scholarly journals Oxidative and non-oxidative active turnover of genomic methylcytosine in distinct pluripotent states

2019 ◽  
Author(s):  
Fabio Spada ◽  
Sarah Schiffers ◽  
Angie Kirchner ◽  
Yingqian Zhang ◽  
Gautier Arista ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Methylation ◽  
Active Component ◽  
Turnover Rate ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Metabolic Labelling ◽  
Turnover Process ◽  
Low Levels ◽  
Epigenetic Plasticity

AbstractEpigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known and the underlying pathways are poorly understood. Here we use metabolic labelling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic methylcytidine (mdC), hydroxymethylcytidine (hmdC) and formylcytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on Tet-mediated mdC oxidation, we unveiled an additional mdC oxidation-independent turnover process based on DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage committed cells. Thus, in pluripotent cells active mdC turnover involves both mdC oxidation-dependent and -independent processes.

scholarly journals Hypermethylation of mismatch repair gene hMSH2 associates with platinum-resistant disease in epithelial ovarian cancer

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Hua Tian ◽  
Li Yan ◽  
Li Xiao-fei ◽  
Sun Hai-yan ◽  
Chen Juan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Ovarian Cancer ◽  
Dna Methylation ◽  
Epithelial Ovarian Cancer ◽  
Upstream Region ◽  
Platinum Resistance ◽  
Maldi Tof Mass Spectrometry ◽  
Platinum Resistant ◽  
Aberrant Dna Methylation ◽  
Low Expression

Abstract Purpose One major reason of the high mortality of epithelial ovarian cancer (EOC) is due to platinum-based chemotherapy resistance. Aberrant DNA methylation may be a potential mechanism underlying the development of platinum resistance in EOC. The purpose of this study is to discover potential aberrant DNA methylation that contributes to drug resistance. Methods By initially screening of 16 platinum-sensitive/resistant samples from EOC patients with reduced representation bisulfite sequencing (RRBS), the upstream region of the hMSH2 gene was discovered hypermethylated in the platinum-resistant group. The effect of hMSH2 methylation on the cellular response to cisplatin was explored by demethylation and knockdown assays in ovarian cancer cell line A2780. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was employed to examine the methylation levels of hMSH2 upstream region in additional 40 EOC patient samples. RT-qPCR and IHC assay was used to detect the hMSH2 mRNA and protein expression in extended 150 patients. Results RRBS assay discovered an upstream region from − 1193 to − 1125 of hMSH2 was significant hypermethylated in resistant EOC patients (P = 1.06 × 10−14). In vitro analysis demonstrated that global demethylation increased cisplatin sensitivity along with a higher expression of the hMSH2 mRNA and protein. Knockdown hMSH2 reduced the cell sensitivity to cisplatin. MALDI-TOF mass spectrometry assay validated the strong association of hypermethylation of hMSH2 upstream region with platinum resistance. Spearman’s correlation analysis revealed a significantly negative connection between methylation level of hMSH2 upstream region and its expression. The Kaplan-Meier analyses showed the high methylation of hMSH2 promoter region, and its low expressions are associated with worse survival. In multivariable models, hMSH2 low expression was an independent factor predicting poor outcome (P = 0.03, HR = 1.91, 95%CI = 1.85–2.31). Conclusion The hypermethylation of hMSH2 upstream region is associated with platinum resistant in EOC, and low expression of hMSH2 may be an index for the poor prognosis.

scholarly journals Isotopologue distributions of peptide product ions by tandem mass spectrometry: Quantitation of low levels of deuterium incorporation

2007 ◽  
Vol 367 (1) ◽  
pp. 40-48 ◽  
Author(s):  
Benlian Wang ◽  
Gang Sun ◽  
David R. Anderson ◽  
Minghong Jia ◽  
Stephen Previs ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
Tandem Mass ◽  
Deuterium Incorporation ◽  
Product Ions ◽  
Low Levels ◽  
Peptide Product

scholarly journals Design, measurement and processing of region-specific DNA methylation assays: the mass spectrometry-based method EpiTYPER

2015 ◽  
Vol 6 ◽  
Author(s):  
H. Eka D. Suchiman ◽  
Roderick C. Slieker ◽  
Dennis Kremer ◽  
P. Eline Slagboom ◽  
Bastiaan T. Heijmans ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Methylation

scholarly journals DNA methylation specifies chromosomal localization of MeCP2.

1996 ◽  
Vol 16 (1) ◽  
pp. 414-421 ◽  
Author(s):  
X Nan ◽  
P Tate ◽  
E Li ◽  
A Bird
Keyword(s):  
Dna Methylation ◽  
Cultured Cells ◽  
Centromeric Heterochromatin ◽  
Intact Protein ◽  
Chromosomal Protein ◽  
Mouse Cells ◽  
Low Levels ◽  
Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

scholarly journals Reversible programming of pluripotent cell differentiation

2000 ◽  
Vol 113 (3) ◽  
pp. 555-566 ◽  
Author(s):  
J. Lake ◽  
J. Rathjen ◽  
J. Remiszewski ◽  
P.D. Rathjen
Keyword(s):  
Gene Expression ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Primitive Endoderm ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Differentiation Potential

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

Epigenomic profiling in polycythaemia vera and essential thrombocythaemia shows low levels of aberrant DNA methylation

2011 ◽  
Vol 64 (11) ◽  
pp. 1010-1013 ◽  
Author(s):  
S. Barrio ◽  
M. Gallardo ◽  
E. Albizua ◽  
A. Jimenez ◽  
I. Rapado ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Polycythaemia Vera ◽  
Essential Thrombocythaemia ◽  
Low Levels ◽  
Aberrant Dna Methylation

Deficiency in Vitamin B12 and Ferritin Is Associated With Epigenetic Alterations in Cancer Patients  

2021 ◽  
Author(s):  
Khaled A. Elawdan ◽  
Sabah Farouk ◽  
Salah Araf ◽  
Hany Khalil
Keyword(s):  
Dna Methylation ◽  
Vitamin B12 ◽  
Cancer Patients ◽  
Promoter Region ◽  
Dna Methyltransferases ◽  
Epigenetic Alterations ◽  
Promoter Regions ◽  
Methylation Sensitive Restriction Enzyme ◽  
Low Levels ◽  
Dna Methylation Analysis

Abstract Background: Cancer is the second-leading cause of death worldwide, caused by several mutations in DNA within the cells including epigenetic alteration. The epigenetic changes are external modifications to the DNA that switch “on” or “off” gene expression. The present study was conducted to investigate the epigenetic modifications and its correlation with the levels of vitamin B12 and ferritin in cancer patients with hepatocellular carcinoma (HCC), breast cancer (BC), lung cancer (LC), or colon cancer (CC). Methods and Results: A total of 200 blood samples were obtained from cancer patients and healthy individuals. The relative expression of DNA methyltransferases (DNMTs), Ten-Eleven translocation (TET), and methionine synthase (MS) was evaluated in patients with the normal level of vitamin B12/ferritin and patients with the deficient levels of them. DNA methylation within the promoter regions was investigated of each indicated genes using the methylation-sensitive restriction enzyme HpaII and bisulfite PCR. Interestingly, the expression of DNMT1, DNMT3a, and DNMT3b was increased in patients with low levels of vitamin B12 and ferritin, while the expression of MS, TET1, and TET3 was significantly decreased. DNA methylation analysis in patients with deficient levels of vitamin B12/ferritin showed a methylated-cytosine within the location 318/CG and 385/CG in the promoter region of TET1 and TET3, respectively. Moreover, the bisulfite PCR assay further confirmed the methylation changes in the promoter region of TET1 and TET3 at the indicated locations. Conclusion: These data indicate that the deficiency in vitamin B12 and ferritin in cancer patients plays a key role in the epigenetic exchanges during cancer development.

scholarly journals Analysis of allergenic residues in wines by triple quadrupole LCMS

2019 ◽  
Vol 12 ◽  
pp. 04012
Author(s):  
F.R. Spinelli ◽  
G.J. Cargnel ◽  
A.P. Drehmer ◽  
C. Blatt ◽  
M. Baptistão ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Triple Quadrupole ◽  
Liquid Chromatography Mass Spectrometry ◽  
Treatment Practices ◽  
Unambiguous Determination ◽  
Allergenic Proteins ◽  
Beta Casein ◽  
Fining Agents ◽  
Low Levels

During the winemaking are used technology coadjuvants, between them: albumin, caseinates and lysozyme. These compounds have great oenological properties, however, the presence of their residues can represent risks to people who are allergic to them because they are derived from eggs and milk. Mass spectrometry methods enables unambiguous determination of allergenic proteins at low levels in wines. Therefore, the aim of this study was to determine the concentrations of ovalbumin, alpha-casein, beta-casein and lysozyme in experimental wines treated with different concentrations of them by triple quadrupole liquid chromatography mass spectrometry with Jet Stream Electrospray Ionization (ESI). The wines were elaborated and treated with different concentrations of albumin, lysozyme and potassium caseinate. Bentonite and decantation were used for the removal of the fining agents. The quantification limits (LOQ) for ovalbumin, a-casein, b-casein and lysozyme were: 0.002 mg/L, 0.24 mg/L, 0.75 mg/L and 0.04 mg/L, respectively. Non residues of the proteins were identified in the experimental wines treated with the different amounts of potassium caseinate, albumin and lysozyme, analyzed in this study. These results provide an evidence of the absence of residues of caseinate, albumin and lysozyme in the concentrations tested in the wines if good treatment practices are followed.

scholarly journals Mechanism of Intrinsic Resistance to Vancomycin in Clostridium innocuum NCIB 10674

2004 ◽  
Vol 186 (11) ◽  
pp. 3415-3422 ◽  
Author(s):  
Véronique David ◽  
Bülent Bozdogan ◽  
Jean-Luc Mainardi ◽  
Raymond Legrand ◽  
Laurent Gutmann ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Pressure ◽  
Amino Group ◽  
Intrinsic Resistance ◽  
Chromosomal Fragment ◽  
High Affinity ◽  
C Terminus ◽  
Low Levels ◽  
Enterococcus Gallinarum ◽  
Level Resistance

ABSTRACT We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 μg/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[d-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of ddlc. innocuum and C. innocuum racemase genes putatively encoding homologues of d-Ala:d-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned ddlc. innocuum and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[d-Ala], the presence of an UDP-MurNAc-pentapeptide[d-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanXYc gene from Enterococcus gallinarum BM4174 which encodes a d,d-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [d-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal ddlc. innocuum and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.

scholarly journals APOL1 polymorphism modulates sphingolipid profile of human podocytes

2020 ◽  
Vol 37 (6) ◽  
pp. 729-744 ◽  
Author(s):  
Manuela Valsecchi ◽  
Valentina Cazzetta ◽  
Ferdinando Oriolo ◽  
Xiqian Lan ◽  
Rocco Piazza ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Plasma Membrane ◽  
High Throughput ◽  
Living Cells ◽  
Bioactive Components ◽  
Wild Type ◽  
Drastic Reduction ◽  
Metabolic Labelling ◽  
Layer Chromatography ◽  
Apolipoprotein L1

AbstractApolipoprotein L1 (APOL1) wild type (G0) plays a role in the metabolism of sphingolipids, glycosphingolipids, sphingomyelin and ceramide, which constitute bioactive components of the lipid rafts (DRM). We asked whether APOL1 variants (APOL1-Vs) G1 and G2 carry the potential to alter the metabolism of sphingolipids in human podocytes. The sphingolipid pattern in HPs overexpressing either APOL1G0 or APOL1-Vs was analysed by using a thin mono- and bi-dimensional layer chromatography, mass-spectrometry and metabolic labelling with [1-3H]sphingosine. HP G0 and G1/G2-Vs exhibit a comparable decrease in lactosylceramide and an increase in the globotriaosylceramide content. An analysis of the main glycohydrolases activity involved in glycosphingolipid catabolism showed an overall decrease in the activeness of the tested enzymes, irrespective of the type of APOL1-Vs expression. Similarly, the high throughput cell live-based assay showed a comparable increased action of the plasma membrane glycosphingolipid-glycohydrolases in living cells independent of the genetic APOL1 expression profile. Importantly, the most significative modification of the sphingolipid pattern induced by APOL1-Vs occurred in DRM resulted with a drastic reduction of radioactivity associated with sphingolipids. G1/G2-Vs present a decrease amount of globotriaosylceramide and globopentaosylceramide compared to G0. Additionally, ceramide at the DRM site and lactosylceramide in general, showed a greatest fall in G1/G2 in comparison with G0. Additionally, the levels of glucosylceramide decreased only in the DRM of human podocytes overexpressing G1/G2-Vs. These findings suggest that altered sphingolipidsprofiles may contribute to the deranged functionality of the plasma membrane in APOL1 risk milieu.

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