In-solution hybrid capture of bisulfite-converted DNA for targeted bisulfite sequencing of 174 ADME genes

DNA methylation is one of the most important epigenetic alterations involved in the control of gene expression. Bisulfite sequencing of genomic DNA is currently the only method to study DNA methylation patterns at single-nucleotide resolution. Hence, next-generation sequencing of bisulfite-converted DNA is the method of choice to investigate DNA methylation profiles at the genome-wide scale. Nevertheless, whole genome sequencing for analysis of human methylomes is expensive, and a method for targeted gene analysis would provide a good alternative in many cases where the primary interest is restricted to a set of genes.Here, we report the successful use of a custom Agilent SureSelect Target Enrichment system for the hybrid capture of bisulfite-converted DNA. We prepared bisulfite-converted next-generation sequencing libraries, which are enriched for the coding and regulatory regions of 174 ADME genes (i.e. genes involved in the metabolism and distribution of drugs). Sequencing of these libraries on Illumina’s HiSeq2000 revealed that the method allows a reliable quantification of methylation levels of CpG sites in the selected genes, and validation of the method using pyrosequencing and the Illumina 450K methylation BeadChips revealed good concordance.     

Eye movements in the process of reading as an indicator of development of reading skill

The parameters of oculomotor activity were investigated using the video recording method when texts of different complexity were read by pupils with various reading skill levels. The parameters of eye movements and their determination have been described. It has been shown that the total reading time, the average duration of fixations, and the total number of regressions decrease, and the amplitude of progressive and regressive saccades increases as the reading skill is formed, which testifies to the perfection of the mechanisms of implementing the cognitive processes that form the basis of reading.     

Early Miocene herpetofaunas from the Greek localities of Aliveri and Karydia – bridging a gap in the knowledge of amphibians and reptiles from the early Neogene of southeastern Europe

Abstract

We here describe new remains of amphibians and reptiles from the early Miocene (MN 4) of two different Greek localities, Aliveri and Karydia. The newly described material consists of urodelans, alytids, indeterminate anurans, turtles, crocodylians, lacertids, indeterminate scincomorphs, anguids, colubrids, viperids, and indeterminate snakes. The presence of the frog Latonia cf. gigantea in Greece is documented for the first time. Additionally, the presence of viperids in Aliveri implies a much wider distribution for these snakes during the early Miocene of Europe. Of special interest is the presence of a peculiar colubrid that seems to possess a hitherto unknown vertebral structure, which is herein defined as the ‘paracentral ridge’. Although incomplete, the new material has important taxonomic and biogeographic implications, as it enhances our understanding of southeastern European herpetofaunas from the early Miocene, a time period that was characterised by major dispersal and extinction events and climatic change that affected the whole continent.     

Models of Monoamine Oxidase A and B Active Sites Obtained by using 3D QSAR with CoMFA Analysis

Abstract

The monoamine oxidase catalyses the oxidative deamination of neuroactive amines. This enzyme exists in two forms A and B, which differ by substrates preference and inhibitors specificity. Investigation of the structures of these enzymes and design new selective inhibitors are of greatly interesting since MAO A inhibitors are used in therapeutic practice as antidepressants and MAO B inhibitors – in the treatment Parkinson's diseases. The three dimension structures of monoamine oxidases are still unknown. Therefore, one of the most perspective approach to define significant features of structure active site is method based on analysis of structure-activity relationship (3D QSAR) with comparison of molecular fields analysis (CoMFA) allowing to get the spatial distribution of important properties affecting the activity.

In present study we investigate the structures of active sites MAO A and B using 16 pyrazinocarbazole derivatives in variant conformation. Majority of pyrazinocarbazole derivatives have a rigit conformation, but three of those is sufficiently flexible. The latters can be in two conformation types: long molecules (substitution accommodate along axis of main structure) and short molecules (substitution accommodate at acute angle about of main structure). Several 3D QSAR and CoMFA models of MAO A and B active sites were design for data sets containing various types of flexible molecules conformation. All obtained models are statistical reliable and have sufficient predictive power for tested compound tetrindole. The best MAO A model that include two flexible molecules in long conformations was obtained, and the longest one of those in short conformation. In contrast, for MAO B model containing all flexible molecules in the short conformations is more preferred.

On the basis of obtained data the schematic models of MAO A and B active sites structures are proposed. According to these models MAO A active site have the narrow long cavity that accommodate long molecules, while MAO B active site is broader and shorter.     

CC/GG Contacts Facilitate the B to A Transition of DMA in Solution

Abstract

Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTG- CAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts.

First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents.

A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex. The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA. The B-phility value was estimated to be 3 kcal/mol of 10-mer.

The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex. The designed 10-mers made it possible to estimate quantitatively the A- phility of CC/GG contact as compared with an average DNA: (F_A-F_B)CC=0.2 Kcal/mol, (F_A-F_B)DNA=0.7 Kcal/mol.     

Peculiarities of the B to A Transition of the λ Phage Regulatory Site OR3 and of Its Fragment

Abstract

Conformations of the synthetic deoxyoligonucleotide 17 base pairs long, which is an OR3 operator of λ phage, and of its 9-b.p. fragment were studied by the circular dichroism method (CD). The regions of stability of the double-stranded state were determined for these duplexes. A comparison of the CD spectra for these oligonucleotides with the CD for a lengthy DNA showed the conformation of these short DNA pieces to belong to the B-family.

A cooperative change in the CD spectra is observed in trifluoroethanol (TFE) solutions at a TFE concentration specific for each oligonucleotide, which is supposed to stem from a B to A transition. The length of the fragment was found to affect the ability for the B-A transition. The transition into the A form is hindered by 13% TFE for the short 9-nucleotide in comparison with the 17-nucleotide. We suggest that this is due to the B form stabilization by terminal base pairs (B-phility of the ends).     

Morphology of the jaw apparatus in 8 species of Patellogastropoda (Mollusca, Gastropoda) with special reference to Testudinalia tesulata (Lottiidae)

The fine structure of the jaw apparatus was studied by scanning electron microscopy in eight species of Patellogastropoda. The jaw apparatus is an unpaired two-layered dorsolateral structure with anterior and posterior wings attached to the odontophore by muscles. The jaw of Testudinalia tesulata (O.F. Müller, 1776) is a derivative of the cuticle typical for the foregut. The tissue forming the jaw is a specialized foregut epithelium (gnathoepithelium), consisting of a special type of cells called gnathoblasts. The jaw grows in areas of the epithelium characterized by high concentration of electron-dense vesicles, ER and long microvilli that penetrate deep into the jaw plate. This indicates that the gnathoblasts take an active part in jaw growth. In most cases, these areas of the gnathoepithelium are highly folded. The main differences between the species studied are form and thickness of the frontal edge of the jaw. These differences do not correlate with the systematic position of the species studied but likely depend more on the feeding mode. The transmission electron microscopy studies yielded new morphological criteria for comparison between various gastropod species and other members of Trochozoa, in particular, Annelida. The jaws of Annelida are cuticular structures formed on the surface of specialized epithelial cells, often also called gnathoblasts. The jaw of Patellogastropoda can be attributed to the first type of annelid jaw formation characterized by an epithelium with long microvilli and continuous growth.     

3D-QSAR with CoMFA Model of Prolylendopeptidase Substrates

Abstract

The prolylendopeptidase (PEP) is the proteolytic enzyme, which plays an essential role in the regulation of some processes in central nervous system, such as memory, learning and behavior. It was shown that PEP activity changes at different diseases, like Parkinsons or Alzheimer's diseases, and some PEP inhibitors are used in therapy. At present time the discovery of new types of PEP inhibitors are the actual task.

In this study the structure of PEP active site was analyzed by 3D-QSAR with CoMFA methods using of 12 PEP substrates. The designed pharmacophore model assumes that substrates interact with PEP active site by pyrrolidol ring of proline residue and by hydrogen bonding.

The 3-D-QSAR + CoMFA model of PEP substrates propose that the hydrophobic bonds play the essential role in substrate interaction with enzyme. This model reveals the important steric and electrostatic areas around the molecules and the presence of substituents controls the PEP activity for substrates. Analysis of obtained data allows to assume, that substrate binding in PEP active site causes essential perturbations of substrate structure. This effect mainly depends on chemical nature of the amino acid side chain, located near to proline.     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

A TRPC1 Protein-dependent Pathway Regulates Osteoclast Formation and Function

Ca²⁺ signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca²⁺ stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ϵ) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ϵ in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, mediating store-operated currents. TRPC1ϵ physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ϵ-Orai1 complex through TRPC1ϵ suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ϵ and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.