM that could be recommended without limitations for use in humans.

  3. Near BPS skyrmions and restricted harmonic maps

    NASA Astrophysics Data System (ADS)

    Speight, J. M.

    2015-06-01

    Motivated by a class of near BPS Skyrme models introduced by Adam, Sánchez-Guillén and Wereszczyński, the following variant of the harmonic map problem is introduced: a map φ :(M, g) →(N, h) between Riemannian manifolds is restricted harmonic if it locally extremizes E2 on its SDiff(M) orbit, where SDiff(M) denotes the group of volume preserving diffeomorphisms of (M, g), and E2 denotes the Dirichlet energy. It is conjectured that near BPS skyrmions tend to restricted harmonic maps in the BPS limit. It is shown that φ is restricted harmonic if and only if φ∗ h has exact divergence, and a linear stability theory of restricted harmonic maps is developed, from which it follows that all weakly conformal maps are stable restricted harmonic. Examples of restricted harmonic maps in every degree class R3 → SU(2) and R2 →S2 are constructed. It is shown that the axially symmetric BPS skyrmions on which all previous analytic studies of near BPS Skyrme models have been based, are not restricted harmonic, casting doubt on the phenomenological predictions of such studies. The problem of minimizing E2 for φ :Rk → N over all linear volume preserving diffeomorphisms is solved explicitly, and a deformed axially symmetric family of Skyrme fields constructed which are candidates for approximate near BPS skyrmions at low baryon number. The notion of restricted harmonicity is generalized to restricted F-criticality where F is any functional on maps (M, g) →(N, h) which is, in a precise sense, geometrically natural. The case where F is a linear combination of E2 and E4, the usual Skyrme term, is studied in detail, and it is shown that inverse stereographic projection R3 →S3 ≡ SU(2) is stable restricted F-critical for every such F.

  4. Rotating restricted Schur polynomials

    NASA Astrophysics Data System (ADS)

    Bornman, Nicholas; de Mello Koch, Robert; Tribelhorn, Laila

    2017-09-01

    Large N but nonplanar limits of 𝒩 = 4 super-Yang-Mills theory can be described using restricted Schur polynomials. Previous investigations demonstrate that the action of the one loop dilatation operator on restricted Schur operators, with classical dimension of order N and belonging to the su(2) sector, is largely determined by the su(2) ℛ symmetry algebra as well as structural features of perturbative field theory. Studies presented so far have used the form of ℛ symmetry generators when acting on small perturbations of half-BPS operators. In this paper as a first step towards going beyond small perturbations of the half-BPS operators, we explain how the exact action of symmetry generators on restricted Schur polynomials can be determined.

  5. Perioperative Fluid Restriction

    PubMed Central

    Bleier, Joshua I.S.; Aarons, Cary B.

    2013-01-01

    Perioperative fluid management of the colorectal surgical patient has evolved significantly over the last five decades. Older notions espousing aggressive hydration have been shown to be associated with increased complications. Newer data regarding fluid restriction has shown an association with improved outcomes. Management of perioperative fluid administration can be considered in three primary phases: In the preoperative phase, data suggests that avoidance of preoperative bowel preparation and avoidance of undue preoperative dehydration can improve outcomes. Although the type of intraoperative fluid given does not have a significant effect on outcome, data do suggest that a restrictive fluid regimen results in improved outcomes. Finally, in the postoperative phase of fluid management, a fluid-restrictive regimen, coupled with early enteral feeding also seems to result in improved outcomes. PMID:24436675

  6. Calorie restriction and stroke

    PubMed Central

    2011-01-01

    Stroke, a major cause of disability and mortality in the elderly, occurs when a cerebral blood vessel is occluded or ruptured, resulting in ischemic damage and death of brain cells. The injury mechanism involves metabolic and oxidative stress, excitotoxicity, apoptosis and inflammatory processes, including activation of glial cells and infiltration of leukocytes. In animal models, dietary energy restriction, by daily calorie reduction (CR) or intermittent fasting (IF), extends lifespan and decreases the development of age-related diseases. Dietary energy restriction may also benefit neurons, as suggested by experimental evidence showing that CR and IF protect neurons against degeneration in animal models. Recent findings by our group and others suggest the possibility that dietary energy restriction may protect against stroke induced brain injury, in part by inducing the expression of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF); protein chaperones, including heat shock protein 70 (Hsp70) and glucose regulated protein 78 (GRP78); antioxidant enzymes, such as superoxide dismutases (SOD) and heme oxygenase-1 (HO-1), silent information regulator T1 (SIRT1), uncoupling proteins and anti-inflammatory cytokines. This article discusses the protective mechanisms activated by dietary energy restriction in ischemic stroke. PMID:21910904

  7. Restricting Grammatical Complexity

    ERIC Educational Resources Information Center

    Frank, Robert

    2004-01-01

    Theories of natural language syntax often characterize grammatical knowledge as a form of abstract computation. This paper argues that such a characterization is correct, and that fundamental properties of grammar can and should be understood in terms of restrictions on the complexity of possible grammatical computation, when defined in terms of…

  8. 50 CFR 660.511 - Catch restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... § 660.511 Catch restrictions. (a) All CPS harvested shoreward of the outer boundary of the EEZ (0-200... trip limit for harvesting vessels fishing in the CPS Limited Entry Zone for CPS other than live bait without a limited entry permit is 5 mt tons of all CPS finfish combined. (c)The trip limit for...

  9. 50 CFR 660.511 - Catch restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... § 660.511 Catch restrictions. (a) All CPS harvested shoreward of the outer boundary of the EEZ (0-200... trip limit for harvesting vessels fishing in the CPS Limited Entry Zone for CPS other than live bait without a limited entry permit is 5 mt tons of all CPS finfish combined. (c)The trip limit for...

  10. Genome-wide DNA methylation profiling by modified reduced representation bisulfite sequencing in Brassica rapa suggests that epigenetic modifications play a key role in polyploid genome evolution

    PubMed Central

    Chen, Xun; Ge, Xianhong; Wang, Jing; Tan, Chen; King, Graham J.; Liu, Kede

    2015-01-01

    Brassica rapa includes some of the most important vegetables worldwide as well as oilseed crops. The complete annotated genome sequence confirmed its paleohexaploid origins and provides opportunities for exploring the detailed process of polyploid genome evolution. We generated a genome-wide DNA methylation profile for B. rapa using a modified reduced representation bisulfite sequencing (RRBS) method. This sampling represented 2.24% of all CG loci (2.5 × 105), 2.16% CHG (2.7 × 105), and 1.68% CHH loci (1.05 × 105) (where H = A, T, or C). Our sampling of DNA methylation in B. rapa indicated that 52.4% of CG sites were present as 5mCG, with 31.8% of CHG and 8.3% of CHH. It was found that genic regions of single copy genes had significantly higher methylation compared to those of two or three copy genes. Differences in degree of genic DNA methylation were observed in a hierarchical relationship corresponding to the relative age of the three ancestral subgenomes, primarily accounted by single-copy genes. RNA-seq analysis revealed that overall the level of transcription was negatively correlated with mean gene methylation content and depended on copy number or was associated with the different subgenomes. These results provide new insights into the role epigenetic variation plays in polyploid genome evolution, and suggest an alternative mechanism for duplicate gene loss. PMID:26500672

  11. Whole-genome bisulfite sequencing maps from multiple human tissues reveal novel CpG islands associated with tissue-specific regulation

    PubMed Central

    Mendizabal, Isabel; Yi, Soojin V.

    2016-01-01

    CpG islands (CGIs) are one of the most widely studied regulatory features of the human genome, with critical roles in development and disease. Despite such significance and the original epigenetic definition, currently used CGI sets are typically predicted from DNA sequence characteristics. Although CGIs are deeply implicated in practical analyses of DNA methylation, recent studies have shown that such computational annotations suffer from inaccuracies. Here we used whole-genome bisulfite sequencing from 10 diverse human tissues to identify a comprehensive, experimentally obtained, single-base resolution CGI catalog. In addition to the unparalleled annotation precision, our method is free from potential bias due to arbitrary sequence features or probe affinity differences. In addition to clarifying substantial false positives in the widely used University of California Santa Cruz (UCSC) annotations, our study identifies numerous novel epigenetic loci. In particular, we reveal significant impact of transposable elements on the epigenetic regulatory landscape of the human genome and demonstrate ubiquitous presence of transcription initiation at CGIs, including alternative promoters in gene bodies and non-coding RNAs in intergenic regions. Moreover, coordinated DNA methylation and chromatin modifications mark tissue-specific enhancers at novel CGIs. Enrichment of specific transcription factor binding from ChIP-seq supports mechanistic roles of CGIs on the regulation of tissue-specific transcription. The new CGI catalog provides a comprehensive and integrated list of genomic hotspots of epigenetic regulation. PMID:26512062

  12. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing

    PubMed Central

    Crampton, Mollee; Sripathi, Venkateswara R.; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar (“Sierra”) using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation. PMID:27199997

  13. Bisulfite Sequencing with Daphnia Highlights a Role for Epigenetics in Regulating Stress Response to Microcystis through Preferential Differential Methylation of Serine and Threonine Amino Acids.

    PubMed

    Asselman, Jana; De Coninck, Dieter Im; Beert, Eline; Janssen, Colin R; Orsini, Luisa; Pfrender, Michael E; Decaestecker, Ellen; De Schamphelaere, Karel Ac

    2017-01-17

    Little is known about the influence that environmental stressors may have on genome-wide methylation patterns, and to what extent epigenetics may be involved in environmental stress response. Yet, studies of methylation patterns under stress could provide crucial insights on stress response and toxicity pathways. Here, we focus on genome-wide methylation patterns in the microcrustacean Daphnia magna, a model organism in ecotoxicology and risk assessment, exposed to the toxic cyanobacterium Microcystis aeruginosa. Bisulfite sequencing of exposed and control animals highlighted differential methylation patterns in Daphnia upon exposure to Microcystis primarily in exonic regions. These patterns are enriched for serine/threonine amino acid codons and genes related to protein synthesis, transport and degradation. Furthermore, we observed that genes with differential methylation corresponded well with genes susceptible to alternative splicing in response to Microcystis stress. Overall, our results suggest a complex mechanistic response in Daphnia characterized by interactions between DNA methylation and gene regulation mechanisms. These results underscore that DNA methylation is modulated by environmental stress and can also be an integral part of the toxicity response in our study species.

  14. Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

    PubMed

    Crampton, Mollee; Sripathi, Venkateswara R; Hossain, Khwaja; Kalavacharla, Venu

    2016-01-01

    Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation.

  15. Frequencies of restriction sites.

    PubMed Central

    Waterman, M S

    1983-01-01

    Restriction sites or other sequence patterns are usually assumed to occur according to a Poisson distribution with mean equal to the reciprocal of the probability of the given site or pattern. For situations where non-overlapping occurrences of patterns, such as restriction sites, are the objects of interest, this note shows that the Poisson assumption is frequently misleading. Both the case of base composition (independent bases) and of dinucleotide frequencies (Markov chains) are treated. Moreover, a new technique is presented which allows treatment of collections of patterns, where the departure from the Poisson assumption is even more striking. This later case includes double digests, and an example of a five enzyme digest is included. PMID:6324109

  16. License restrictions at Barnwell

    SciTech Connect

    Autry, V.R.

    1991-12-31

    The State of South Carolina was delegated the authority by the US Nuclear Regulatory Commission to regulate the receipt, possession, use and disposal of radioactive material as an Agreement State. Since 1970, the state has been the principal regulatory authority for the Barnwell Low-Level Waste Disposal Facility operated by Chem-Nuclear Systems, Inc. The radioactive material license issued authorizing the receipt and disposal of low-level waste contains numerous restrictions to ensure environmental protection and compliance with shallow land disposal performance criteria. Low-level waste has evolved from minimally contaminated items to complex waste streams containing high concentrations of radionuclides and processing chemicals which necessitated these restrictions. Additionally, some waste with their specific radionuclides and concentration levels, many classified as low-level radioactive waste, are not appropriate for shallow land disposal unless additional precautions are taken. This paper will represent a number of these restrictions, the rationale for them, and how they are being dealt with at the Barnwell disposal facility.

  17. A systematic review of the separate and combined effects of energy restriction and exercise on fat-free mass in middle-aged and older adults: implications for sarcopenic obesity.

    PubMed

    Weinheimer, Eileen M; Sands, Laura P; Campbell, Wayne W

    2010-07-01

    The systematic review presented here assessed the effects of energy restriction (ER) and exercise (EX) on fat-free mass (FFM) in overweight and obese middle-aged and older adults. PubMed was searched using the key words "weight loss or energy restriction" AND "skeletal muscle or body composition," with limitations set for "human" and "middle-aged and aged." Results from 52 studies are reported as the percentages of EX (mainly aerobic training), ER, or ER+EX groups that had a specified change in body weight and FFM, since insufficient data were available for a meta-analysis. The EX groups had modest body weight and FFM changes. Eighty-one percent and 39% of the ER and ER+EX groups, respectively, lost > or = 15% of body weight as FFM. These findings suggest that exercise is an effective tool to help men and postmenopausal women aged > or = 50 years, with a BMI greater than 25 kg/m(2) preserve FFM after moderate ER-induced weight loss, which is important for combating sarcopenic obesity.

  18. Is fluid restriction needed in heart failure?

    PubMed

    Castro-Gutiérrez, Victoria; Rada, Gabriel

    2017-01-09

    Fluid restriction is usually recommended in chronic heart failure. However, the evidence base to support this is not that clear. Searching in Epistemonikos database, which is maintained by screening multiple databases, we identified five systematic reviews evaluating 11 studies addressing the question of this article, including seven randomized trials. We extracted data, combined the evidence using meta-analysis and generated a summary of findings table following the GRADE approach. We concluded fluid restriction probably decreases hospital readmission in chronic heart failure and might decrease mortality, but the certainty of the evidence for the latter is low.

  19. APOBECs and Virus Restriction

    PubMed Central

    Harris, Reuben S.; Dudley, Jaquelin P.

    2015-01-01

    The APOBEC family of single-stranded DNA cytosine deaminases comprises a formidable arm of the vertebrate innate immune system. Pre-vertebrates express a single APOBEC, whereas some mammals produce as many as eleven enzymes. The APOBEC3 subfamily displays both copy number variation and polymorphisms, consistent with ongoing pathogenic pressures. These enzymes restrict the replication of many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. APOBEC1 and activation-induced cytosine deaminase (AID) have specialized functions in RNA editing and antibody gene diversification, respectively, whereas APOBEC2 and APOBEC4 appear to have different functions. Nevertheless, the APOBEC family protects against both periodic viral zoonoses as well as exogenous and endogenous parasite replication. This review highlights viral pathogens that are restricted by APOBEC enzymes, but manage to escape through unique mechanisms. The sensitivity of viruses that lack counterdefense measures highlights the need to develop APOBEC-enabling small molecules as a new class of anti-viral drugs. PMID:25818029

  20. Microorganisms with Novel Dissimilatory (Bi)Sulfite Reductase Genes Are Widespread and Part of the Core Microbiota in Low-Sulfate Peatlands ▿ †

    PubMed Central

    Steger, Doris; Wentrup, Cecilia; Braunegger, Christina; Deevong, Pinsurang; Hofer, Manuel; Richter, Andreas; Baranyi, Christian; Pester, Michael; Wagner, Michael; Loy, Alexander

    2011-01-01

    Peatlands of the Lehstenbach catchment (Germany) house as-yet-unidentified microorganisms with phylogenetically novel variants of the dissimilatory (bi)sulfite reductase genes dsrAB. These genes are characteristic of microorganisms that reduce sulfate, sulfite, or some organosulfonates for energy conservation but can also be present in anaerobic syntrophs. However, nothing is currently known regarding the abundance, community dynamics, and biogeography of these dsrAB-carrying microorganisms in peatlands. To tackle these issues, soils from a Lehstenbach catchment site (Schlöppnerbrunnen II fen) from different depths were sampled at three time points over a 6-year period to analyze the diversity and distribution of dsrAB-containing microorganisms by a newly developed functional gene microarray and quantitative PCR assays. Members of novel, uncultivated dsrAB lineages (approximately representing species-level groups) (i) dominated a temporally stable but spatially structured dsrAB community and (ii) represented “core” members (up to 1% to 1.7% relative abundance) of the autochthonous microbial community in this fen. In addition, denaturing gradient gel electrophoresis (DGGE)- and clone library-based comparisons of the dsrAB diversity in soils from a wet meadow, three bogs, and five fens of various geographic locations (distance of ∼1 to 400 km) identified that one Syntrophobacter-related and nine novel dsrAB lineages are widespread in low-sulfate peatlands. Signatures of biogeography in dsrB-based DGGE data were not correlated with geographic distance but could be explained largely by soil pH and wetland type, implying that the distribution of dsrAB-carrying microorganisms in wetlands on the scale of a few hundred kilometers is not limited by dispersal but determined by local environmental conditions. PMID:21169452

  1. Restrictive chest wall disorders.

    PubMed

    Donath, Joseph; Miller, Albert

    2009-06-01

    Hypoventilation can be caused by diseases of the chest wall. Any anatomical or functional abnormality of the bony thorax increases dead space ventilation and the work of breathing, whether congenital or acquired, acute or chronic, and whether its cause is infectious, traumatic, environmental, iatrogenic, or unknown. In this article, we discuss these heterogeneous disorders from the viewpoint of the practicing nonpediatric pulmonary physician, only briefly touching on surgical, pediatric, rheumatologic, and other nonpulmonary ramifications. Emphasis is on the most common and the best researched forms of chest wall restriction, including kyphoscoliosis, fibrothorax, thoracoplasty, flail chest, and ankylosing spondylitis. Other diseases such as osteoporosis with its less well known pulmonary effects, and some rarely seen entities, are briefly discussed.

  2. Restriction-modification mediated barriers to exogenous DNA uptake and incorporation employed by Prevotella intermedia.

    PubMed

    Johnston, Christopher D; Skeete, Chelsey A; Fomenkov, Alexey; Roberts, Richard J; Rittling, Susan R

    2017-01-01

    Prevotella intermedia, a major periodontal pathogen, is increasingly implicated in human respiratory tract and cystic fibrosis lung infections. Nevertheless, the specific mechanisms employed by this pathogen remain only partially characterized and poorly understood, largely due to its total lack of genetic accessibility. Here, using Single Molecule, Real-Time (SMRT) genome and methylome sequencing, bisulfite sequencing, in addition to cloning and restriction analysis, we define the specific genetic barriers to exogenous DNA present in two of the most widespread laboratory strains, P. intermedia ATCC 25611 and P. intermedia Strain 17. We identified and characterized multiple restriction-modification (R-M) systems, some of which are considerably divergent between the two strains. We propose that these R-M systems are the root cause of the P. intermedia transformation barrier. Additionally, we note the presence of conserved Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) systems in both strains, which could provide a further barrier to exogenous DNA uptake and incorporation. This work will provide a valuable resource during the development of a genetic system for P. intermedia, which will be required for fundamental investigation of this organism's physiology, metabolism, and pathogenesis in human disease.

  3. Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII

    PubMed Central

    Pawlak, Sebastian D.; Radlinska, Monika; Chmiel, Agnieszka A.; Bujnicki, Janusz M.; Skowronek, Krzysztof J.

    2005-01-01

    Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5′GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5′-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity. PMID:15684412

  4. A combination of probiotics and whey proteins enhances anti-obesity effects of calcium and dairy products during nutritional energy restriction in aP2-agouti transgenic mice.

    PubMed

    Yoda, Kazutoyo; Sun, Xiaocum; Kawase, Manabu; Kubota, Akira; Miyazawa, Kenji; Harata, Gaku; Hosoda, Masataka; Hiramatsu, Masaru; He, Fang; Zemel, Michael B

    2015-06-14

    Lactobacillus rhamnosus GG, Lactobacillus paracasei TMC0409, Streptococcus thermophilus TMC1543 and whey proteins were used to prepare fermented milk. For the experiment aP2- agouti transgenic mice were pre-treated with a high-sucrose/high-fat diet for 6 weeks to induce obesity. The obese mice were fed a diet containing 1·2% Ca and either non-fat dried milk (NFDM) or probiotic-fermented milk (PFM) with nutritional energy restriction for 6 weeks. The animals were examined after the treatment for changes in body weight, fat pad weight, fatty acid synthase (FAS) activity, lypolysis, the expression levels of genes related to lipid metabolism, insulin sensitivity in adipocytes and skeletal muscle and the presence of biomarkers for oxidative and inflammatory stress in plasma. It was found that the PFM diet significantly reduced body weight, fat accumulation, and adipocyte FAS activity, and increased adipocyte lipolysis as compared with the effects of the NFDM diet (P<0·05). The adipose tissue gene expression of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) was significantly suppressed in mice that were fed PFM as compared with those that were fed NFDM (P<0·05). PFM caused a greater up-regulation of skeletal muscle PPARα, PPARδ, uncoupling protein 3 (UCP3) and GLUT4 expression and a significant decrease in the plasma concentration of insulin, malondialdehyde, TNF-α, monocyte chemotactic protein-1 and C-reactive protein as compared with the effects of NFDM (P<0·05). Fermentation of milk with selected probiotics and supplementation of milk with whey proteins may thus enhance anti-obesity effects of Ca and dairy products by the suppression of adipose tissue lipogenesis, activation of fat oxidation in skeletal muscle and reduction of oxidative and inflammatory stress.

  5. Protein deficiency and energy restriction in young cebus monkeys.

    PubMed

    Samonds, K W; Hegsted, D M

    1978-03-01

    Infant cebus monkeys (Cebus albifrons) were fed liquid formulas that were limited in protein, energy, or a combination of the two restrictions. Weight gain, food intake, hematological development, and plasma protein and cholesterol levels were monitored over a 20-week period. The animals restricted in protein developed the classical signs of protein deficiency--reductions in plasma albumin, a mild anemia, accumulation of fat in the liver, and, in a few cases, facial edema. These animals maintained a relatively high energy intake, and apparently wasted energy when compared to similarly non-growing energy-restricted animals. Energy-restricted animals did not exhibit these symptoms, even when their daily protein intake was reduced to match that of protein-restricted monkeys. It is concluded that an energy restriction superimposed upon a limited protein intake did not increase protein requirements or precipitate protein deficiency.

  6. Protein deficiency and energy restriction in young cebus monkeys.

    PubMed Central

    Samonds, K W; Hegsted, D M

    1978-01-01

    Infant cebus monkeys (Cebus albifrons) were fed liquid formulas that were limited in protein, energy, or a combination of the two restrictions. Weight gain, food intake, hematological development, and plasma protein and cholesterol levels were monitored over a 20-week period. The animals restricted in protein developed the classical signs of protein deficiency--reductions in plasma albumin, a mild anemia, accumulation of fat in the liver, and, in a few cases, facial edema. These animals maintained a relatively high energy intake, and apparently wasted energy when compared to similarly non-growing energy-restricted animals. Energy-restricted animals did not exhibit these symptoms, even when their daily protein intake was reduced to match that of protein-restricted monkeys. It is concluded that an energy restriction superimposed upon a limited protein intake did not increase protein requirements or precipitate protein deficiency. Images PMID:418417

  7. Restrictive vs. non-restrictive composition: a magnetoencephalography study

    PubMed Central

    Leffel, Timothy; Lauter, Miriam; Westerlund, Masha; Pylkkänen, Liina

    2014-01-01

    Recent research on the brain mechanisms underlying language processing has implicated the left anterior temporal lobe (LATL) as a central region for the composition of simple phrases. Because these studies typically present their critical stimuli without contextual information, the sensitivity of LATL responses to contextual factors is unknown. In this magnetoencephalography (MEG) study, we employed a simple question-answer paradigm to manipulate whether a prenominal adjective or determiner is interpreted restrictively, i.e., as limiting the set of entities under discussion. Our results show that the LATL is sensitive to restriction, with restrictive composition eliciting higher responses than non-restrictive composition. However, this effect was only observed when the restricting element was a determiner, adjectival stimuli showing the opposite pattern, which we hypothesise to be driven by the special pragmatic properties of non-restrictive adjectives. Overall, our results demonstrate a robust sensitivity of the LATL to high level contextual and potentially also pragmatic factors. PMID:25379512

  8. Retroviral restriction: nature's own solution.

    PubMed

    Jones, Christopher E; McKnight, Áine

    2016-12-01

    The present review will discuss recent advances in the development of anti-HIV therapies inspired by studies of the mechanisms of host restriction factor-mediated resistance to HIV infection. Manipulating the interplay between host cell restriction factors and viral accessory factors that overcome them can potentially be therapeutically useful. Preliminarily successful therapies - some of which are entering clinical trials - either inhibit the ability of virus to evade restriction factor-mediated immunity, or promote intracellular levels of restriction factors. These aims are achieved by multiple means, which are discussed. Many restriction factors appear to provide potentially useful targets for anti-HIV therapies, so time and interest should be invested in investigating ways to successfully therapeutically manipulate restriction factor-mediated immunity.

  9. Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring.

    PubMed

    Zhang, Qian; Sun, Xiaofang; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

    2017-01-01

    Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet) or a low chromium diet (LC, 0.14 mg chromium/kg diet) during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON), while others remained on the same diet (CON-CON, or LC-LC) for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA methylation which

  10. Dietary Chromium Restriction of Pregnant Mice Changes the Methylation Status of Hepatic Genes Involved with Insulin Signaling in Adult Male Offspring

    PubMed Central

    Zhang, Qian; Sun, Xiaofang; Xiao, Xinhua; Zheng, Jia; Li, Ming; Yu, Miao; Ping, Fan; Wang, Zhixin; Qi, Cuijuan; Wang, Tong; Wang, Xiaojing

    2017-01-01

    Maternal undernutrition is linked with an elevated risk of diabetes mellitus in offspring regardless of the postnatal dietary status. This is also found in maternal micro-nutrition deficiency, especial chromium which is a key glucose regulator. We investigated whether maternal chromium restriction contributes to the development of diabetes in offspring by affecting DNA methylation status in liver tissue. After being mated with control males, female weanling 8-week-old C57BL mice were fed a control diet (CON, 1.19 mg chromium/kg diet) or a low chromium diet (LC, 0.14 mg chromium/kg diet) during pregnancy and lactation. After weaning, some offspring were shifted to the other diet (CON-LC, or LC-CON), while others remained on the same diet (CON-CON, or LC-LC) for 29 weeks. Fasting blood glucose, serum insulin, and oral glucose tolerance test was performed to evaluate the glucose metabolism condition. Methylation differences in liver from the LC-CON group and CON-CON groups were studied by using a DNA methylation array. Bisulfite sequencing was carried out to validate the results of the methylation array. Maternal chromium limitation diet increased the body weight, blood glucose, and serum insulin levels. Even when switched to the control diet after weaning, the offspring also showed impaired glucose tolerance and insulin resistance. DNA methylation profiling of the offspring livers revealed 935 differentially methylated genes in livers of the maternal chromium restriction diet group. Pathway analysis identified the insulin signaling pathway was the main process affected by hypermethylated genes. Bisulfite sequencing confirmed that some genes in insulin signaling pathway were hypermethylated in livers of the LC-CON and LC-LC group. Accordingly, the expression of genes in insulin signaling pathway was downregulated. There findings suggest that maternal chromium restriction diet results in glucose intolerance in male offspring through alterations in DNA methylation which

  11. Characterization of the primary structure of T cell receptor beta chains in cells infiltrating the salivary gland in the sicca syndrome of HIV-1 infection. Evidence of antigen-driven clonal selection suggested by restricted combinations of V beta J beta gene segment usage and shared somatically encoded amino acid residues.

    PubMed Central

    Dwyer, E; Itescu, S; Winchester, R

    1993-01-01

    Infection with HIV-1 occasionally results in a sicca syndrome, termed the diffuse infiltrative lymphocytosis syndrome, characterized by infiltration of the salivary glands with a predominance of CD8 T cells. This response is strongly associated with certain MHC class I and class II alleles. To define the salivary gland T cell receptor (TCR) repertoire, the primary structure of the TCR beta-chains was determined using in situ cDNA synthesis followed by the "anchored" polymerase chain reaction. The sequences of 59 beta-chains from five individuals with diffuse infiltrative lymphocytosis syndrome shared structural features suggesting antigenic clonal selection. Certain combinations of V beta J beta gene segments were selectively overrepresented in the repertoire sample, demonstrating a common restricted usage of certain V beta and J beta gene segments. The beta-chains derived from these overrepresented V beta J beta combinations revealed a preference for specific amino acids at position 97 in the third complementarity-determining region, a residue postulated to contact peptide antigen. Moreover, the nucleotides encoding this position were not germline in origin. TCR beta-chains in nonoverrepresented V beta J beta combinations did not exhibit preferential usage of selected somatically encoded residues. The pattern of TCR beta-chains expressed in the salivary gland of a control person with primary Sjögren's syndrome was considerably more heterogeneous and different from that found in diffuse infiltrative lymphocytosis syndrome. Images PMID:8392093

  12. AGU Sonar Data Restriction Panel

    NASA Astrophysics Data System (ADS)

    The AGU Council accepted the report of the panel set up in February to study the issue of restriction by the U.S. Navy of access to high-resolution sonar data for the U.S. Exclusive Economic Zone. Panel chairman John Bossier announced that “the Navy has acted in the best interests of the nation” in lifting the restriction order. Only two areas, egress routes to two submarine bases (see “Navy Defines Areas Under Sonar Ban,” in News, this issue), remain restricted.Panel members were Bruce Douglas, Alexander Malahoff, Donald Piepgras, Paul Richards, David Smith and Manik Talwani.

  13. A restriction mapping engine using constraint logic programming

    SciTech Connect

    Dix, T.I.; Yee, C.N.

    1994-12-31

    Restriction mapping generally requires the application of information from various digestions by restriction enzymes to find solution sets. We use both the predicate calculus and constraint solving capabilities of CLP(R) to develop an engine for restriction mapping. Many of the techniques employed by biologists to manually find solutions are supported by the engine in a consistent manner. We provide generalized pipeline and cross-multiply operators for combining sub-maps. Our approach encourages the building of maps iteratively. We show how other techniques can be readily incorporated.

  14. Novel subtype of type IIs restriction enzymes. BfiI endonuclease exhibits similarities to the EDTA-resistant nuclease Nuc of Salmonella typhimurium.

    PubMed

    Sapranauskas, R; Sasnauskas, G; Lagunavicius, A; Vilkaitis, G; Lubys, A; Siksnys, V

    2000-10-06

    The type IIs restriction enzyme BfiI recognizes the non-palindromic nucleotide sequence 5'-ACTGGG-3' and cleaves complementary DNA strands 5/4 nucleotides downstream of the recognition sequence. The genes coding for the BfiI restriction-modification (R-M) system were cloned/sequenced and biochemical characterization of BfiI restriction enzyme was performed. The BfiI R-M system contained three proteins: two N4-methylcytosine methyltransferases and a restriction enzyme. Sequencing of bisulfite-treated methylated DNA indicated that each methyltransferase modifies cytosines on opposite strands of the recognition sequence. The N-terminal part of the BfiI restriction enzyme amino acid sequence revealed intriguing similarities to an EDTA-resistant nuclease of Salmonella typhimurium. Biochemical analyses demonstrated that BfiI, like the nuclease of S. typhimurium, cleaves DNA in the absence of Mg(2+) ions and hydrolyzes an artificial substrate bis(p-nitrophenyl) phosphate. However, unlike the nonspecific S. typhimurium nuclease, BfiI restriction enzyme cleaves DNA specifically. We propose that the DNA-binding specificity of BfiI stems from the C-terminal part of the protein. The catalytic N-terminal subdomain of BfiI radically differs from that of type II restriction enzymes and is presumably similar to the EDTA-resistant nonspecific nuclease of S. typhimurium; therefore, BfiI did not require metal ions for catalysis. We suggest that BfiI represents a novel subclass of type IIs restriction enzymes that differs from the archetypal FokI endonuclease by the fold of its cleavage domain, the domain location, and reaction mechanism.

  15. Methionine restriction and lifespan control

    PubMed Central

    Lee, Byung Cheon; Kaya, Alaattin; Gladyshev, Vadim N.

    2016-01-01

    Dietary restriction (DR) without malnutrition is associated with longevity in various organisms. However, it has also been shown that reduced calorie intake is often ineffective in extending lifespan. Selecting optimal dietary regimens for DR studies is complicated, as the same regimen may lead to different outcomes depending on genotype and environmental factors. Recent studies suggested that interventions such as moderate protein restriction with/without adequate nutrition (e.g. particular amino acids or carbohydrates) may have additional beneficial effects mediated by certain metabolic and hormonal factors implicated in the biology of aging, regardless of total calorie intake. In particular, it was shown that restriction of a single amino acid, methionine, can mimic the effects of DR and extend lifespan in various model organisms. We discuss beneficial effects of methionine-restricted (MR) diet, the molecular pathways involved, and the use of this regimen in longevity interventions. PMID:26663138

  16. Using Pinochle to Motivate the Restricted Combinations with Repetitions Problem

    ERIC Educational Resources Information Center

    Gorman, Patrick S.; Kunkel, Jeffrey D.; Vasko, Francis J.

    2011-01-01

    A standard example used in introductory combinatoric courses is to count the number of five-card poker hands possible from a straight deck of 52 distinct cards. A more interesting problem is to count the number of distinct hands possible from a Pinochle deck in which there are multiple, but obviously limited, copies of each type of card (two…

  17. Genetics Home Reference: familial restrictive cardiomyopathy

    MedlinePlus

    ... Home Health Conditions familial restrictive cardiomyopathy familial restrictive cardiomyopathy Printable PDF Open All Close All Enable Javascript ... view the expand/collapse boxes. Description Familial restrictive cardiomyopathy is a genetic form of heart disease. For ...

  18. 25 CFR 152.25 - Negotiated sales, gifts and exchanges of trust or restricted lands.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... restricted land, or a combination of such land and other things of value, for other lands or combinations of land and other things of value. The value of the consideration received by the Indian in the exchange... value. With the approval of the Secretary, Indian owners may convey trust or restricted land, for...

  19. Maternal food restrictions during breastfeeding

    PubMed Central

    Jeong, Goun; Park, Sung Won; Lee, Yeon Kyung; Ko, Sun Young

    2017-01-01

    Purpose This study investigated self-food restriction during breastfeeding, reviewed the literature showing the effect of maternal diet on the health of breast-fed infants, and explored the validity of dietary restrictions. Methods Questionnaire data were collected from breastfeeding Korean mothers who visited the pediatric clinic of Cheil General Hospital & Women's Healthcare Center from July 2015 through August 2015. The survey included items assessing maternal age, number of children, maternal educational attainment, household income, degree of difficulty with self-food restriction, types of self-restricted foods, dietary customs during breastfeeding, and sources of information about breastfeeding. Results The questionnaire was completed by 145 mothers. More than a third (n=56, 39%) had discomfort from and usually avoided 4–5 types of food (mean, 4.92). Mothers younger than 40 years had more discomfort (odds ratio [OR], 12.762; P=0.017). Primiparas felt less discomfort than multiparas (OR, 0.436; P=0.036). Dietary practices were not influenced by maternal educational attainment or household income. The most common self-restricted foods were caffeine (n=131, 90.3%), spicy foods (n=124, 85.5%), raw foods (n=109, 75.2%), cold foods (n=100, 69%), and sikhye (traditional sweet Korean rice beverage) (n=100, 69%). Most mothers (n=122, 84.1%) avoided foods for vague reasons. Conclusion Most mothers restricted certain foods unnecessarily. Literature review identified no foods that mothers should absolutely avoid during breastfeeding unless the infant reacts negatively to the food. PMID:28392822

  20. Generalized Pump-restriction Theorem

    SciTech Connect

    Sinitsyn, Nikolai A; Chernyak, Vladimir Y

    2008-01-01

    We formulate conditions under which periodic modulations of parameters on a finite graph with stochastic transitions among its nodes do not lead to overall pump currents through any given link. Our theorem unifies previously known results with the new ones and provides a universal approach to explore futher restrictions on stochastic pump effect in non-adiabatically driven systems with detailed balance.

  1. Controlling Pandemic Flu: The Value of International Air Travel Restrictions

    PubMed Central

    Epstein, Joshua M.; Goedecke, D. Michael; Yu, Feng; Morris, Robert J.; Wagener, Diane K.; Bobashev, Georgiy V.

    2007-01-01

    Background Planning for a possible influenza pandemic is an extremely high priority, as social and economic effects of an unmitigated pandemic would be devastating. Mathematical models can be used to explore different scenarios and provide insight into potential costs, benefits, and effectiveness of prevention and control strategies under consideration. Methods and Findings A stochastic, equation-based epidemic model is used to study global transmission of pandemic flu, including the effects of travel restrictions and vaccination. Economic costs of intervention are also considered. The distribution of First Passage Times (FPT) to the United States and the numbers of infected persons in metropolitan areas worldwide are studied assuming various times and locations of the initial outbreak. International air travel restrictions alone provide a small delay in FPT to the U.S. When other containment measures are applied at the source in conjunction with travel restrictions, delays could be much longer. If in addition, control measures are instituted worldwide, there is a significant reduction in cases worldwide and specifically in the U.S. However, if travel restrictions are not combined with other measures, local epidemic severity may increase, because restriction-induced delays can push local outbreaks into high epidemic season. The per annum cost to the U.S. economy of international and major domestic air passenger travel restrictions is minimal: on the order of 0.8% of Gross National Product. Conclusions International air travel restrictions may provide a small but important delay in the spread of a pandemic, especially if other disease control measures are implemented during the afforded time. However, if other measures are not instituted, delays may worsen regional epidemics by pushing the outbreak into high epidemic season. This important interaction between policy and seasonality is only evident with a global-scale model. Since the benefit of travel restrictions

  2. 5 CFR 300.604 - Restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT (GENERAL) Time-In-Grade Restrictions § 300.604 Restrictions. The following time-in-grade restrictions must be met unless... may be advanced without time restriction to positions up to GS-5 if the position to be filled is...

  3. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 5 2010-01-01 2010-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as amended...

  4. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 14 Aeronautics and Space 5 2012-01-01 2012-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as amended...

  5. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 5 2011-01-01 2010-01-01 true Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as amended...

  6. 14 CFR 1203.305 - Restricted data.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 14 Aeronautics and Space 5 2013-01-01 2013-01-01 false Restricted data. 1203.305 Section 1203.305... Classification Principles and Considerations § 1203.305 Restricted data. Restricted Data or Formerly Restricted Data is so classified when originated, as required by the Atomic Energy Act of 1954, as amended...

  7. 30 CFR 57.11008 - Restricted clearance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Restricted clearance. 57.11008 Section 57.11008... Escapeways Travelways-Surface and Underground § 57.11008 Restricted clearance. Where restricted clearance creates a hazard to persons, the restricted clearance shall be conspicuously marked....

  8. 30 CFR 56.11008 - Restricted clearance.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Restricted clearance. 56.11008 Section 56.11008 Mineral Resources MINE SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR METAL AND NONMETAL MINE... Restricted clearance. Where restricted clearance creates a hazard to persons, the restricted clearance...

  9. 5 CFR 300.604 - Restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT (GENERAL) Time-In-Grade Restrictions § 300.604 Restrictions. The following time-in-grade restrictions must be met unless... may be advanced without time restriction to positions up to GS-5 if the position to be filled is no...

  10. 5 CFR 300.604 - Restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT (GENERAL) Time-In-Grade Restrictions § 300.604 Restrictions. The following time-in-grade restrictions must be met unless... may be advanced without time restriction to positions up to GS-5 if the position to be filled is no...

  11. 5 CFR 300.604 - Restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT (GENERAL) Time-In-Grade Restrictions § 300.604 Restrictions. The following time-in-grade restrictions must be met unless... may be advanced without time restriction to positions up to GS-5 if the position to be filled is no...

  12. 5 CFR 300.604 - Restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT CIVIL SERVICE REGULATIONS EMPLOYMENT (GENERAL) Time-In-Grade Restrictions § 300.604 Restrictions. The following time-in-grade restrictions must be met unless... may be advanced without time restriction to positions up to GS-5 if the position to be filled is no...

  13. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 4 2011-10-01 2011-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  14. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  15. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  16. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  17. 49 CFR 215.203 - Restricted cars.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 4 2013-10-01 2013-10-01 false Restricted cars. 215.203 Section 215.203..., DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Restricted Equipment § 215.203 Restricted cars. (a) This section restricts the operation of any railroad freight car that is— (1) More than...

  18. Overcoming Communication Restrictions in Collectives

    NASA Technical Reports Server (NTRS)

    Tumer, Kagan; Agogino, Adrian K.

    2004-01-01

    Many large distributed system are characterized by having a large number of components (eg., agents, neurons) whose actions and interactions determine a %orld utility which rates the performance of the overall system. Such collectives are often subject to communication restrictions, making it difficult for components which try to optimize their own private utilities, to take actions that also help optimize the world utility. In this article we address that coordination problem and derive four utility functions which present different compromises between how aligned a component s private utility is with the world utility and how readily that component can determine the actions that optimize its utility. The results show that the utility functions specifically derived to operate under communication restrictions outperform both traditional methods and previous collective-based methods by up to 75%.

  19. Temperature based Restricted Boltzmann Machines

    NASA Astrophysics Data System (ADS)

    Li, Guoqi; Deng, Lei; Xu, Yi; Wen, Changyun; Wang, Wei; Pei, Jing; Shi, Luping

    2016-01-01

    Restricted Boltzmann machines (RBMs), which apply graphical models to learning probability distribution over a set of inputs, have attracted much attention recently since being proposed as building blocks of multi-layer learning systems called deep belief networks (DBNs). Note that temperature is a key factor of the Boltzmann distribution that RBMs originate from. However, none of existing schemes have considered the impact of temperature in the graphical model of DBNs. In this work, we propose temperature based restricted Boltzmann machines (TRBMs) which reveals that temperature is an essential parameter controlling the selectivity of the firing neurons in the hidden layers. We theoretically prove that the effect of temperature can be adjusted by setting the parameter of the sharpness of the logistic function in the proposed TRBMs. The performance of RBMs can be improved by adjusting the temperature parameter of TRBMs. This work provides a comprehensive insights into the deep belief networks and deep learning architectures from a physical point of view.

  20. Temperature based Restricted Boltzmann Machines.

    PubMed

    Li, Guoqi; Deng, Lei; Xu, Yi; Wen, Changyun; Wang, Wei; Pei, Jing; Shi, Luping

    2016-01-13

    Restricted Boltzmann machines (RBMs), which apply graphical models to learning probability distribution over a set of inputs, have attracted much attention recently since being proposed as building blocks of multi-layer learning systems called deep belief networks (DBNs). Note that temperature is a key factor of the Boltzmann distribution that RBMs originate from. However, none of existing schemes have considered the impact of temperature in the graphical model of DBNs. In this work, we propose temperature based restricted Boltzmann machines (TRBMs) which reveals that temperature is an essential parameter controlling the selectivity of the firing neurons in the hidden layers. We theoretically prove that the effect of temperature can be adjusted by setting the parameter of the sharpness of the logistic function in the proposed TRBMs. The performance of RBMs can be improved by adjusting the temperature parameter of TRBMs. This work provides a comprehensive insights into the deep belief networks and deep learning architectures from a physical point of view.

  1. Purpose Restrictions on Information Use

    DTIC Science & Technology

    2013-06-03

    solutions. In Proceedings of the International Conference on Automated Planning and Scheduling (2011), F. Bacchus, C . Domshlak, S . Edelkamp, and M. Helmert...TITLE AND SUBTITLE Purpose Restrictions on Information Use 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) 5d...PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME( S ) AND ADDRESS(ES) Carnegie Mellon University,CyLab,Pittsburgh,PA

  2. Fetal growth restriction: current knowledge.

    PubMed

    Nardozza, Luciano Marcondes Machado; Caetano, Ana Carolina Rabachini; Zamarian, Ana Cristina Perez; Mazzola, Jaqueline Brandão; Silva, Carolina Pacheco; Marçal, Vivian Macedo Gomes; Lobo, Thalita Frutuoso; Peixoto, Alberto Borges; Araujo Júnior, Edward

    2017-05-01

    Fetal growth restriction (FGR) is a condition that affects 5-10% of pregnancies and is the second most common cause of perinatal mortality. This review presents the most recent knowledge on FGR and focuses on the etiology, classification, prediction, diagnosis, and management of the condition, as well as on its neurological complications. The Pubmed, SCOPUS, and Embase databases were searched using the term "fetal growth restriction". Fetal growth restriction (FGR) may be classified as early or late depending on the time of diagnosis. Early FGR (<32 weeks) is associated with substantial alterations in placental implantation with elevated hypoxia, which requires cardiovascular adaptation. Perinatal morbidity and mortality rates are high. Late FGR (≥32 weeks) presents with slight deficiencies in placentation, which leads to mild hypoxia and requires little cardiovascular adaptation. Perinatal morbidity and mortality rates are lower. The diagnosis of FGR may be clinical; however, an arterial and venous Doppler ultrasound examination is essential for diagnosis and follow-up. There are currently no treatments to control FGR; the time at which pregnancy is interrupted is of vital importance for protecting both the mother and fetus. Early diagnosis of FGR is very important, because it enables the identification of the etiology of the condition and adequate monitoring of the fetal status, thereby minimizing risks of premature birth and intrauterine hypoxia.

  3. Postoperative activity restrictions: any evidence?

    PubMed

    Weir, Larissa F; Nygaard, Ingrid E; Wilken, Jason; Brandt, Debra; Janz, Kathleen F

    2006-02-01

    Because of a widespread but untested belief that increased intra-abdominal pressure contributes to pelvic floor disorders, physicians commonly restrict various activities postoperatively. Our aim was to describe intra-abdominal pressures during common physical activities. Thirty women of wide age and weight ranges who were not undergoing treatment for pelvic floor disorders performed 3 repetitions of various activities while intra-abdominal pressures (baseline and maximal) were approximated via microtip rectal catheters. We calculated median peak and net pressures (centimeters of H(2)O). We assessed correlations between abdominal pressures and body mass index, abdominal circumference, and grip strength (a proxy for overall strength). P < .025 was considered significant. Median peak abdominal pressures ranged from 48 (lifting 8 lb from a counter) to 150 (lifting 35 lb from the floor), with much variation. Many activities did not raise the intra-abdominal pressure more than simply getting out of a chair, including lifting 8, 13, and 20 lb from a counter, lifting 8 or 13 lb from the floor, climbing stairs, walking briskly, or doing abdominal crunches. Body mass index and abdominal circumference each correlated positively with peak, but not net, pressures. Age and grip strength were not associated with abdominal pressure. Some activities commonly restricted postoperatively have no greater effect on intra-abdominal pressures than unavoidable activities like rising from a chair. How lifting is done impacts intra-abdominal pressure. Many current postoperative guidelines are needlessly restrictive. Further research is needed to determine whether increased intra-abdominal pressure truly promotes pelvic floor disorders. III.

  4. Restricted Transport in Small Pores

    PubMed Central

    Anderson, John L.; Quinn, John A.

    1974-01-01

    The basic hydrodynamic equations governing transport in submicron pores are reexamined. Conditions necessary for a simplified, one-dimensional treatment of the diffusion/convection process are established. Steric restrictions and Brownian motion are incorporated directly into the resulting model. Currently available fluid mechanical results are used to evaluate an upper limit on hindered diffusion; this limit is valid for small particle-to-pore ratios. Extensions of the analysis are shown to depend on numerical solutions of the related hydrodynamic problem, that of asymmetrical particle motion in a bounded fluid. PMID:4813157

  5. 9 CFR 78.20 - General restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Bison Because of Brucellosis § 78.20 General restrictions. Bison may not be...

  6. 9 CFR 78.5 - General restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS BRUCELLOSIS Restrictions on Interstate Movement of Cattle Because of Brucellosis § 78.5 General restrictions. Cattle may not be...

  7. A fluorescence method for detection of DNA and DNA methylation based on graphene oxide and restriction endonuclease HpaII.

    PubMed

    Wei, Wei; Gao, Chunyan; Xiong, Yanxiang; Zhang, Yuanjian; Liu, Songqin; Pu, Yuepu

    2015-01-01

    DNA methylation plays an important role in many biological events and is associated with various diseases. Most traditional methods for detection of DNA methylation are based on the complex and expensive bisulfite method. In this paper, we report a novel fluorescence method to detect DNA and DNA methylation based on graphene oxide (GO) and restriction endonuclease HpaII. The skillfully designed probe DNA labeled with 5-carboxyfluorescein (FAM) and optimized GO concentration keep the probe/target DNA still adsorbed on the GO. After the cleavage action of HpaII the labeled FAM is released from the GO surface and its fluorescence recovers, which could be used to detect DNA in the linear range of 50 pM-50 nM with a detection limit of 43 pM. DNA methylation induced by transmethylase (Mtase) or other chemical reagents prevents HpaII from recognizing and cleaving the specific site; as a result, fluorescence cannot recover. The fluorescence recovery efficiency is closely related to the DNA methylation level, which can be used to detect DNA methylation by comparing it with the fluorescence in the presence of intact target DNA. The method for detection of DNA and DNA methylation is simple, reliable and accurate. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Temperature based Restricted Boltzmann Machines

    PubMed Central

    Li, Guoqi; Deng, Lei; Xu, Yi; Wen, Changyun; Wang, Wei; Pei, Jing; Shi, Luping

    2016-01-01

    Restricted Boltzmann machines (RBMs), which apply graphical models to learning probability distribution over a set of inputs, have attracted much attention recently since being proposed as building blocks of multi-layer learning systems called deep belief networks (DBNs). Note that temperature is a key factor of the Boltzmann distribution that RBMs originate from. However, none of existing schemes have considered the impact of temperature in the graphical model of DBNs. In this work, we propose temperature based restricted Boltzmann machines (TRBMs) which reveals that temperature is an essential parameter controlling the selectivity of the firing neurons in the hidden layers. We theoretically prove that the effect of temperature can be adjusted by setting the parameter of the sharpness of the logistic function in the proposed TRBMs. The performance of RBMs can be improved by adjusting the temperature parameter of TRBMs. This work provides a comprehensive insights into the deep belief networks and deep learning architectures from a physical point of view. PMID:26758235

  9. Restrictive management of neonatal polycythemia.

    PubMed

    Morag, Iris; Strauss, Tzipora; Lubin, Daniel; Schushan-Eisen, Irit; Kenet, Gili; Kuint, Jacob

    2011-10-01

    Partial exchange transfusion (PET) is traditionally suggested as treatment for neonates diagnosed with polycythemia. Nevertheless, justification of this treatment is controversial. We evaluated the risk for short-term complications associated with a restrictive treatment protocol for neonatal polycythemia. A retrospective cross-sectional analytical study was conducted. Three treatment groups were defined and managed according to their degree of polycythemia, defined by capillary tube filled with venous blood and manually centrifuged hematocrit: group 1, hematocrit 65 to 69% and no special treatment was recommended; group 2, hematocrit 70 to 75% and intravenous fluids were given and feedings were withheld until hematocrit decreased to < 70%; and group 3, hematocrit ≥ 76% or symptomatic neonates and PET was recommended. During the study period, 190 neonates were diagnosed with polycythemia. The overall rate of short-term complications was 15% (28 neonates). Seizures, proven necrotizing enterocolitis, or thrombosis did not occur in any participating neonates. PET was performed in 31 (16%) neonates. The groups did not differ in their rate of early neonatal morbidities or length of hospitalization. Restrictive treatment for neonatal asymptomatic polycythemia is not associated with an increased risk of short-term complications.

  10. Restricted sample variance reduces generalizability.

    PubMed

    Lakes, Kimberley D

    2013-06-01

    One factor that affects the reliability of observed scores is restriction of range on the construct measured for a particular group of study participants. This study illustrates how researchers can use generalizability theory to evaluate the impact of restriction of range in particular sample characteristics on the generalizability of test scores and to estimate how changes in measurement design could improve the generalizability of the test scores. An observer-rated measure of child self-regulation (Response to Challenge Scale; Lakes, 2011) is used to examine scores for 198 children (Grades K through 5) within the generalizability theory (GT) framework. The generalizability of ratings within relatively developmentally homogeneous samples is examined and illustrates the effect of reduced variance among ratees on generalizability. Forecasts for g coefficients of various D study designs demonstrate how higher generalizability could be achieved by increasing the number of raters or items. In summary, the research presented illustrates the importance of and procedures for evaluating the generalizability of a set of scores in a particular research context.

  11. Identification of novel methylation markers in cervical cancer using restriction landmark genomic scanning.

    PubMed

    Wang, Sophia S; Smiraglia, Dominic J; Wu, Yue-Zhong; Ghosh, Srimoyee; Rader, Janet S; Cho, Kathleen R; Bonfiglio, Thomas A; Nayar, Ritu; Plass, Christoph; Sherman, Mark E

    2008-04-01

    Aberrant methylation of CpG islands in gene promoters often represents an early clonal event in carcinogenesis. Accordingly, defining methylation profiles may be useful for developing marker panels for early detection or predicting the risk of cancer precursors. To identify specific genes frequently methylated in cervical cancer, we conducted methylation profiling of 20 primary human cervical cancers using NotI-based restriction landmark genomic scanning (RLGS). Of 2,172 RLGS fragments analyzed (average, 1,753 CpG islands per patient), 186 RLGS fragments were lost in at least one tumor and 40 were lost in three or more. Methylation was identified in 19 (95%) of 20 tumor samples compared with normal DNA. Bisulfite sequencing was conducted to confirm RLGS results. Of the confirmed markers frequently methylated, we developed Methylight assays for two corresponding genes, nucleolar protein 4 (NOL4), and lipoma HMGIC fusion partner-like protein 4 (LHFPL4), which were methylated in 85% and 55% of cancers, respectively. Using these assays, we further confirmed frequent CpG island methylation in the original cancers and in another independent series of 15 cervical cancers. We also showed methylation at a reduced frequency in a set of carefully reviewed cytology specimens demonstrating cells exfoliated from cancer precursor lesions. In summary, we identified, for the first time, NOL4 and LHFPL4 as novel methylation targets specific for cervical cancer. Inclusion of NOL4 and LHFPL4 in evaluating methylation panels for early detection, risk prediction, and etiologic research on cervical cancer is warranted.

  12. 40 CFR 1033.740 - Credit restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 34 2013-07-01 2013-07-01 false Credit restrictions. 1033.740 Section... CONTROL OF EMISSIONS FROM LOCOMOTIVES Averaging, Banking, and Trading for Certification § 1033.740 Credit restrictions. Use of emission credits generated under this part 1033 or 40 CFR part 92 is restricted...

  13. 40 CFR 1033.740 - Credit restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Credit restrictions. 1033.740 Section... CONTROL OF EMISSIONS FROM LOCOMOTIVES Averaging, Banking, and Trading for Certification § 1033.740 Credit restrictions. Use of emission credits generated under this part 1033 or 40 CFR part 92 is restricted...

  14. 50 CFR 216.35 - Permit restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 10 2013-10-01 2013-10-01 false Permit restrictions. 216.35 Section 216.35 Wildlife and Fisheries NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC... MAMMALS Special Exceptions § 216.35 Permit restrictions. The following restrictions shall apply to all...

  15. 50 CFR 648.163 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Gear restrictions. 648.163 Section 648.163... Bluefish Fishery § 648.163 Gear restrictions. If the Council determines through its annual review or framework adjustment process that gear restrictions are necessary to assure that the fishing mortality...

  16. Restriction Enzyme Mapping: A Simple Student Practical.

    ERIC Educational Resources Information Center

    Higgins, Stephen J.; And Others

    1990-01-01

    An experiment that uses the recombinant plasmid pX1108 to illustrate restriction mapping is described. The experiment involves three restriction enzymes and employs single and double restriction enzyme digestions. A list of needed materials, procedures, safety precautions, results, and discussion are included. (KR)

  17. Problem-Solving Test: Restriction Endonuclease Mapping

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  18. 14 CFR 73.13 - Restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Restrictions. 73.13 Section 73.13 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRSPACE SPECIAL USE AIRSPACE Restricted Areas § 73.13 Restrictions. No person may operate an aircraft within...

  19. Problem-Solving Test: Restriction Endonuclease Mapping

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2011-01-01

    The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…

  20. Restricted Schur polynomials and finite N counting

    SciTech Connect

    Collins, Storm

    2009-01-15

    Restricted Schur polynomials have been posited as orthonormal operators for the change of basis from N=4 SYM to type IIB string theory. In this paper we briefly expound the relationship between the restricted Schur polynomials and the operators forwarded by Brown, Heslop, and Ramgoolam. We then briefly examine the finite N counting of the restricted Schur polynomials.

  1. Restriction Enzyme Mapping: A Simple Student Practical.

    ERIC Educational Resources Information Center

    Higgins, Stephen J.; And Others

    1990-01-01

    An experiment that uses the recombinant plasmid pX1108 to illustrate restriction mapping is described. The experiment involves three restriction enzymes and employs single and double restriction enzyme digestions. A list of needed materials, procedures, safety precautions, results, and discussion are included. (KR)

  2. 21 CFR 203.20 - Sales restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 4 2010-04-01 2010-04-01 false Sales restrictions. 203.20 Section 203.20 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DRUGS: GENERAL PRESCRIPTION DRUG MARKETING Sales Restrictions § 203.20 Sales restrictions. Except as provided in § 203.22...

  3. 9 CFR 92.3 - Movement restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Movement restrictions. 92.3 Section 92... ANIMAL PRODUCTS: PROCEDURES FOR REQUESTING RECOGNITION OF REGIONS § 92.3 Movement restrictions. Whenever... exist and the EC imposes prohibitions or other restrictions on the movement of animals or animal...

  4. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Restricted hazelnuts. 982.14 Section 982.14... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  5. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  6. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  7. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Restricted hazelnuts. 982.14 Section 982.14... AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  8. 7 CFR 982.14 - Restricted hazelnuts.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Restricted hazelnuts. 982.14 Section 982.14... Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE HAZELNUTS GROWN IN OREGON AND WASHINGTON Order Regulating Handling Definitions § 982.14 Restricted hazelnuts. Restricted hazelnuts...

  9. 50 CFR 648.23 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Mackerel, Squid, and Butterfish Fisheries § 648.23 Gear restrictions. (a) Mesh restrictions and exemptions...°45.0′ 74°53.0′ M24 35°28.0′ 74°52.0′ (4) Mackerel, squid, and butterfish bottom trawling restricted areas. (i) Oceanographer Canyon. No permitted mackerel, squid, or butterfish vessel may fish with...

  10. 40 CFR 1033.740 - Credit restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Credit restrictions. 1033.740 Section... CONTROL OF EMISSIONS FROM LOCOMOTIVES Averaging, Banking, and Trading for Certification § 1033.740 Credit restrictions. Use of emission credits generated under this part 1033 or 40 CFR part 92 is restricted depending...

  11. DNA hypermethylation of CD3(+) T cells from cord blood of infants exposed to intrauterine growth restriction.

    PubMed

    Williams, Lyda; Seki, Yoshinori; Delahaye, Fabien; Cheng, Alex; Fuloria, Mamta; Hughes Einstein, Francine; Charron, Maureen J

    2016-08-01

    Intrauterine growth restriction (IUGR) is associated with increased susceptibility to obesity, metabolic syndrome and type 2 diabetes. Although the mechanisms underlying the developmental origins of metabolic disease are poorly understood, evidence suggests that epigenomic alterations play a critical role. We sought to identify changes in DNA methylation patterns that are associated with IUGR in CD3(+) T cells purified from umbilical cord blood obtained from male newborns who were appropriate for gestational age (AGA) or who had been exposed to IUGR. CD3(+) T cells were isolated from cord blood obtained from IUGR and AGA infants. The genome-wide methylation profile in eight AGA and seven IUGR samples was determined using the HELP tagging assay. Validation analysis using targeted bisulfite sequencing and bisulfite massARRAY was performed on the original cohort as well as biological replicates consisting of two AGA and four IUGR infants. The Segway algorithm was used to identify methylation changes within regulatory regions of the genome. A global shift towards hypermethylation in IUGR was seen compared with AGA (89.8% of 4,425 differentially methylated loci), targeted to regulatory regions of the genome, specifically promoters and enhancers. Pathway analysis identified dysregulation of pathways involved in metabolic disease (type 2 diabetes mellitus, insulin signalling, mitogen-activated protein kinase signalling) and T cell development, regulation and activation (T cell receptor signalling), as well as transcription factors (TCF3, LEF1 and NFATC) that regulate T cells. Furthermore, bump-hunting analysis revealed differentially methylated regions in PRDM16 and HLA-DPB1, genes important for adipose tissue differentiation, stem cell maintenance and function and T cell activation. Our findings suggest that the alterations in methylation patterns observed in IUGR CD3(+) T cells may have functional consequences in targeted genes, regulatory regions and transcription

  12. Pleasant Events, Activity Restriction, and Blood Pressure in Dementia Caregivers

    PubMed Central

    Chattillion, Elizabeth A.; Ceglowski, Jenni; Roepke, Susan K.; von Känel, Roland; Losada, Andres; Mills, Paul J.; Romero-Moreno, Rosa; Grant, Igor; Patterson, Thomas L.; Mausbach, Brent T.

    2012-01-01

    Objective A combination of high engagement in pleasurable activities and low perceived activity restriction is potentially protective for a number of health and quality of life outcomes. This study tests the newly proposed Pleasant Events and Activity Restriction (PEAR) model to explain level of blood pressure (BP) in a sample of elderly dementia caregivers. Methods This cross-sectional study included 66 caregivers, ≥ 55 years of age, providing in-home care to a relative with dementia. Planned comparisons were made to assess group differences in BP between caregivers reporting high engagement in pleasant events plus low perceived activity restriction (HPLR; N = 22) to those with low pleasure plus high restriction (LPHR; N = 23) or those with either high pleasure plus high restriction or low pleasure plus low restriction (HPHR/LPLR; N = 21). Results After adjustments for age, sex, body mass index, use of anti-hypertensive medication, physical activity, and number of health problems, HPLR participants (86.78 mm Hg) had significantly lower mean arterial pressure compared to LPHR participants (94.70 mm Hg) (p = .01, Cohen’s d=0.89) and HPHR/LPLR participants (94.84 mm Hg) (p = .023, d=0.91). Similar results were found in post-hoc comparisons of both systolic and diastolic BP. Conclusions This study extends support for the PEAR model to physical health outcomes. Differences in BP between the HPLR group and other groups were of large magnitude and thus clinically meaningful. The findings may inform intervention studies aimed at investigating whether increasing pleasant events and lowering perceived activity restriction may lower BP. PMID:22888824

  13. Systematic identification and annotation of human methylation marks based on bisulfite sequencing methylomes reveals distinct roles of cell type-specific hypomethylation in the regulation of cell identity genes.

    PubMed

    Liu, Hongbo; Liu, Xiaojuan; Zhang, Shumei; Lv, Jie; Li, Song; Shang, Shipeng; Jia, Shanshan; Wei, Yanjun; Wang, Fang; Su, Jianzhong; Wu, Qiong; Zhang, Yan

    2016-01-08

    DNA methylation is a key epigenetic mark that is critical for gene regulation in multicellular eukaryotes. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understand the complex regulatory network for cell fate determination. In this study, we proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757 887 genome segments. Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, we identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in cell type-specific manner. In particular, we observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation.

  14. Neurodevelopment after fetal growth restriction.

    PubMed

    Baschat, Ahmet A

    2014-01-01

    Fetal growth restriction (FGR) can emerge as a complication of placental dysfunction and increases the risk for neurodevelopmental delay. Marked elevations of umbilical artery (UA) Doppler resistance that set the stage for cardiovascular and biophysical deterioration with subsequent preterm birth characterize early-onset FGR. Minimal, or absent UA Doppler abnormalities and isolated cerebral Doppler changes with subtle deterioration and a high risk for unanticipated term stillbirth are characteristic for late-onset FGR. Nutritional deficiency manifested in lagging head growth is the most powerful predictor of developmental delay in all forms of FGR. Extremes of blood flow resistance and cardiovascular deterioration, prematurity and intracranial hemorrhage increase the risks for psychomotor delay and cerebral palsy. In late-onset FGR, regional cerebral vascular redistribution correlates with abnormal behavioral domains. Irrespective of the phenotype of FGR, prenatal tests that provide precise and independent stratification of risks for adverse neurodevelopment have yet to be determined.

  15. An Infinite Restricted Boltzmann Machine.

    PubMed

    Côté, Marc-Alexandre; Larochelle, Hugo

    2016-07-01

    We present a mathematical construction for the restricted Boltzmann machine (RBM) that does not require specifying the number of hidden units. In fact, the hidden layer size is adaptive and can grow during training. This is obtained by first extending the RBM to be sensitive to the ordering of its hidden units. Then, with a carefully chosen definition of the energy function, we show that the limit of infinitely many hidden units is well defined. As with RBM, approximate maximum likelihood training can be performed, resulting in an algorithm that naturally and adaptively adds trained hidden units during learning. We empirically study the behavior of this infinite RBM, showing that its performance is competitive to that of the RBM, while not requiring the tuning of a hidden layer size.

  16. Restrictive dermopathy and fetal behaviour.

    PubMed

    Mulder, E J; Beemer, F A; Stoutenbeek, P

    2001-07-01

    We report three siblings from consecutive pregnancies affected with restrictive dermopathy (RD). During the second pregnancy, fetal behavioural development and growth were studied extensively using ultrasound at 1-4 week intervals. Dramatic and sudden changes occurred in fetal body movements and growth but not until the end of the second trimester of pregnancy. Prominent at that time were prolonged periods of fetal quiescence and very low heart rate variability, together with abnormally executed body movements of short duration. Retarded femoral development and jerky abrupt fetal body movements (abnormal movement quality) were already present in the early second trimester of pregnancy. Facial anomalies emerged despite the presence of fetal mouth movements. The clinical features of RD were only partly explained by present knowledge of skin development and the fetal akinesia deformation sequence hypothesis. Quantitative assessment of fetal movements proved to be a poor early marker for antenatal diagnosis of this disorder.

  17. Stillbirth and fetal growth restriction.

    PubMed

    Bukowski, Radek

    2010-09-01

    The association between stillbirth and fetal growth restriction is strong and supported by a large body of evidence and clinically employed for the stillbirth prediction. However, although assessment of fetal growth is a basis of clinical practice, it is not trivial. Essentially, fetal growth is a result of the genetic growth potential of the fetus and placental function. The growth potential is the driving force of fetal growth, whereas the placenta as the sole source of nutrients and oxygen might become the rate limiting element of fetal growth if its function is impaired. Thus, placental dysfunction may prevent the fetus from reaching its full genetically determined growth potential. In this sense fetal growth and its aberration provides an insight into placental function. Fetal growth is a proxy for the test of the effectiveness of placenta, whose function is otherwise obscured during pregnancy.

  18. Parenting and restrictions in childhood epilepsy.

    PubMed

    Rodenburg, Roos; Meijer, Anne Marie; Scherphof, Charlotte; Carpay, Johannes A; Augustijn, Paul; Aldenkamp, A P; Deković, Maja

    2013-06-01

    From the overprotection literature, the predictive and interactional (moderation) effects of controlling and indulgent parenting on restrictions in children with epilepsy were examined. Parents of 73 children with epilepsy completed questionnaires on parenting, restrictions, and functional status. Predictive and moderation effects were tested using multiple regression analysis. Moderation was tested with interactive computational methods. Restrictions were significantly (R(2)=.38, FΔ=6.59***, p<.001) predicted from seizure frequency (β=.24*, p<.05), functional status (β=-.42***, p<.001), and interaction between controlling and indulgent parenting (β=.28**, p<.01). Moderation occurred predominantly for high values of control: controlling parents who were not indulgent imposed fewer restrictions. In contrast, controlling parents who were indulgent imposed more restrictions. Parents who were controlling and more indulgent imposed more restrictions. Clinicians should ask parents about parenting and restrictions. Future research should examine whether the current study's findings can be replicated. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Cardiac MRI in restrictive cardiomyopathy.

    PubMed

    Gupta, A; Singh Gulati, G; Seth, S; Sharma, S

    2012-02-01

    Restrictive cardiomyopathy (RCM) is a specific group of heart muscle disorders characterized by inadequate ventricular relaxation during diastole. This leads to diastolic dysfunction with relative preservation of systolic function. Although short axis systolic function is usually preserved in RCM, the long axis systolic function may be severely impaired. Confirmation of diagnosis and information regarding aetiology, extent of myocardial damage, and response to treatment requires imaging. Importantly, differentiation from constrictive pericarditis (CCP) is needed, as only the latter is managed surgically. Echocardiography is the initial cardiac imaging technique but cannot reliably suggest a tissue diagnosis; although recent advances, especially tissue Doppler imaging and spectral tracking, have improved its ability to differentiate RCM from CCP. Cardiac catheterization is the reference standard, but is invasive, two-dimensional, and does not aid myocardial characterization. Cardiac magnetic resonance (CMR) is a versatile technique providing anatomical, morphological and functional information. In recent years, it has been shown to provide important information regarding disease mechanisms, and also been found useful to guide treatment, assess its outcome and predict patient prognosis. This review describes the CMR features of RCM, appearances in various diseases, its overall role in patient management, and how it compares with other imaging techniques.

  20. [Constrictive pericarditis and restrictive myocardiopathy].

    PubMed

    Espínola Zavaleta, N; Maribel Vogel, L; Isaac Tazar, J; Yánac Chávez, P; Romero Cárdenas, A; Vargas Barrón, J

    1999-01-01

    The purpose of this study was to assess the clinical and echocardiographic characteristics of constrictive pericarditis (CP) and restrictive cardiomyopathy (RC) and to compare them with the results obtained with cardiac catheterization. Clinical history, electrocardiogram and X-ray were taken in all patients, and transthoracic and transesophageal echocardiography were performed. Cardiac catheterization with transmyocardial biopsy was performed on only 5 patients. Wall thickness and left ventricular dimensions were normal in all patients with CP. Wall thickness was increased in those with RC. No patients demonstrated alterations in segmental wall movement. The pericardium was thickened and abnormally bright in the 3 patients with CP. In patients with CP the percentage of atrioventricular, semilunar, pulmonary and hepatic flow changes with respiration were more than 10%. In patients with RC this flow variation was less notable. However, the percentage of systolic and diastolic flow velocity increase of hepatic veins during expiration was greater than in CP. We can conclude that M-mode, two dimensional and Doppler echocardiography is extremely useful noninvasive method to differentiate CP and RC with good correlation with cardiac catheterization.

  1. Prenatal Depression Restricts Fetal Growth

    PubMed Central

    Diego, Miguel A.; Field, Tiffany; Hernandez-Reif, Maria; Schanberg, Saul; Kuhn, Cynthia; Gonzalez-Quintero, Victor Hugo

    2009-01-01

    Objective To identify whether prenatal depression is a risk factor for fetal growth restriction. Methods Midgestation (18-20 weeks GA) estimated fetal weight and urine cortisol and birth weight and gestational age at birth data were collected on a sample of 40 depressed and 40 non-depressed women. Estimated fetal weight and birthweight data were then used to compute fetal growth rates. Results Depressed women had a 13% greater incidence of premature delivery (Odds Ratio (OR) = 2.61) and 15% greater incidence of low birthweight (OR = 4.75) than non-depressed women. Depressed women also had elevated prenatal cortisol levels (p = .006) and fetuses who were smaller (p = .001) and who showed slower fetal growth rates (p = .011) and lower birthweights (p = .008). Mediation analyses further revealed that prenatal maternal cortisol levels were a potential mediator for the relationship between maternal symptoms of depression and both gestational age at birth and the rate of fetal growth. After controlling for maternal demographic variables, prenatal maternal cortisol levels were associated with 30% of the variance in gestational age at birth and 14% of the variance in the rate of fetal growth. Conclusion Prenatal depression was associated with adverse perinatal outcomes, including premature delivery and slower fetal growth rates. Prenatal maternal cortisol levels appear to play a role in mediating these outcomes. PMID:18723301

  2. Placental Adaptations in Growth Restriction

    PubMed Central

    Zhang, Song; Regnault, Timothy R.H.; Barker, Paige L.; Botting, Kimberley J.; McMillen, Isabella C.; McMillan, Christine M.; Roberts, Claire T.; Morrison, Janna L.

    2015-01-01

    The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions. PMID:25580812

  3. Osmotic pressure beyond concentration restrictions.

    PubMed

    Grattoni, Alessandro; Merlo, Manuele; Ferrari, Mauro

    2007-10-11

    Osmosis is a fundamental physical process that involves the transit of solvent molecules across a membrane separating two liquid solutions. Osmosis plays a role in many biological processes such as fluid exchange in animal cells (Cell Biochem. Biophys. 2005, 42, 277-345;1 J. Periodontol. 2007, 78, 757-7632) and water transport in plants. It is also involved in many technological applications such as drug delivery systems (Crit. Rev. Ther. Drug. 2004, 21, 477-520;3 J. Micro-Electromech. Syst. 2004, 13, 75-824) and water purification. Extensive attention has been dedicated in the past to the modeling of osmosis, starting with the classical theories of van't Hoff and Morse. These are predictive, in the sense that they do not involve adjustable parameters; however, they are directly applicable only to limited regimes of dilute solute concentrations. Extensions beyond the domains of validity of these classical theories have required recourse to fitting parameters, transitioning therefore to semiempirical, or nonpredictive models. A novel approach was presented by Granik et al., which is not a priori restricted in concentration domains, presents no adjustable parameters, and is mechanistic, in the sense that it is based on a coupled diffusion model. In this work, we examine the validity of predictive theories of osmosis, by comparison with our new experimental results, and a meta-analysis of literature data.

  4. Establishment of diagnosis by bisulfite-treated methylation-specific PCR method and analysis of clinical characteristics of pseudohypoparathyroidism type 1b.

    PubMed

    Kinoshita, Kaori; Minagawa, Masanori; Takatani, Tomozumi; Takatani, Rieko; Ohashi, Mika; Kohno, Yoichi

    2011-01-01

    Pathogenesis of pseudohypoparathyroidism type 1b (PHP-1b) is related to the loss of methylation at the GNAS exon A/B region, which is combined with epigenetic defects at other differentially methylated GNAS regions in most sporadic cases. In this study, we established a method for evaluating the methylation status of a CpG island in exon A/B using a methylation-specific polymerase chain reaction (MSPCR). We designed two primer pairs specific for the methylated and unmethylated alleles and evaluated the methylation status of GNAS exon A/B in samples from PHP-1b patients and normal controls. We examined 20 Japanese patients and 20 normal controls, and one primer set was found to be appropriate for diagnosis. Furthermore, we evaluated the clinical data of patients. Weight and height of patients were not significantly different from the normal population. Short stature (adult height ≤ 2SD) was observed in one patient and short metacarpals in two. Obesity was observed in six patients, and no patient showed ectopic subcutaneous calcification. Seven patients showed subclinical hypothyroidism because of resistance to thyroid stimulating hormone, but no patient had an abnormally low free thyroxine level, and none received oral thyroid hormone replacement. For diagnosis of PHP-1b, only clinical data is not sufficient because a few PHP-1b patients show clinical features similar to PHP-1a, and hence, molecular biology techniques are required for correct diagnosis. We concluded that MSPCR is applicable for rapid molecular diagnosis of PHP-1b.

  5. Assessing restrictiveness of national alcohol marketing policies.

    PubMed

    Esser, Marissa B; Jernigan, David H

    2014-01-01

    To develop an approach for monitoring national alcohol marketing policies globally, an area of the World Health Organization's (WHO) Global Alcohol Strategy. Data on restrictiveness of alcohol marketing policies came from the 2002 and 2008 WHO Global Surveys on Alcohol and Health. We included four scales in a sensitivity analysis to determine optimal weights to score countries on their marketing policies and applied the selected scale to assess national marketing policy restrictiveness. Nearly, 36% of countries had no marketing restrictions. The overall restrictiveness levels were not significantly different between 2002 and 2008. The number of countries with strict marketing regulations did not differ across years. This method of monitoring alcohol marketing restrictiveness helps track progress towards implementing WHO'S Global Alcohol Strategy. Findings indicate a consistent lack of restrictive policies over time, making this a priority area for national and global action. © The Author 2014. Medical Council on Alcohol and Oxford University Press. All rights reserved.

  6. Critical role of oxalate restriction in association with calcium restriction to decrease the probability of being a stone former: insufficient effect in idiopathic hypercalciuria.

    PubMed

    Bataille, P; Pruna, A; Gregoire, I; Charransol, G; de Fremont, J F; Coevoet, B; Galy, C; Fournier, A

    1983-01-01

    The probability of being a stone former (PSF) was calculated according to the method of Robertson in three groups of idiopathic calcium stone formers (normocalciuria (NCa), dietary hypercalciuria (DH) and idiopathic hypercalciuria (IH] during four conditions: on a free diet; on a calcium and oxalate restricted diet for four days and after an oxalate load (200 g of spinach) while on a calcium unrestricted or calcium restricted diet. Combined calciuria (Ca) and oxaluria (Ox) restriction significantly decreased PSF only in NCa and DH whereas the decrease was not significant in IH because of a concomitant significant increase in oxalate excretion. Increase of PSF with the oxalate load was significantly greater on calcium restricted than on calcium unrestricted diets in all groups of patients (4-6-12 times greater in NCa, DH and IH respectively). This shows the critical role of oxalate restriction when calcium is restricted in order to decrease the PSF. Combined restriction is not sufficient in idiopathic hypercalciuric patients to decrease their probability of stone formation.

  7. The Benefits of Calorie Restriction and Calorie Restriction Mimetics as Related to the Eye

    PubMed Central

    Anekonda, T.S.

    2010-01-01

    The effects of calorie restriction without malnutrition seem to possess many beneficial effects in numerous disease states. Recently, studies related to calorie restriction mimetics that biochemically mimic the effects of calorie restriction are also becoming increasingly popular. Both calorie restriction and calorie restriction mimetics trigger an adaptive response reminiscent of mild-stress or low-dose toxic response, which is frequently referred to as hormesis in the toxicology literature. Although some benefits of calorie restriction and calorie restriction mimetics have been studied, the role of hormesis-related pathways in the eye has not been given a special attention. This review will present the current literature on calorie restriction and calorie restriction mimetics as related to most prominent eye diseases and provide insights on the therapeutic role of hormesis in eye diseases. PMID:20844606

  8. Sleep in patients with restrictive lung disease.

    PubMed

    Won, Christine H J; Kryger, Meir

    2014-09-01

    Restrictive lung disease leads to ventilatory defects and diffusion impairments. These changes may contribute to abnormal nocturnal pathophysiology, including sleep architecture disruption and impaired ventilation and oxygenation. Patients with restrictive lung disease may suffer significant daytime fatigue and dysfunction. Hypercarbia and hypoxemia during sleep may impact progression of lung disease and related symptoms. Little is known about the impact of treatment of sleep disruption on sleep quality and overall prognosis in restrictive lung disease. This review discusses the pathophysiology of sleep and comorbid sleep disorders in restrictive lung diseases including interstitial lung disease, neuromuscular disease, and obesity hypoventilation syndrome. Published by Elsevier Inc.

  9. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for the...

  10. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for the...

  11. 42 CFR 73.13 - Restricted experiments.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...) Restricted experiments: (1) Experiments utilizing recombinant DNA that involve the deliberate transfer of a... agriculture. (2) Experiments involving the deliberate formation of recombinant DNA containing genes for the...

  12. Restricted licensing among older drivers in Iowa.

    PubMed

    Braitman, Keli A; Chaudhary, Neil K; McCartt, Anne T

    2010-12-01

    To determine whether Iowa's license restriction program identifies older drivers who appear to be at greater crash risk and to assess compliance with license restrictions. A total of 522 drivers 70 and older who were attempting to renew their driver's licenses at licensing offices in Iowa participated in two telephone surveys: one shortly after renewal to discuss driving before renewal and another 6months later to assess any changes. Surveys assessed driving behavior, crashes, and violations as well as self-reported visual impairments, prescription medications, and physical mobility limitations. Of the 522 drivers, 232 renewed their licenses without having to take a road test (Group 1), and 290 were required to take a road test; of the drivers taking a road test, 191 renewed without restrictions (Group 2), 93 received restrictions (Group 3), and 6 had their licenses suspended (Group 4). The small number of drivers with suspensions precluded including this group in analyses. There were clear distinctions among drivers in the first three groups at the initial survey. Driver age increased across Groups 1-3, as did some visual impairments, number of prescription medications, and physical mobility limitations. Many drivers who received restrictions (Group 3) already were driving fewer miles than drivers in Groups 1-2, and were driving less often at night and on high-speed roads. Following license renewal, reported average weekly mileage decreased more among drivers with license restrictions (Group 3) (36%) than among drivers without restrictions (Groups 1-2) (4% each). For all license restriction types (headlight, geographic area, or speed), decreases in the likelihood of driving during these restricted conditions were greater for drivers with the relevant restrictions than without. Most drivers complied with restrictions. Iowa's license restriction program identifies drivers with more self-reported visual impairments, prescription medications, and physical mobility

  13. Permanganate/bisulfite (PM/BS) conditioning-horizontal electro-dewatering (HED) of activated sludge: Effect of reactive Mn(III) species.

    PubMed

    Guo, Xinxin; Wang, Yili; Wang, Dongsheng

    2017-11-01

    A novel activated sludge (AS) conditioning method through permanganate/bisulfate (PM/BS) process was proposed. The method involved a new conditioner of reactive Mn(III) intermediate. Moreover, a Mn(III) conditioning-horizontal electro-dewatering (Mn(III) C-HED) process was established to improve AS dewatering performance. Underlying mechanisms were unraveled by investigating changes in physicochemical characteristics, scanning electron microscope (SEM) morphology, and transformation of water and organic matters. The optimum dewatering conditions for Mn(III) C-HED process with the final water content of 86.94% were determined as the combination of KMnO4 0.01 mol/L AS and NaHSO3 0.05 mol/L AS at 20 V for 120 min. Results showed that Mn(III) C-HED process effectively reduced free water and bound water with the corresponding removal ratios of 51.68% and 87.62% at the anode-side as well as 36.55% and 85.08% at the cathode-side, respectively. During the PM/BS process, the produced Mn(III), Mn(2+), and MnO2 exerted chemical and physical effects on AS conditioning and dewatering. Mn(III) disintegrated extracellular polymeric substances (EPS) fractions and cells in AS, as well as induced partial bound water release. Additionally, flocculation effect induced by Mn(2+) and MnO2 skeleton building also benefited AS dewatering. AS cells were further disrupted under the effect of a horizontal electric field. Accordingly, EPS within the AS matrix was solubilized, tightly bound (TB)-EPS or loosely bound (LB)-EPS was converted to their corresponding outer EPS fractions, and AS dewaterability improved. Additionally, changes in pH and temperature at HED stage damaged the AS cells and changed the floc properties, thereby leading to easy separation of liquid and AS particles. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Restricted surface matching: a new registration method for medical images

    NASA Astrophysics Data System (ADS)

    Gong, JianXing; Zamorano, Lucia J.; Jiang, Zhaowei; Nolte, Lutz P.; Diaz, Fernando

    1998-06-01

    Since its introduction to neurological surgery in the early 1980's, computer assisted surgery (CAS) with and without robotics navigation has been applied to several medical fields. The common issue all CAS systems is registration between two pre-operative 3D image modalities (for example, CT/MRI/PET et al) and the 3D image references of the patient in the operative room. In Wayne State University, a new way is introduced for medical image registration, which is different from traditional fiducial point registration and surface registration. We call it restricted surface matching (RSM). The method fast, convenient, accurate and robust. It combines the advantages from two registration methods mentioned before. Because of a penalty function introduced in its cost function, it is called `RSM'. The surface of a 3D image modality is pre-operatively extracted using segmentation techniques, and a distance map is created from such surface. The surface of another 3D reference is presented by a cloud of 3D points. At least three rough landmarks are used to restrict a registration not far away from global minimum. The local minimum issue is solved by use of a restriction for in the cost function and larger number of random starting points. The accuracy of matching is achieved by gradually releasing the restriction and limiting the influence of outliers. It only needs about half a minute to find the global minimum (for 256 X 256 X 56 images) in a SunSparc 10 station.

  15. Improving computer security for authentication of users: influence of proactive password restrictions.

    PubMed

    Proctor, Robert W; Lien, Mei-Ching; Vu, Kim-Phuong L; Schultz, E Eugene; Salvendy, Gavriel

    2002-05-01

    Entering a username-password combination is a widely used procedure for identification and authentication in computer systems. However, it is a notoriously weak method, in that the passwords adopted by many users are easy to crack. In an attempt to improve security, proactive password checking may be used, in which passwords must meet several criteria to be more resistant to cracking. In two experiments, we examined the influence of proactive password restrictions on the time that it took to generate an acceptable password and to use it subsequently to long in. The required length was a minimum of five characters in Experiment 1 and eight characters in Experiment 2. In both experiments, one condition had only the length restriction, and the other had additional restrictions. The additional restrictions greatly increased the time it took to generate the password but had only a small effect on the time it took to use it subsequently to long in. For the five-character passwords, 75% were cracked when no other restrictions were imposed, and this was reduced to 33% with the additional restrictions. For the eight-character passwords, 17% were cracked with no other restrictions, and 12.5% with restrictions. The results indicate that increasing the minimum character length reduces crackability and increases security, regardless of whether additional restrictions are imposed.

  16. 12 CFR 575.11 - Operating restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY MUTUAL HOLDING COMPANIES § 575.11 Operating restrictions. (a) Activities restrictions. A mutual holding company may engage in any business activity specified in 12 U.S.C. 1467a(c)(2) or (c)(9)(A)(ii). In addition, the business...

  17. 46 CFR 119.405 - Fuel restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 4 2011-10-01 2011-10-01 false Fuel restrictions. 119.405 Section 119.405 Shipping... Machinery Requirements § 119.405 Fuel restrictions. The use of a fuel, other than diesel fuel, as an alternative fuel for an internal combustion engine, except gasoline when used as a fuel for outboard motors...

  18. 46 CFR 119.405 - Fuel restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 4 2013-10-01 2013-10-01 false Fuel restrictions. 119.405 Section 119.405 Shipping... Machinery Requirements § 119.405 Fuel restrictions. The use of a fuel, other than diesel fuel, as an alternative fuel for an internal combustion engine, except gasoline when used as a fuel for outboard motors...

  19. 46 CFR 182.405 - Fuel restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 7 2012-10-01 2012-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed...

  20. 46 CFR 119.405 - Fuel restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 4 2012-10-01 2012-10-01 false Fuel restrictions. 119.405 Section 119.405 Shipping... Machinery Requirements § 119.405 Fuel restrictions. The use of a fuel, other than diesel fuel, as an alternative fuel for an internal combustion engine, except gasoline when used as a fuel for outboard motors...

  1. 46 CFR 182.405 - Fuel restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 7 2011-10-01 2011-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed...

  2. 46 CFR 182.405 - Fuel restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 7 2014-10-01 2014-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed...

  3. 46 CFR 182.405 - Fuel restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 7 2013-10-01 2013-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed...

  4. 46 CFR 119.405 - Fuel restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 4 2014-10-01 2014-10-01 false Fuel restrictions. 119.405 Section 119.405 Shipping... Machinery Requirements § 119.405 Fuel restrictions. The use of a fuel, other than diesel fuel, as an alternative fuel for an internal combustion engine, except gasoline when used as a fuel for outboard motors...

  5. 7 CFR 3550.118 - Grant restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Grant restrictions. 3550.118 Section 3550.118... AGRICULTURE DIRECT SINGLE FAMILY HOUSING LOANS AND GRANTS Section 504 Origination and Section 306C Water and Waste Disposal Grants § 3550.118 Grant restrictions. (a) Maximum grant. Lifetime assistance to any...

  6. 7 CFR 3550.118 - Grant restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Grant restrictions. 3550.118 Section 3550.118... AGRICULTURE DIRECT SINGLE FAMILY HOUSING LOANS AND GRANTS Section 504 Origination and Section 306C Water and Waste Disposal Grants § 3550.118 Grant restrictions. (a) Maximum grant. Lifetime assistance to any...

  7. 7 CFR 3555.102 - Loan restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Loan restrictions. 3555.102 Section 3555.102... AGRICULTURE GUARANTEED RURAL HOUSING PROGRAM (Eff. 9-1-14) Loan Requirements § 3555.102 Loan restrictions. A guarantee will not be issued if loan funds are to be used for: (a) Existing manufactured homes. Purchase...

  8. 50 CFR 660.506 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 660.506 Section 660.506 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 660.506 Gear restrictions. The only fishing gear authorized for use in the reduction fishery...

  9. 50 CFR 665.104 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.104 Section 665... § 665.104 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for American Samoa bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  10. 50 CFR 665.605 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.605 Section 665... Fisheries § 665.605 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for PRIA bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  11. 50 CFR 665.464 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.464 Section 665.464 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.464 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  12. 50 CFR 665.664 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.664 Section 665.664 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Fisheries § 665.664 Gear restrictions. Only selective gear may be used to harvest coral from any...

  13. 50 CFR 665.664 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.664 Section 665.664 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Fisheries § 665.664 Gear restrictions. Only selective gear may be used to harvest coral from any...

  14. 50 CFR 665.264 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.264 Section 665.264 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  15. 50 CFR 665.264 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.264 Section 665.264 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  16. 50 CFR 665.164 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.164 Section 665.164 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.164 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  17. 50 CFR 665.164 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.164 Section 665.164 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.164 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  18. 50 CFR 665.104 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.104 Section 665... § 665.104 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for American Samoa bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  19. 50 CFR 665.664 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.664 Section 665.664 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Fisheries § 665.664 Gear restrictions. Only selective gear may be used to harvest coral from any...

  20. 50 CFR 665.605 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.605 Section 665... Fisheries § 665.605 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for PRIA bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  1. 50 CFR 665.664 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.664 Section 665.664 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Fisheries § 665.664 Gear restrictions. Only selective gear may be used to harvest coral from any...

  2. 50 CFR 660.506 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 660.506 Section 660.506 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 660.506 Gear restrictions. The only fishing gear authorized for use in the reduction fishery...

  3. 50 CFR 665.605 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.605 Section 665... Fisheries § 665.605 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for PRIA bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  4. 50 CFR 665.406 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.406 Section 665... § 665.406 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for bottomfish with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear. Possession of a bottom...

  5. 50 CFR 665.406 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.406 Section 665... § 665.406 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for bottomfish with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear. Possession of a bottom...

  6. 50 CFR 665.664 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Gear restrictions. 665.664 Section 665.664 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION... § 665.664 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  7. 50 CFR 665.264 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.264 Section 665.264 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  8. 50 CFR 648.203 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Gear restrictions. 648.203 Section 648... Atlantic Herring Fishery § 648.203 Gear restrictions. (a) Midwater trawl gear may only be used by a vessel... Nantucket Lightship Area as described in § 648.81(c)(1), provided it complies with the midwater trawl...

  9. 50 CFR 665.464 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.464 Section 665.464 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.464 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  10. 50 CFR 665.164 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.164 Section 665.164 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.164 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  11. 50 CFR 665.264 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Gear restrictions. 665.264 Section 665.264..., DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Hawaii Fisheries § 665.264 Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  12. 50 CFR 665.104 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.104 Section 665... § 665.104 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for American Samoa bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  13. 50 CFR 665.164 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.164 Section 665.164 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.164 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  14. 50 CFR 648.203 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Gear restrictions. 648.203 Section 648.203... Herring Fishery § 648.203 Gear restrictions. (a) Midwater trawl gear may only be used by a vessel issued a... Lightship Area as described in § 648.81(c)(1), provided it complies with the midwater trawl gear...

  15. 50 CFR 665.104 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.104 Section 665... § 665.104 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for American Samoa bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  16. 50 CFR 665.464 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Gear restrictions. 665.464 Section 665.464 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION... Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  17. 50 CFR 665.605 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.605 Section 665... Fisheries § 665.605 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for PRIA bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear....

  18. 50 CFR 665.605 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Gear restrictions. 665.605 Section 665.605... § 665.605 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for PRIA bottomfish MUS with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear. Possession of...

  19. 50 CFR 665.464 - Gear restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Gear restrictions. 665.464 Section 665.464 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.464 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  20. 50 CFR 665.406 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Gear restrictions. 665.406 Section 665... § 665.406 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for bottomfish with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear. Possession of a bottom...

  1. 50 CFR 665.164 - Gear restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Gear restrictions. 665.164 Section 665.164..., DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC American Samoa Fisheries § 665.164 Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  2. 50 CFR 665.264 - Gear restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Gear restrictions. 665.264 Section 665.264 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... Gear restrictions. Only selective gear may be used to harvest coral from any precious coral permit area....

  3. 50 CFR 648.203 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Gear restrictions. 648.203 Section 648... Atlantic Herring Fishery § 648.203 Gear restrictions. (a) Midwater trawl gear may only be used by a vessel... Nantucket Lightship Area as described in § 648.81(c)(1), provided it complies with the midwater trawl...

  4. 50 CFR 648.203 - Gear restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Gear restrictions. 648.203 Section 648... Atlantic Herring Fishery § 648.203 Gear restrictions. (a) Midwater trawl gear may only be used by a vessel... Nantucket Lightship Area as described in § 648.81(c)(1), provided it complies with the midwater trawl...

  5. 50 CFR 665.464 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.464 Section 665.464 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC... § 665.464 Gear restrictions. Only selective gear may be used to harvest coral from any precious...

  6. 50 CFR 665.406 - Gear restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Gear restrictions. 665.406 Section 665... § 665.406 Gear restrictions. (a) Bottom trawls and bottom set gillnets. Fishing for bottomfish with bottom trawls and bottom set gillnets is prohibited. (b) Possession of gear. Possession of a bottom...

  7. 24 CFR 850.151 - Project restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... DISABILITIES PROGRAM) HOUSING DEVELOPMENT GRANTS Project Management § 850.151 Project restrictions. (a) Owner... 24 Housing and Urban Development 4 2014-04-01 2014-04-01 false Project restrictions. 850.151... owner shall not convert the units in the project to condominium ownership or to a form of...

  8. 24 CFR 850.151 - Project restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... DISABILITIES PROGRAM) HOUSING DEVELOPMENT GRANTS Project Management § 850.151 Project restrictions. (a) Owner... 24 Housing and Urban Development 4 2013-04-01 2013-04-01 false Project restrictions. 850.151... owner shall not convert the units in the project to condominium ownership or to a form of...

  9. 24 CFR 850.151 - Project restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... DISABILITIES PROGRAM) HOUSING DEVELOPMENT GRANTS Project Management § 850.151 Project restrictions. (a) Owner... 24 Housing and Urban Development 4 2011-04-01 2011-04-01 false Project restrictions. 850.151... owner shall not convert the units in the project to condominium ownership or to a form of...

  10. 24 CFR 850.151 - Project restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... DISABILITIES PROGRAM) HOUSING DEVELOPMENT GRANTS Project Management § 850.151 Project restrictions. (a) Owner... 24 Housing and Urban Development 4 2012-04-01 2012-04-01 false Project restrictions. 850.151... owner shall not convert the units in the project to condominium ownership or to a form of...

  11. 10 CFR 110.29 - Restricted destinations.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 2 2012-01-01 2012-01-01 false Restricted destinations. 110.29 Section 110.29 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.29 Restricted destinations. Afghanistan Andorra Angola Burma (Myanmar) Djibouti India Israel...

  12. 10 CFR 110.29 - Restricted destinations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 2 2011-01-01 2011-01-01 false Restricted destinations. 110.29 Section 110.29 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.29 Restricted destinations. Afghanistan Andorra Angola Burma (Myanmar) Djibouti India Israel...

  13. 10 CFR 110.29 - Restricted destinations.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 2 2014-01-01 2014-01-01 false Restricted destinations. 110.29 Section 110.29 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.29 Restricted destinations. Afghanistan Andorra Angola Burma (Myanmar) Djibouti India Israel...

  14. 10 CFR 110.29 - Restricted destinations.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 10 Energy 2 2013-01-01 2013-01-01 false Restricted destinations. 110.29 Section 110.29 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.29 Restricted destinations. Afghanistan Andorra Angola Burma (Myanmar) Djibouti India Israel...

  15. 10 CFR 110.29 - Restricted destinations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 2 2010-01-01 2010-01-01 false Restricted destinations. 110.29 Section 110.29 Energy NUCLEAR REGULATORY COMMISSION (CONTINUED) EXPORT AND IMPORT OF NUCLEAR EQUIPMENT AND MATERIAL Licenses § 110.29 Restricted destinations. Afghanistan Andorra Angola Burma (Myanmar) Djibouti India Israel Libya...

  16. 7 CFR 3560.576 - Occupancy restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... restrictions. (a) Restrictions on conditions of occupancy. (1) No borrower or grantee will be permitted to... condition of occupancy of the housing. (2) Tenant selection should be in accordance with the loan agreement... country. For migrant farmworkers living in seasonal housing the actual dollars earned from farm labor by...

  17. 7 CFR 3560.576 - Occupancy restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... restrictions. (a) Restrictions on conditions of occupancy. (1) No borrower or grantee will be permitted to... condition of occupancy of the housing. (2) Tenant selection should be in accordance with the loan agreement... country. For migrant farmworkers living in seasonal housing the actual dollars earned from farm labor by...

  18. 7 CFR 3560.576 - Occupancy restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... restrictions. (a) Restrictions on conditions of occupancy. (1) No borrower or grantee will be permitted to... condition of occupancy of the housing. (2) Tenant selection should be in accordance with the loan agreement... country. For migrant farmworkers living in seasonal housing the actual dollars earned from farm labor by...

  19. 7 CFR 3560.576 - Occupancy restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... restrictions. (a) Restrictions on conditions of occupancy. (1) No borrower or grantee will be permitted to... condition of occupancy of the housing. (2) Tenant selection should be in accordance with the loan agreement... country. For migrant farmworkers living in seasonal housing the actual dollars earned from farm labor by...

  20. 7 CFR 3430.705 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.705 Section 3430.705 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Biomass Research and Development Initiative § 3430.705 Funding restrictions. (a...

  1. 7 CFR 3430.505 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.505 Section 3430.505 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... Funding restrictions. (a) Construction. Funds made available for grants under this subsection shall not be...

  2. 7 CFR 3430.205 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.205 Section 3430.205 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Specialty Crop Research Initiative § 3430.205 Funding restrictions. (a) Prohibition...

  3. 7 CFR 3430.705 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.705 Section 3430.705 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Biomass Research and Development Initiative § 3430.705 Funding restrictions. (a...

  4. 7 CFR 3430.905 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.905 Section 3430.905 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS New Era Rural Technology Competitive Grants Program § 3430.905 Funding restrictions. (a...

  5. 7 CFR 3430.605 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.605 Section 3430.605 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Beginning Farmer and Rancher Development Program § 3430.605 Funding restrictions. (a...

  6. 7 CFR 3430.405 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.405 Section 3430.405 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Organic Agriculture Research and Extension Initiative § 3430.405 Funding restrictions...

  7. 7 CFR 3430.605 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.605 Section 3430.605 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Beginning Farmer and Rancher Development Program § 3430.605 Funding restrictions. (a...

  8. 7 CFR 3430.505 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.505 Section 3430.505 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... Funding restrictions. (a) Construction. Funds made available for grants under this subsection shall not be...

  9. 7 CFR 3430.605 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.605 Section 3430.605 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Beginning Farmer and Rancher Development Program § 3430.605 Funding restrictions. (a...

  10. 7 CFR 3430.705 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.705 Section 3430.705 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Biomass Research and Development Initiative § 3430.705 Funding restrictions. (a...

  11. 7 CFR 3430.305 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.305 Section 3430.305 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Agriculture and Food Research Initiative § 3430.305 Funding restrictions. (a...

  12. 7 CFR 3430.205 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.205 Section 3430.205 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Specialty Crop Research Initiative § 3430.205 Funding restrictions. (a) Prohibition...

  13. 7 CFR 3430.205 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.205 Section 3430.205 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Specialty Crop Research Initiative § 3430.205 Funding restrictions. (a) Prohibition...

  14. 7 CFR 3430.605 - Funding restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Funding restrictions. 3430.605 Section 3430.605 Agriculture Regulations of the Department of Agriculture (Continued) COOPERATIVE STATE RESEARCH, EDUCATION... § 3430.605 Funding restrictions. (a) Facility costs. Funds made available under this subpart shall not be...

  15. 7 CFR 3430.205 - Funding restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 15 2010-01-01 2010-01-01 false Funding restrictions. 3430.205 Section 3430.205 Agriculture Regulations of the Department of Agriculture (Continued) COOPERATIVE STATE RESEARCH, EDUCATION... Funding restrictions. (a) Prohibition against construction. Funds made available under this subpart shall...

  16. 7 CFR 3430.305 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.305 Section 3430.305 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Agriculture and Food Research Initiative § 3430.305 Funding restrictions. (a...

  17. 7 CFR 3430.205 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.205 Section 3430.205 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Specialty Crop Research Initiative § 3430.205 Funding restrictions. (a) Prohibition...

  18. 7 CFR 3430.405 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.405 Section 3430.405 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Organic Agriculture Research and Extension Initiative § 3430.405 Funding restrictions...

  19. 7 CFR 3430.605 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.605 Section 3430.605 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Beginning Farmer and Rancher Development Program § 3430.605 Funding restrictions. (a...

  20. 7 CFR 3430.305 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.305 Section 3430.305 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Agriculture and Food Research Initiative § 3430.305 Funding restrictions. (a...

  1. 7 CFR 3430.505 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.505 Section 3430.505 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... Funding restrictions. (a) Construction. Funds made available for grants under this subsection shall not be...

  2. 7 CFR 3430.905 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.905 Section 3430.905 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS New Era Rural Technology Competitive Grants Program § 3430.905 Funding restrictions. (a...

  3. 7 CFR 3430.305 - Funding restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 15 2011-01-01 2011-01-01 false Funding restrictions. 3430.305 Section 3430.305 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Agriculture and Food Research Initiative § 3430.305 Funding restrictions. (a...

  4. 7 CFR 3430.905 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.905 Section 3430.905 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS New Era Rural Technology Competitive Grants Program § 3430.905 Funding restrictions. (a...

  5. 7 CFR 3430.405 - Funding restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 15 2012-01-01 2012-01-01 false Funding restrictions. 3430.405 Section 3430.405 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Organic Agriculture Research and Extension Initiative § 3430.405 Funding restrictions...

  6. 7 CFR 3430.405 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.405 Section 3430.405 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Organic Agriculture Research and Extension Initiative § 3430.405 Funding restrictions...

  7. 7 CFR 3430.705 - Funding restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 15 2014-01-01 2014-01-01 false Funding restrictions. 3430.705 Section 3430.705 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... ADMINISTRATIVE PROVISIONS Biomass Research and Development Initiative § 3430.705 Funding restrictions. (a...

  8. 7 CFR 3430.505 - Funding restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 15 2013-01-01 2013-01-01 false Funding restrictions. 3430.505 Section 3430.505 Agriculture Regulations of the Department of Agriculture (Continued) NATIONAL INSTITUTE OF FOOD AND... Funding restrictions. (a) Construction. Funds made available for grants under this subsection shall not be...

  9. 18 CFR 1304.410 - Navigation restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 2 2012-04-01 2012-04-01 false Navigation restrictions... Miscellaneous § 1304.410 Navigation restrictions. (a) Except for the placement of riprap along the shoreline... established for commercial navigation. (b) Structures shall not be located in such a way as to block...

  10. 18 CFR 1304.410 - Navigation restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 2 2014-04-01 2014-04-01 false Navigation restrictions... Miscellaneous § 1304.410 Navigation restrictions. (a) Except for the placement of riprap along the shoreline... established for commercial navigation. (b) Structures shall not be located in such a way as to block...

  11. 18 CFR 1304.410 - Navigation restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Navigation restrictions... Miscellaneous § 1304.410 Navigation restrictions. (a) Except for the placement of riprap along the shoreline... established for commercial navigation. (b) Structures shall not be located in such a way as to block...

  12. 18 CFR 1304.410 - Navigation restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 2 2013-04-01 2012-04-01 true Navigation restrictions... Miscellaneous § 1304.410 Navigation restrictions. (a) Except for the placement of riprap along the shoreline... established for commercial navigation. (b) Structures shall not be located in such a way as to block...

  13. 18 CFR 1304.410 - Navigation restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Navigation restrictions... Miscellaneous § 1304.410 Navigation restrictions. (a) Except for the placement of riprap along the shoreline... established for commercial navigation. (b) Structures shall not be located in such a way as to block...

  14. 7 CFR 400.407 - Restricted access.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 6 2010-01-01 2010-01-01 false Restricted access. 400.407 Section 400.407 Agriculture Regulations of the Department of Agriculture (Continued) FEDERAL CROP INSURANCE CORPORATION, DEPARTMENT OF... Social Security Account Numbers and Employer Identification Numbers § 400.407 Restricted access. The...

  15. 25 CFR 163.12 - Harvesting restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Harvesting restrictions. 163.12 Section 163.12 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER GENERAL FORESTRY REGULATIONS Forest Management and Operations § 163.12 Harvesting restrictions. (a) Harvesting timber on commercial forest land...

  16. 25 CFR 163.12 - Harvesting restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Harvesting restrictions. 163.12 Section 163.12 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER GENERAL FORESTRY REGULATIONS Forest Management and Operations § 163.12 Harvesting restrictions. (a) Harvesting timber on commercial forest land...

  17. 25 CFR 163.12 - Harvesting restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Harvesting restrictions. 163.12 Section 163.12 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER GENERAL FORESTRY REGULATIONS Forest Management and Operations § 163.12 Harvesting restrictions. (a) Harvesting timber on commercial forest land...

  18. 25 CFR 163.12 - Harvesting restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Harvesting restrictions. 163.12 Section 163.12 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER GENERAL FORESTRY REGULATIONS Forest Management and Operations § 163.12 Harvesting restrictions. (a) Harvesting timber on commercial forest land...

  19. 25 CFR 163.12 - Harvesting restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Harvesting restrictions. 163.12 Section 163.12 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAND AND WATER GENERAL FORESTRY REGULATIONS Forest Management and Operations § 163.12 Harvesting restrictions. (a) Harvesting timber on commercial forest land...

  20. 46 CFR 182.405 - Fuel restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on... 46 Shipping 7 2010-10-01 2010-10-01 false Fuel restrictions. 182.405 Section 182.405...

  1. 46 CFR 119.405 - Fuel restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... Machinery Requirements § 119.405 Fuel restrictions. The use of a fuel, other than diesel fuel, as an alternative fuel for an internal combustion engine, except gasoline when used as a fuel for outboard motors as... 46 Shipping 4 2010-10-01 2010-10-01 false Fuel restrictions. 119.405 Section 119.405...

  2. 12 CFR 1807.904 - Lobbying restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 12 Banks and Banking 9 2013-01-01 2013-01-01 false Lobbying restrictions. 1807.904 Section 1807.904 Banks and Banking COMMUNITY DEVELOPMENT FINANCIAL INSTITUTIONS FUND, DEPARTMENT OF THE TREASURY CAPITAL MAGNET FUND Terms and Conditions of Assistance § 1807.904 Lobbying restrictions. No...

  3. 12 CFR 1807.904 - Lobbying restrictions.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 12 Banks and Banking 10 2014-01-01 2014-01-01 false Lobbying restrictions. 1807.904 Section 1807.904 Banks and Banking COMMUNITY DEVELOPMENT FINANCIAL INSTITUTIONS FUND, DEPARTMENT OF THE TREASURY CAPITAL MAGNET FUND Terms and Conditions of Assistance § 1807.904 Lobbying restrictions. No...

  4. 12 CFR 1807.904 - Lobbying restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 7 2011-01-01 2011-01-01 false Lobbying restrictions. 1807.904 Section 1807.904 Banks and Banking COMMUNITY DEVELOPMENT FINANCIAL INSTITUTIONS FUND, DEPARTMENT OF THE TREASURY CAPITAL MAGNET FUND Terms and Conditions of Assistance § 1807.904 Lobbying restrictions. No...

  5. 12 CFR 1807.904 - Lobbying restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 12 Banks and Banking 9 2012-01-01 2012-01-01 false Lobbying restrictions. 1807.904 Section 1807.904 Banks and Banking COMMUNITY DEVELOPMENT FINANCIAL INSTITUTIONS FUND, DEPARTMENT OF THE TREASURY CAPITAL MAGNET FUND Terms and Conditions of Assistance § 1807.904 Lobbying restrictions. No...

  6. 40 CFR 258.28 - Liquids restrictions.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 26 2013-07-01 2013-07-01 false Liquids restrictions. 258.28 Section... MUNICIPAL SOLID WASTE LANDFILLS Operating Criteria § 258.28 Liquids restrictions. (a) Bulk or noncontainerized liquid waste may not be placed in MSWLF units unless: (1) The waste is household waste other...

  7. 28 CFR 804.3 - Restrictions.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 28 Judicial Administration 2 2012-07-01 2012-07-01 false Restrictions. 804.3 Section 804.3 Judicial Administration COURT SERVICES AND OFFENDER SUPERVISION AGENCY FOR THE DISTRICT OF COLUMBIA ACCEPTANCE OF GIFTS § 804.3 Restrictions. (a) The Agency is not authorized to accept gifts of money, stock...

  8. 28 CFR 804.3 - Restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Restrictions. 804.3 Section 804.3 Judicial Administration COURT SERVICES AND OFFENDER SUPERVISION AGENCY FOR THE DISTRICT OF COLUMBIA ACCEPTANCE OF GIFTS § 804.3 Restrictions. (a) The Agency is not authorized to accept gifts of money, stock...

  9. 32 CFR 763.4 - Entry restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 5 2011-07-01 2011-07-01 false Entry restrictions. 763.4 Section 763.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY ISLANDS UNDER NAVY JURISDICTION RULES GOVERNING PUBLIC ACCESS Entry Regulations for Kaho'olawe Island, Hawaii § 763.4 Entry restrictions. (a...

  10. 32 CFR 763.4 - Entry restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 5 2010-07-01 2010-07-01 false Entry restrictions. 763.4 Section 763.4 National Defense Department of Defense (Continued) DEPARTMENT OF THE NAVY ISLANDS UNDER NAVY JURISDICTION RULES GOVERNING PUBLIC ACCESS Entry Regulations for Kaho'olawe Island, Hawaii § 763.4 Entry restrictions. (a...

  11. 12 CFR 575.11 - Operating restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Banks and Banking OFFICE OF THRIFT SUPERVISION, DEPARTMENT OF THE TREASURY MUTUAL HOLDING COMPANIES § 575.11 Operating restrictions. (a) Activities restrictions. A mutual holding company may engage in any... of subsidiaries of mutual holding companies may include the activities specified in § 575.10(a)(6) of...

  12. 50 CFR 648.102 - Time restrictions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 10 2011-10-01 2011-10-01 false Time restrictions. 648.102 Section 648... Summer Flounder Fisheries § 648.102 Time restrictions. Link to an amendment published at 76 FR 60626... flounder from May 1 through September 30. This time period may be adjusted pursuant to the procedures in...

  13. 50 CFR 648.142 - Time restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Time restrictions. 648.142 Section 648.142... Fishery § 648.142 Time restrictions. Vessels that are not eligible for a moratorium permit under § 648.4(a... bass from May 22 through October 11 and November 1 through December 31, unless this time period is...

  14. 50 CFR 648.102 - Time restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 8 2010-10-01 2010-10-01 false Time restrictions. 648.102 Section 648.102... Flounder Fisheries § 648.102 Time restrictions. Unless otherwise specified pursuant to § 648.107, vessels... possession limit may fish for summer flounder from May 1 through September 30. This time period may be...

  15. An Event Restriction Interval Theory of Tense

    ERIC Educational Resources Information Center

    Beamer, Brandon Robert

    2012-01-01

    This dissertation presents a novel theory of tense and tense-like constructions. It is named after a key theoretical component of the theory, the event restriction interval. In Event Restriction Interval (ERI) Theory, sentences are semantically evaluated relative to an index which contains two key intervals, the evaluation interval and the event…

  16. 14 CFR 73.83 - Restrictions.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 2 2010-01-01 2010-01-01 false Restrictions. 73.83 Section 73.83 Aeronautics and Space FEDERAL AVIATION ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) AIRSPACE SPECIAL USE AIRSPACE Prohibited Areas § 73.83 Restrictions. No person may operate an aircraft within...

  17. Restricted Interests and Teacher Presentation of Items

    ERIC Educational Resources Information Center

    Stocco, Corey S.; Thompson, Rachel H.; Rodriguez, Nicole M.

    2011-01-01

    Restricted and repetitive behavior (RRB) is more pervasive, prevalent, frequent, and severe in individuals with autism spectrum disorders (ASDs) than in their typical peers. One subtype of RRB is restricted interests in items or activities, which is evident in the manner in which individuals engage with items (e.g., repetitious wheel spinning),…

  18. 49 CFR 236.812 - Speed, restricted.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 4 2012-10-01 2012-10-01 false Speed, restricted. 236.812 Section 236.812 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Speed, restricted. A speed that will permit stopping within one-half the range of vision, but not...

  19. 49 CFR 236.812 - Speed, restricted.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 4 2010-10-01 2010-10-01 false Speed, restricted. 236.812 Section 236.812 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Speed, restricted. A speed that will permit stopping within one-half the range of vision, but not...

  20. 49 CFR 236.812 - Speed, restricted.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 4 2014-10-01 2014-10-01 false Speed, restricted. 236.812 Section 236.812 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Speed, restricted. A speed that will permit stopping within one-half the range of vision, but not...