Lineage-specific regulation of epigenetic modifier genes in human liver and brain.

Despite an abundance of studies on chromatin states and dynamics, there is an astonishing dearth of information on the expression of genes responsible for regulating histone and DNA modifications. We used here a set of 156 defined epigenetic modifier genes (EMG) and profiled their expression pattern in cells of different lineages. As reference value, expression data from human embryonic stem cells (hESC) were used. Hepatocyte-like cells were generated from hESC, and their EMG expression was compared to primary human liver cells. In parallel, we generated postmitotic human neurons (Lu d6), and compared their relative EMG expression to human cortex (Ctx). Clustering analysis of all cell types showed that neuronal lineage samples grouped together (94 similarly regulated EMG), as did liver cells (61 similarly-regulated), while the two lineages were clearly distinct. The general classification was followed by detailed comparison of the major EMG groups; genes that were higher expressed in differentiated cells than in hESC included the acetyltransferase KAT2B and the methyltransferase SETD7. Neuro-specific EMGs were the histone deacetylases HDAC5 and HDAC7, and the arginine-methyltransferase PRMT8. Comparison of young (Lu d6) and more aged (Ctx) neuronal samples suggested a maturation-dependent switch in the expression of functionally homologous proteins. For instance, the ratio of the histone H3 K27 methyltransfereases, EZH1 to EZH2, was high in Ctx and low in Lu d6. The same was observed for the polycomb repressive complex 1 (PRC1) subunits CBX7 and CBX8. A large proportion of EMGs in differentiated cells was very differently expressed than in hESC, and absolute levels were significantly higher in neuronal samples than in hepatic cells. Thus, there seem to be distinct qualitative and quantitative differences in EMG expression between cell lineages.

From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects.

The superordinate principles governing the transcriptome response of differentiating cells exposed to drugs are still unclear. Often, it is assumed that toxicogenomics data reflect the immediate mode of action (MoA) of drugs. Alternatively, transcriptome changes could describe altered differentiation states as indirect consequence of drug exposure. We used here the developmental toxicants valproate and trichostatin A to address this question. Neurally differentiating human embryonic stem cells were treated for 6 days. Histone acetylation (primary MoA) increased quickly and returned to baseline after 48 h. Histone H3 lysine methylation at the promoter of the neurodevelopmental regulators PAX6 or OTX2 was increasingly altered over time. Methylation changes remained persistent and correlated with neurodevelopmental defects and with effects on PAX6 gene expression, also when the drug was washed out after 3-4 days. We hypothesized that drug exposures altering only acetylation would lead to reversible transcriptome changes (indicating MoA), and challenges that altered methylation would lead to irreversible developmental disturbances. Data from pulse-chase experiments corroborated this assumption. Short drug treatment triggered reversible transcriptome changes; longer exposure disrupted neurodevelopment. The disturbed differentiation was reflected by an altered transcriptome pattern, and the observed changes were similar when the drug was washed out during the last 48 h. We conclude that transcriptome data after prolonged chemical stress of differentiating cells mainly reflect the altered developmental stage of the model system and not the drug MoA. We suggest that brief exposures, followed by immediate analysis, are more suitable for information on immediate drug responses and the toxicity MoA.

Quantitative analysis for breast density estimation in low dose chest CT scans.

A computational method was developed for the measurement of breast density using chest computed tomography (CT) images and the correlation between that and mammographic density. Sixty-nine asymptomatic Asian women (138 breasts) were studied. With the marked lung area and pectoralis muscle line in a template slice, demons algorithm was applied to the consecutive CT slices for automatically generating the defined breast area. The breast area was then analyzed using fuzzy c-mean clustering to separate fibroglandular tissue from fat tissues. The fibroglandular clusters obtained from all CT slices were summed then divided by the summation of the total breast area to calculate the percent density for CT. The results were compared with the density estimated from mammographic images. For CT breast density, the coefficient of variations of intraoperator and interoperator measurement were 3.00 % (0.59 %-8.52 %) and 3.09 % (0.20 %-6.98 %), respectively. Breast density measured from CT (22 ± 0.6 %) was lower than that of mammography (34 ± 1.9 %) with Pearson correlation coefficient of r=0.88. The results suggested that breast density measured from chest CT images correlated well with that from mammography. Reproducible 3D information on breast density can be obtained with the proposed CT-based quantification methods.

Stilbene levels and antioxidant activity of Vranec and Merlot wines from Macedonia: effect of variety and enological practices.

The content of the stilbenes trans-resveratrol and piceid as well as the antioxidant activity of Macedonian red wines from the two main grape varieties Vranec and Merlot have been evaluated. Тhe effects of time of maceration, type of yeast and the level of sulphur dioxide applied on stilbene content and antioxidant activity have been studied. The most important factor in winemaking technology is the maceration time since the highest concentrations of trans-resveratrol, piceid and highest antioxidant activity were found following 6 and 10 days of maceration. Concerning the yeast type, higher concentrations of trans-resveratrol and piceid have been obtained with French yeast "Levuline CHP" in comparison to Macedonian yeast "Vinalco". In contrast, the higher antioxidant activity of wines from both varieties of grapes was observed by application of Macedonian yeast "Vinalco".

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

Extensive transcriptional regulation of chromatin modifiers during human neurodevelopment.

Epigenetic changes, including histone modifications or chromatin remodeling are regulated by a large number of human genes. We developed a strategy to study the coordinate regulation of such genes, and to compare different cell populations or tissues. A set of 150 genes, comprising different classes of epigenetic modifiers was compiled. This new tool was used initially to characterize changes during the differentiation of human embryonic stem cells (hESC) to central nervous system neuroectoderm progenitors (NEP). qPCR analysis showed that more than 60% of the examined transcripts were regulated, and >10% of them had a >5-fold increased expression. For comparison, we differentiated hESC to neural crest progenitors (NCP), a distinct peripheral nervous system progenitor population. Some epigenetic modifiers were regulated into the same direction in NEP and NCP, but also distinct differences were observed. For instance, the remodeling ATPase SMARCA2 was up-regulated >30-fold in NCP, while it remained unchanged in NEP; up-regulation of the ATP-dependent chromatin remodeler CHD7 was increased in NEP, while it was down-regulated in NCP. To compare the neural precursor profiles with those of mature neurons, we analyzed the epigenetic modifiers in human cortical tissue. This resulted in the identification of 30 regulations shared between all cell types, such as the histone methyltransferase SETD7. We also identified new markers for post-mitotic neurons, like the arginine methyl transferase PRMT8 and the methyl transferase EZH1. Our findings suggest a hitherto unexpected extent of regulation, and a cell type-dependent specificity of epigenetic modifiers in neurodifferentiation.

Effect of winemaking treatment and wine aging on phenolic content in Vranec wines.

Phenolic compounds and colour stability of red wines produced from Vranec Vitis vinifera L. grape variety were investigated by means of different maceration times (3, 6 and 10 days), two doses of SO2 (30 and 70 mg/L SO2), two yeasts for fermentation (Vinalco and Levuline), temperature of storage and time of aging (3, 6 and 16 months). In general, maceration time influenced the phenolics extraction from the grapes into the wine. Highest concentrations of phenolic components were observed in the wines produced with 6 days of maceration, except for the flavan-3-ols which were present in highest amounts in the wines macerated for 10 days. Higher doses of SO2 increased the extraction of polyphenols, preventing the wines from oxidation, while the effect of yeast on phenolics extraction was not significant. Wine aging affected the phenolic content of wines produced with 3 days of maceration and caused intensive decrease of anthocyanins during the storage period. Wines aged at higher temperature showed lower anthocyanin levels and less intense coloration. Principal component analysis revealed that separation of the wines was performed according to the hue value in correlation with the maceration time and time of wine aging.

HPLC method for determination of verapamil in human plasma after solid-phase extraction.

A simple, rapid and precise HPLC method has been developed for the assay of verapamil in human plasma. The clean up of the plasma samples was tested using several adsorbents for solid-phase extraction and best recovery was obtained using mixed-mode cartridges (HLB - hydrophilic-lipophilic balance) ranging between 94.70 and 103.71%. HPLC separation was performed with isocratic elution on Lichrospher 60 RP-select B column (250 mm x 4 mm I.D., 5 microm particle size). The mobile phase was 40% acetonitrile and 0.025 mol/L KH2PO4 with pH 2.5 at flow rate of 1 mL/min. Diltiazem was used as internal standard and the detection wavelength was 200 nm. The calibration curves were linear in the range of 10-500 ng/mL. The developed method is convenient for routine analysis of verapamil in human plasma.