Isocitrate Dehydrogenase 2 Dysfunction Contributes to 5-hydroxymethylcytosine Depletion in Gastric Cancer Cells.

The isocitrate dehydrogenase (IDH) family of enzymes comprises of the key functional metabolic enzymes in the Krebs cycle that catalyze the conversion of isocitrate to α-ketoglutarate (α-KG). α-KG acts as a cofactor in the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). However, the relationship between 5hmC and IDH in gastric cancer remains unclear. Our study revealed that the 5hmC level was substantially lower and 5mC level was slightly higher in gastric cancer tissues; however, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) levels did not change significantly in these tissues. We further examined the expression levels of IDH1 and IDH2 in gastric cancer tissues and observed that IDH2 levels were significantly lower in gastric cancer tissues than in the adjacent normal tissues. The ectopic expression of IDH2 can increase 5hmC levels in gastric cancer cells. In conclusion, our results suggested that IDH2 dysfunction is involved in 5hmC depletion during gastric cancer progression.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) deposited on silica nanoparticles constructs hierarchically ordered nanocapsules: curcumin conjugated nanocapsules as a novel strategy to amplify guanine selectivity among nucleobases.

Poly (9-(2-diallylaminoethyl)adenine HCl-co-sulfur dioxide) (Poly A) deposited on silica nanoparticles self-assembles to form hierarchically ordered nanocapsules. These nanocapsules can be conjugated with curcumin. The curcumin-conjugated nanocapsules are found to be spherical in size and their size ranges between 200 and 600 nm. We found that curcumin conjugated with silica nanoparticles marginally shows a selectivity (∼20%) for guanine over adenine, cytosine, thymine and uracil, but this selectivity is extraordinarily amplified to more than 500% in curcumin-conjugated nanocapsules prepared from the above procedure. FT-IR spectra along with lifetime measurements suggest that specific interaction between adenine moieties of Poly A nanocapsules and thymine/uracil does not affect the fluorescence of poly A nanocapsules. Thus, the sensitivity and selectivity for guanine estimation is due to hydrophobic interactions, which are assisted by the low water solubility of guanine as compared to the other nucleobases. The present method illustrates a wider linear dynamic range in the higher concentration range as compared to the reported methods. Finally, the degradation study proves that stability of curcumin is improved dramatically in such nanocapsules demonstrating that nanotechnology could be a viable method to improve selectivity of specific analyte and robustness of probe molecule during fluorescence based bio-sensing.

The relative importance of the adsorption and partitioning mechanisms in hydrophilic interaction liquid chromatography.

We propose an original model of effective diffusion along packed beds of mesoporous particles for HILIC developed by combining Torquatos model for heterogeneous beds (external eluent+particles), Landauers model for porous particles (solid skeleton+internal eluent), and the time-averaged model for the internal eluent (bulk phase+diffuse water (W) layer+rigid W layer). The new model allows to determine the analyte concentration in rigid and diffuse W layer from the experimentally determined retention factor and intra-particle diffusivity and thus to distinguish the retentive contributions from adsorption and partitioning. We apply the model to investigate the separation of toluene (TO, as a non-retained compound), nortriptyline (NT), cytosine (CYT), and niacin (NA) on an organic ethyl/inorganic silica hybrid adsorbent. Elution conditions are varied through the choice of a third solvent (W, ethanol, tetrahydrofuran (THF), acetonitrile (ACN), or n-hexane) in a mobile phase (MP) of ACN/aqueous acetate buffer (pH 5)/third solvent (90/5/5, v/v/v). Whereas NA and CYT retention factors increase monotonously from W to n-hexane as third solvent, NT retention reaches its maximum with polar aprotic third solvents. The involved equilibrium constants for adsorption and partitioning, however, do not follow the same trends as the overall retention factors. NT retention is dominated by partitioning and NA retention by adsorption, while CYT retention is controlled by adsorption rather than partitioning. Our results reveal that the relative importance of adsorption and partitioning mechanisms depends in a complex way from analyte properties and experimental parameters and cannot be predicted generally.

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.

Error rates for nanopore discrimination among cytosine, methylcytosine, and hydroxymethylcytosine along individual DNA strands.

Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ~3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5' and 3' of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context.

How to distinguish methyl-cytosine from cytosine with high fidelity.

Methylation of cytosines in the DNA is central to the epigenetic code. The patterns along the DNA formed by these chemical marks instruct the cell which proteins to express and their faithful maintenance after replication are vital to the organism's life. Although Dnmt1 is the enzyme catalyzing the methylation reaction, it was found that UHRF1 (ubiquitin-like, containing PHD and RING finger domain 1) is the protein that actually recognizes hemi-methylated CpG sites. Nevertheless, the physical mechanism driving the strikingly robust distinction between hemi-methylated and unmethylated sites is not known. In this paper, we show that the large difference in the binding affinities of UHRF1 to these sites is possible not due to the presence of the methyl group itself but is a result of the accompanying changes in the distribution of the electrons around the cytosine ring. In particular, methylation reduces the dipole moment of cytosine and, as a consequence, unmethylated DNA in its unbound state in water is more stable than hemi-methylated DNA. Furthermore, the interaction energy of hemi-methylated DNA bound to UHRF1 with its surrounding is stronger than that of unmethylated DNA. Thus, the change in the electronic structure of cytosine upon methylation destabilizes the unbound state and stabilizes the bound state rendering discrimination with high fidelity possible.

Selective capture of 5-hydroxymethylcytosine from genomic DNA.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer(1). Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells(2-4). 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases(2, 3). 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development(2, 5-10). To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC(11). To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically(12). Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by ß-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing. Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

[Separation of bases, phenols and pharmaceuticals on ionic liquid-modified silica stationary phase with pure water as mobile phase].

N-methylimidazolium ionic liquid (IL) -modified silica was prepared with the reaction of 3-chloropropyl modified silica and N-methylimidazole using toluene as solvent. Based on the multiple interactions between N-methylimidazolium IL-modified silica and analytes such as hydrophobic interaction, electrostatic attraction, repulsion interaction, hydrogen-bonding, etc., the bases (cytosine, thymine, 2-aminopyrimidine and 6-chloroguanine), phenols (m-aminophenol, resorcinol and m-nitrophenol) and three pharmaceuticals (moroxydine hydrochloride, acyclovir and cephalexin hydrate) were separated successfully with only pure water as the mobile phase. These chromatographic separations are environmental friendly, economical and convenient, without any organic solvent or buffer additive. The retention mechanism of these samples on the stationary phase was also investigated.

Genome-wide mapping of 5-hydroxymethylcytosine in embryonic stem cells.

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC). 5hmC and TET proteins have been implicated in stem cell biology and cancer, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the 'poised' chromatin signature found at developmentally-regulated genes in ES cells.

Fast hydrophilic interaction liquid chromatographic separations on bonded zwitterionic stationary phase.

Separation science is an art of obtaining adequate resolution of the desired compounds in minimum time, and with minimum effort in terms of sample preparation and data evaluation. In LC, where selectivity is a main driving force for separation, the availability of different separation modes capable of operating at high flow rates is a way to make combined optimal use of selectivity, efficiency, and speed. The separation of polar and hydrophilic compounds is problematic in RP LC due to the poor retention. Hydrophilic interaction liquid chromatography (HILIC) is a more straightforward separation mode to address this problem. Herein, it is shown that separations in HILIC mode are equally efficient as for RP, providing a potential for very fast separations on short columns. This is not only facilitated by the low viscosity of the mobile phase compositions used, compared to typical RP eluents, but also due to higher column permeability. To exemplify this, baseline separations of uracil and cytosine are shown in less than 4 s and of Tamiflu and its main metabolite in less than 40 s, both under isocratic conditions. HILIC must therefore be considered having potential for high throughput purposes, and being an attractive candidate as the second separation dimension in 2-D HPLC.

Accelerated multiple-pass moving average: a novel algorithm for baseline estimation in CE and its application to baseline correction on real-time bases.

We present a novel algorithm for baseline estimation in CE. The new algorithm which we have named as accelerated multiple-pass moving average (AMPMA) is combined to three preexisting low-pass filters, spike-removal, moving average, and multi-pass moving average filter, to achieve real-time baseline correction with commercial instrumentation. The successful performance of AMPMA is demonstrated with simulated and experimental data. Straightforward comparison of experimental data clearly shows the improvement AMPMA provides to the linear fitting, LOD, and accuracy (absolute error) of CE analysis.

A comparative study of void volume markers in immobilized-artificial-membrane and reversed-phase liquid chromatography.

A comparative study of 10 void volume marker candidates on C(8), C(18), and IAM columns was described under several pH (3.0, 4.8, and 7.3) buffer conditions. Pycnometric analysis has been performed on the columns for comparison. In acidic conditions, small organic carboxylic acids suggested by the manufacturer did not necessarily give reliable and consistent estimates of the void volume for IAM column. However, changes in temperature and organic modifier composition led to small and predictable effects. The results presented here provide minimal but practical guidelines for the use of void volume markers in IAM chromatography.

Retention behavior of small polar compounds on polar stationary phases in hydrophilic interaction chromatography.

Retention characteristics of four polar stationary phases (i.e., amide, amino, silica and sulfobetaine) were studied by using a group of small polar compounds in hydrophilic interaction chromatography (HILIC). Different polar stationary phases shared certain degrees of similarity, but also exhibited differences in retentivity and selectivity for the model compounds. Among the four columns studied, HILIC Silica column had the least retention for the model compounds, but also showed different selectivity from other three columns. Experimental data also provided some evidences that functional groups on the stationary phases might have certain degrees of influence on selectivity possibly through secondary interactions with the model compounds. The retention of the acids on the amino phase decreased with increasing salt concentration in the mobile phase due to the ion-exchange effect, and the retention process was endothermic as opposed to exothermic on other phases. This study also systematically investigated the effect of various experimental factors on the retention of the polar stationary phases, such as acetonitrile content, column temperature, buffer pH, salt type and concentration in the mobile phase.

[Separation and determination of purine bases and pyrimidine bases from nucleic acid hydrolysis by HPLC on BDS column].

The hydrolysates of nucleic acid, six purine bases and pyrimidine bases (cytosine, uracil, guanine, hypoxanthine, adenine and thymine) were separated and determined by using HPLC. It is discussed how the column and mobile phase affect the separation. The peaks of cytosine and adenine are tailed on ordinary C18 column, and they are very good on BDS-C18 column. The KH2PO4-H3PO4 buffer can be used in separation the hydrolysates of RNA and DNA, and the NaAc-HAc buffer is only used in DNA. In addition, pH value is a very important factor for separation. With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant. The chosen mobile phase was 0.1 mol/L KH2PO4-H3PO4 buffer, with a pH value of 4.05. It was detected at UV 260 nm. The determination was completed within 10 min. The RSDs were all less than 3% and the recoveries were in the range of 82%-114%. The method has been applied to the detection of yeast hydrolysates.

Isolation and identification of a metabolite of cidofovir from rat kidney.

Cidofovir is an acyclic nucleotide analog with potent and broad-spectrum antiviral activity against adenoviruses and herpesviruses including cytomegalovirus (CMV). Cidofovir undergoes intracellular phosphorylation by host enzymes to cidofovir phosphate and cidofovir diphosphate (the active form). An unidentified metabolite has been observed previously in rat tissues and in urine of rabbits, rats and monkeys dosed with cidofovir. In the present study, this metabolite was isolated from rat kidney following an intravenous dose of 100 mg kg-1 cidofovir. The metabolite (metabolite I) was separated from cidofovir and impurities using extraction on anion-exchange resin followed by preparative normal and reversed-phase high-performance liquid chromatography (HPLC). The isolated metabolite I was subjected to proton, 13C and phosphorus nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization mass spectroscopy, and confirmed to be cidofovir-phosphocholine. The uptake of cidofovir by rat kidney was saturated at an intravenous dose of 100 mg kg-1, probably as a result of saturation of the renal tubular secretion pathway. However, the relative abundance of cidofovir phosphocholine was not affected by dose.

Liquid chromatographic separation of hexopyranosylated cytosine nucleosides from their degradation products.

Development of a liquid chromatographic method which can separate each of a series of hexopyranosylated cytosine nucleosides from their degradation products formed at acid, neutral and basic pH is described. Both silica-based reverse-phase and polymer columns were examined. Influence of the mobile phase pH, ion-pairing agent, concentration of the buffer and type and concentration of organic modifier were systematically investigated. The concentration of the ion-pairing agent and the buffer were found to have a major effect on selectivity. Samples were finally analyzed on a poly(styrene-divinylbenzene), PLRP-S 100 A (8 microns) 250 x 4.6 mm I.D. column at 60 degrees C and with a mobile phase consisting of acetonitrile-sodium octanesulphonate (pH 2.5; 0.02 M)-potassium phosphate buffer (pH 2.5; 0.2 M)-water (X:25:50:25-X, v/v, where X is variable).

Cytosinine: pyridoxal phosphate tautomerase, a new enzyme in the blasticidin S biosynthetic pathway.

Cytosinine--the nucleoside portion of blasticidin S--and pyridoxal phosphate were incubated with cell-free extracts of Streptomyces griseochromogenes prepared in D2O. 2H NMR analysis of recovered cytosinine showed it to contain deuterium enrichments at H-4' and H-2'. No exchange was observed with either boiled extract or from cytosinine and pyridoxal phosphate alone. These results reveal the presence of a tautomerase activity that contributes to the net transamination at C-4' in the conversion of cytosylglucuronic acid to blasticidin S, and its discovery supports the role of cytosinine as a biosynthetic intermediate.

Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases.

Lipid peroxidation (LPO) products are known to interact with DNA, yielding several types of adduct with nucleobases. In this study, we demonstrate the formation of two ethenobase adducts, 1,N6-ethenoadenine and 3,N4-ethenocytosine, by reaction of LPO products with nucleic acid bases. Rat liver microsomes were incubated at 37 degrees C for 30 min in the presence of inducers of LPO [Fe(II) or cumene hydroperoxide] and adenine or cytosine nucleotides or nucleosides, followed by further heating at 80 degrees C for 30 min to complete the reactions. The etheno adducts detected after immunoaffinity chromatography were 1,N6-etheno-cAMP and 1,N6-etheno-2'-deoxyadenosine (HPLC/fluorimetry), 3,N4-etheno-2'-deoxycytidine (competitive radioimmunoassay), and 1,N6-etheno-2'-deoxyadenosine 3'-monophosphate and 3,N4-etheno-2'-deoxycytidine 3'-monophosphate (32P-postlabeling). Incubation of arachidonic acid supplemented with Fe(II) also led to the formation of the 1,N6-etheno adduct from cAMP. LPO intermediates that may be involved are discussed. These data suggest that etheno adducts may be markers of DNA damage associated with LPO.