Singlet Oxygen Oxidation of Guanine, 9-Methylguanine and Guanine-Cytosine Base Pair: Dynamics and Kinetics Revealed by Parallel Gas- and Solution-Phase Experiments and Computations

Singlet oxygen (1O2) oxidatively generated damage of DNA gives rise to mutagenesis, carcinogenesis, and cellular death. Guanine is the most susceptible DNA target of 1O2. The related process has been studied over three decades but the mechanism has remained elusive. My thesis research has focused on reaction mechanism, dynamics and kinetics of 1O2 oxidation of guanine, 9-methylguanine and guanine-cytosine base pair, from the gas-phase bare ions, through hydrated clusters, to aqueous solution. Various techniques have been adapted in the work, including 1O2 generation and detection, guided-ion beam tandem mass spectrometry, gas-phase ion-molecule scattering, and on-line spectroscopy and mass spectrometry measurement of solution kinetics. Experimental measurements, corroborated by electronic structure calculations, Rice-Ramsperger-Kassel-Marcus (RRKM) theory and direct dynamics trajectory simulations, have provided insights into the 1O2 oxidation chemistry of guanine. Four projects have been completed, and each of which is described below. In the first project, ion-molecule scattering mass spectrometry was utilized to capture unstable endoperoxides in the collisions of hydrated guanine ions (protonated or deprotonated) with 1O2 at ambient temperature. Theoretical calculations have strongly supported an intermediate structure of 5,8-endoperoxide rather than 4,8-endoperoxide was proposed in literature. Protonation and deprotonation of reactants in the gas phase, vis-à-vis acidic and basic media in solution reactions, lead to different oxidation chemistries starting from initial stage. This project has pieced together reaction mechanisms and dynamics data concerning the early stage of 1O2 induced guanine oxidation, which is missing from conventional condensed-phase studies. In the second experiment of this thesis, gas-phase dry and monohydrated 9-methylguanine (9MG) was utilized as a model compound to examine the early stage oxidation mechanism and dynamics of the guanine nucleoside. Different levels of theory, including Multi-referential CASSCF and CASMP2, were applied for a reliable description of the early-stage reaction potential surface (PES). The oxidation of protonated 9MG is initiated by the formation of a 5,8-endoperoxide via a concerted cycloaddition as protonated guanine. In contrast, the initial stage of deprotonated 9MG oxidation switches to an addition of O2 to the C8 position only. The comparison between the 1O2 oxidation of ionized guanine and 9-methylguanine indicates that the N9-substitution not only affects the reaction mechanism but inhibits the reactivity of guanine toward 1O2. In the third project, a solution-phase kinetic and mechanistic study of 1O2 oxidation of guanine and 9MG was examined at pH 3.0, 7.0 and 10.0, respectively. Oxidation products and the branching ratio were determined, with each structure inferred from collision-induced dissociation (CID) mass spectra. In basic and neutral solutions, the oxidation products of guanine and 9MG are dominated by spiroiminodihydantoin (Sp), whereas in acidic solution guanidinohydantoin (Gh) is the favored product, showing strong pH dependence of oxidation. gem-diol intermediate, which serves as the precursor for the formation of Gh, was detected. On the basis of solution compositions at each pH, first-order rate constants for individual oxidizable species were extracted. That is 3.2 - 3.6 ´ 106 M-1∙s-1 for deprotonated guanine, 1.1 ´ 106 and 4.6 - 4.9 ´ 107 M-1∙s-1 for neutral and deprotonated 9MG, respectively. Guided by density functional theory-calculated reaction potential energy surfaces, transition state theory (TST) was applied to evaluate the kinetics of the 1O2 addition to guanine and 9MG. The comparison of TST predictions with experiment assures that initial 1O2 addition is the rate-limiting for oxidation, and all of the end products evolve from ensuring endoperoxides and/or peroxides which form at an efficiency of £ 2.5% based on previous measurements of the same systems in the gas phase. In the last project, an experimental and trajectory study was reported, focusing on the 1O2 oxidation of gas-phase deprotonated guanine-cytosine base pair [G·C – H]– that is composed of 9HG·[C – H]– and 7HG·[C – H]– (pairing 9H- or 7H-guanine with N1-deprotonated cytosine), and 9HG·[C – H]–_PT and 7HG·[C – H]–_PT (formed by intra-base-pair proton transfer from guanine N1 to the N3 of [C – H]–). Guided-ion-beam mass spectrometry was used to measure the conformer-averaged product and cross section for [G·C – H]– + 1O2. 1O2 collision dynamics with each of the four conformers was simulated at B3LYP/6-31G(d), to explicate conformation-specific reactivities and changes upon and after oxidation. Trajectories showed that 9HG-containing base pairs favor stepwise formation of 4,8-endoperoxide of guanine, whereas 7HG-containing base pairs prefer concerted formation of guanine 5,8-endoperoxide. Oxidation entangles with intra-base-pair proton transfer, and prefers to occur during the time when the base pair adopts a proton-transferred structure. Guided by trajectories, reaction PESs were established using spin-projected density functional theory. PESs indicate that proton-transferred base-pair conformers have lower barriers for oxidation than non-proton-transferred counterparts

EpCAM Is an Endoderm-Specific Wnt Derepressor that Licenses Hepatic Development

SummaryMechanisms underlying cell-type-specific response to morphogens or signaling molecules during embryonic development are poorly understood. To learn how response to the liver-inductive Wnt2bb signal is achieved, we identify an endoderm-enriched, single transmembrane protein, epithelial-cell-adhesion-molecule (EpCAM), as an endoderm-specific Wnt derepressor in zebrafish. hi2151/epcam mutants exhibit defective liver development similar to prt/wnt2bb mutants. EpCAM directly binds to Kremen1 and disrupts the Kremen1-Dickkopf2 (Dkk2) interaction, which prevents Kremen1-Dkk2-mediated removal of Lipoprotein-receptor-related protein 6 (Lrp6) from the cell surface. These data lead to a model in which EpCAM derepresses Lrp6 and cooperates with Wnt ligand to activate Wnt signaling through stabilizing membrane Lrp6 and allowing Lrp6 clustering into active signalosomes. Thus, EpCAM cell autonomously licenses and cooperatively activates Wnt2bb signaling in endodermal cells. Our results identify EpCAM as the key molecule and its functional mechanism to confer endodermal cells the competence to respond to the liver-inductive Wnt2bb signal

Enhancing Spoofing Speech Detection Using Rhythm Information

Current spoofing speech detection systems need more convincing evidence. In this paper, the flaws of rhythm information inherent in the TTS-generated speech are analyzed to increase the reliability of detection systems. TTS models take text as input and utilize acoustic models to predict rhythm information, which introduces artifacts in the rhythm information. By filtering out vocal tract response, the remaining glottal flow with rhythm information retains detection ability for TTS-generated speech. Based on these analyses, a rhythm perturbation module is proposed to enhance the copy-synthesis data augmentation method. Fake utterances generated by the proposed method force the detecting model to pay attention to the artifacts in rhythm information and effectively improve the ability to detect TTS-generated speech of the anti-spoofing countermeasures.Comment: Five pages, two figure