Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

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

Thiopurine S-Methyltransferase Gene Polymorphisms in a Healthy Slovak Population and Pediatric Patients with Inflammatory Bowel Disease

Thiopurine methyltransferase (TPMT) is a key component in thiopurine metabolism. There is an insufficient evidence about the distribution of the genotype frequencies of TPMT variants and frequencies of TPMT alleles associated with intermediate and deficient activity in a healthy Slovak population and pediatric patients with inflammatory bowel disease (IBD). TPMT variant alleles (*1,*2, *3A, *3B, and *3C) were determined in 114 children treated for IBD and in 281 healthy volunteers. Mutant alleles were present in 9/114 (7.89%) in the IBD patients and in 23/281 (8.19%) of probands. The distribution of the most frequent variants of TPMT gene was similar in a healthy population and patients with IBD.     

Activation and inhibition of cyclin-dependent kinase-2 by phosphorylation; a molecular dynamics study reveals the functional importance of the glycine-rich loop

Nanoseconds long molecular dynamics (MD) trajectories of differently active complexes of human cyclin-dependent kinase 2 (inactive CDK2/ATP, semiactive CDK2/Cyclin A/ATP, fully active pT160-CDK2/Cyclin A/ATP, inhibited pT14-; pY15-; and pT14,pY15,pT160-CDK2/Cyclin A/ATP) were compared. The MD simulations results of CDK2 inhibition by phosphorylation at T14 and/or Y15 sites provide insight into the structural aspects of CDK2 deactivation. The inhibitory sites are localized in the glycine-rich loop (G-loop) positioned opposite the activation T-loop. Phosphorylation of T14 and both inhibitory sites T14 and Y15 together causes ATP misalignment for phosphorylation and G-loop conformational change. This conformational change leads to the opening of the CDK2 substrate binding box. The phosphorylated Y15 residue negatively affects substrate binding or its correct alignment for ATP terminal phospho-group transfer to the CDK2 substrate. The MD simulations of the CDK2 activation process provide results in agreement with previous X-ray data.     

Phytosociological affiliation of Annex II species Tephroseris longifolia subsp. moravica in comparison with two related Tephroseris species with overlapping distribution

The phytosociological affiliation of Tephroseris longifolia subsp. moravica, species of European importance, was studied in relation to two closely related species of the genus Tephroseris which have overlapping distribution within the Western Carpathian Mts: T. intergrifolia and T. crispa. The main aim was to compare plant communities inhabited by the three taxa, to assess the major environmental gradients responsible for variation in their distribution and to estimate ecological indicator values for Tephroseris longifolia subsp. moravica. T. longifolia subsp. moravica was recorded in nine localities in the Slovakia and Czech Republic where it occurs in very specific site conditions of ecotone habitats. Its phytosociological affiliation is restricted to grasslands of the alliances Bromion erecti and Arrhenatherion elatioris and to the ecotone vegetation between these grasslands and beech forests. T. integrifolia occurs most frequently in the Diantho lumnitzeri-Seslerion, Bromion erecti and Quercion pubescenti-petraeae alliances. T. crispa occurs predominantly in communities of the Calthion palustris alliance and Scheuchzerio-Caricetea fuscae, Mulgedio-Aconitetea and Montio-Cardaminetea classes. The major gradient responsible for variation in species composition of communities inhabited by the studied taxa was associated with moisture and nutrient content. The vascular plant-based ecological indicator values for Tephroseris longifolia subsp. moravica calculated from phytosociological relevés with its occurrence were set for light — 6, temperature — 5, continentality — 4, moisture — 5, soil reaction — 6 and nutrients — 5. We conclude that the studied taxon has intermediate relationship to the most of the studied factors in comparison with two related species, T. crispa and T. integrifolia.     

Variation in Trill Characteristics in Tree Pipit Songs: Different Trills for Different Use?

Transmitting information about singer's quality is an important function of song in many bird species, and this information should be useful in territorial interactions. Fast trills, being physically demanding song structures, are particularly suitable candidates for signalling of quality or aggressive motivation. We have evaluated trill characteristics in songs within a population of the Tree Pipit, a common European songbird with no sexual dimorphism, in which song apparently plays a key role in territory defence as well as mate choice. Two types of relatively fast trills (each of them in multiple variants differing in complexity) were commonly observed in repertoires of Tree Pipit males. Trill rates significantly differed among individuals, suggesting that these song structures may carry information about male quality in this species. We tested by playback experiments whether both trill types are used in territorial encounters. Only one of the trill types was sung by males in response to playback, regardless on the trill type played to them. In an immediate response to playback, they increased the frequency of use of this trill, and also significantly increased the trill rate in comparison with spontaneous songs. This confirmed field observations, suggesting that this trill is important in male–male interactions. On the contrary, the use of the fastest, apparently more demanding, trill type actually decreased after the simulated territorial intrusion. We hypothesize that the latter one is more directed towards females, and that while performance of both trill types may reflect male quality, they are primarily used in different contexts.     

Wine polyphenols improve cardiovascular remodeling and vascular function in NO-deficient hypertension

The effects of the red wine polyphenolic compounds (Provinol) on hypertension, left ventricular hypertrophy, myocardial fibrosis, and vascular remodeling were investigated after chronic inhibition of nitric oxide (NO) synthase by administration of N(G)-nitro-L-arginine methyl ester (L-NAME) to rats. Rats were divided into four groups: a control group, a group treated for 4 wk with L-NAME (40 mg x kg(-1) x day(-1)), and two groups treated with L-NAME followed by 3 wk of either spontaneous recovery or recovery with Provinol treatment (40 mg x kg(-1) x day(-1)). Administration of Provinol produced a greater readiness of the decrease in blood pressure than that in the spontaneous recovery group. Provinol significantly depressed myocardial fibrosis and expedited the decrease in aortic cross-sectional area, the increase in endothelium-dependent relaxation, and the decrease in contraction of the aorta. These effects of Provinol were associated with a greater increase of NO synthase activity in the left ventricle and the aorta. The present study provides evidence that Provinol accelerates the regression of blood pressure and improves structural and functional cardiovascular changes produced by chronic inhibition of NO synthesis.     

Dual Effects of Ketoconazole cis-Enantiomers on CYP3A4 in Human Hepatocytes and HepG2 Cells

Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (−)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (−)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, K_i 0.92 µM for testosterone, IC₅₀ 1.46 µM, K_i 2.52 µM for midazolam; (−)-ketoconazole IC₅₀ 0.90 µM, K_i 0.17 µM for testosterone, IC₅₀ 1.04 µM, K_i 1.51 µM for midazolam).     

Towards Clinical Molecular Diagnosis of Inherited Cardiac Conditions: A Comparison of Bench-Top Genome DNA Sequencers

Background

Molecular genetic testing is recommended for diagnosis of inherited cardiac disease, to guide prognosis and treatment, but access is often limited by cost and availability. Recently introduced high-throughput bench-top DNA sequencing platforms have the potential to overcome these limitations.

Methodology/Principal Findings

We evaluated two next-generation sequencing (NGS) platforms for molecular diagnostics. The protein-coding regions of six genes associated with inherited arrhythmia syndromes were amplified from 15 human samples using parallelised multiplex PCR (Access Array, Fluidigm), and sequenced on the MiSeq (Illumina) and Ion Torrent PGM (Life Technologies). Overall, 97.9% of the target was sequenced adequately for variant calling on the MiSeq, and 96.8% on the Ion Torrent PGM. Regions missed tended to be of high GC-content, and most were problematic for both platforms. Variant calling was assessed using 107 variants detected using Sanger sequencing: within adequately sequenced regions, variant calling on both platforms was highly accurate (Sensitivity: MiSeq 100%, PGM 99.1%. Positive predictive value: MiSeq 95.9%, PGM 95.5%). At the time of the study the Ion Torrent PGM had a lower capital cost and individual runs were cheaper and faster. The MiSeq had a higher capacity (requiring fewer runs), with reduced hands-on time and simpler laboratory workflows. Both provide significant cost and time savings over conventional methods, even allowing for adjunct Sanger sequencing to validate findings and sequence exons missed by NGS.

Conclusions/Significance

MiSeq and Ion Torrent PGM both provide accurate variant detection as part of a PCR-based molecular diagnostic workflow, and provide alternative platforms for molecular diagnosis of inherited cardiac conditions. Though there were performance differences at this throughput, platforms differed primarily in terms of cost, scalability, protocol stability and ease of use. Compared with current molecular genetic diagnostic tests for inherited cardiac arrhythmias, these NGS approaches are faster, less expensive, and yet more comprehensive.     

Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization

Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry-based proteomic approaches, we identify centrosomal and mitotic spindle proteins as interactors of ILK. alpha- and beta-tubulin, ch-TOG (XMAP215), and RUVBL1 associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A-TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation.