Targeting of CYP2E1 by miRNAs in alcohol-induced intestine injury.

Although binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol (EtOH)-inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in EtOH metabolism, promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that EtOH promotes CYP2E1 induction and activation, the mechanisms that regulate CYP2E1 expression in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR-378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to EtOH-induced intestinal injury. Here, we have shown that CYP2E1 expression is regulated post-transcriptionally through miRNA-mediated degradation, as follows: (1) the RNA-binding protein AU-binding factor 1 (AUF1) binds mature miRNAs, including CYP2E1-targeting miRNAs, and this binding modulates the degradation of corresponding target mRNAs upon EtOH treatment; (2) the serine/threonine kinase mammalian Ste20-like kinase 1 (MST1) mediates oxidative stress-induced phosphorylation of AUF1. Those findings suggest that reactive oxygen species-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Thus, our study demonstrates the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of EtOH, and the relationship of this pathway to subsequent intestinal injury.

Thermodynamic Reversal and Structural Correlation of 24-Crown-8/Protonated Tryptophan and 24-Crown 8/Protonated Serine Noncovalent Complexes in the Gas Phase vs in Solution: Quantum Chemical Analysis.

We investigate the structures of 24-crown-8/H+/l-tryptophan (CR/TrpH+) and 24-crown-8/H+/l-serine (CR/SerH+) noncovalent host-guest complex both in the gas phase and in an aqueous solution by quantum chemical methods. The Gibbs free energies of the complex in the two phases are calculated to determine the thermodynamically most favorable conformer in each phase. Our predictions indicate that both the carboxyl and the ammonium in CR/TrpH+ and the ammonium in the CR/SerH+ complexes in the lowest Gibbs free energy configurations form hydrogen bonds (H-bonds) with the CR host in the gas phase, while the conformer with the "naked" (devoid of H-bond with the CR host) -CO2H (and/or -OH) is much less favorable (Gibbs free energy higher by >3.6 kcal/mol). In the solution phase, however, a "thermodynamic reversal" occurs, making the higher Gibbs free energy gas-phase CR/TrpH+ and CR/SerH+ conformers thermodynamically more favorable under the influence of solvent molecules. Consequently, the global minimum Gibbs free energy structure in solution is structurally correlated with the thermodynamically much less gas-phase conformer. Discussions are provided concerning the possibility of elucidating host-guest-solvent interactions in solution from the gas-phase host-guest configurations in molecular detail.

Distinguishing gas phase lactose and lactulose complexed with sodiated L-arginine by IRMPD spectroscopy and DFT calculations.

We present the origin of the observed differentiation of lactose and lactulose achieved by complexation with sodiated L-arginine (ArgNa+). We find that the infrared multiphoton dissociation (IRMPD) bands in 3600-3650 and >3650 cm-1 regimes for gas phase lactose and lactulose, respectively, vanish when forming host-guest complexes with ArgNa+. We interpret these differences in the IRMPD spectra by scrutinizing the interactions between the functional groups (guanidium, -CO2-Na+) in ArgNa+ and -OHs in lactose/lactulose. Our calculated structures and infrared spectra of lactose/ArgNa+ and lactulose/ArgNa+ host-guest pairs indicate that the functional groups interact with the low- and high-frequency -OH stretch modes of lactose and lactulose, respectively, in the 3600-3720 cm-1 window.

Mechanistic Investigation of WWOX Function in NF-kB-Induced Skin Inflammation in Psoriasis.

Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperproliferation, aberrant differentiation of keratinocytes, and dysregulated immune responses. WW domain-containing oxidoreductase (WWOX) is a non-classical tumor suppressor gene that regulates multiple cellular processes, including proliferation, apoptosis, and migration. This study aimed to explore the possible role of WWOX in the pathogenesis of psoriasis. Immunohistochemical analysis showed that the expression of WWOX was increased in epidermal keratinocytes of both human psoriatic lesions and imiquimod-induced mice psoriatic model. Immortalized human epidermal keratinocytes were transduced with a recombinant adenovirus expressing microRNA specific for WWOX to downregulate its expression. Inflammatory responses were detected using Western blotting, real-time quantitative reverse transcription polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay. In human epidermal keratinocytes, WWOX knockdown reduced nuclear factor-kappa B signaling and levels of proinflammatory cytokines induced by polyinosinic: polycytidylic acid [(poly(I:C)] in vitro. Furthermore, calcium chelator and protein kinase C (PKC) inhibitors significantly reduced poly(I:C)-induced inflammatory reactions. WWOX plays a role in the inflammatory reaction of epidermal keratinocytes by regulating calcium and PKC signaling. Targeting WWOX could be a novel therapeutic approach for psoriasis in the future.

Functional and molecular dissection of HCMV long non-coding RNAs.

Small, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs); RNA1.2, RNA2.7, RNA4.9, and RNA5.0. Little is known about the function of these lncRNAs in the virus life cycle. Here, we dissected the functional and molecular landscape of HCMV lncRNAs. We found that HCMV lncRNAs occupy ~ 30% and 50-60% of total and poly(A)+viral transcriptome, respectively, throughout virus life cycle. RNA1.2, RNA2.7, and RNA4.9, the three abundantly expressed lncRNAs, appear to be essential in all infection states. Among these three lncRNAs, depletion of RNA2.7 and RNA4.9 results in the greatest defect in maintaining latent reservoir and promoting lytic replication, respectively. Moreover, we delineated the global post-transcriptional nature of HCMV lncRNAs by nanopore direct RNA sequencing and interactome analysis. We revealed that the lncRNAs are modified with N6-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth analysis demonstrated that m6A machineries stabilize HCMV lncRNAs, which could account for the overwhelming abundance of viral lncRNAs. Our study lays the groundwork for understanding the viral lncRNA-mediated regulation of host-virus interaction throughout the HCMV life cycle.

Ionic Liquids as Organocatalysts for Nucleophilic Fluorination: Concepts and Perspectives.

Besides their extremely useful properties as solvent, ionic liquids (ILs) are now considered to be highly instructive tools for enhancing the rates of chemical reactions. The ionic nature of the IL anion and cation seems to be the origin of this fascinating function of ILs as organocatalyst/promoter through their strong Coulombic forces on other ionic species in the reaction and also through the formation of hydrogen bonds with various functional groups in substrates. It is now possible to tailor-make ILs for specific purposes as solvent/promoters in a variety of situations by carefully monitoring these interactions. Despite the enormous potentiality, it seems that the application of ILs as organocatalysts/promoters for chemical reactions have not been fully achieved so far. Herein, we review recent developments of ILs for promoting the nucleophilic reactions, focusing on fluorination. Various aspects of the processes, such as organocatalytic capability, reaction mechanisms and salt effects, are discussed.

Unveiling host-guest-solvent interactions in solution by identifying highly unstable host-guest configurations in thermal non-equilibrium gas phase.

We propose a novel scheme of examining the host-guest-solvent interactions in solution from their gas phase structures. By adopting the permethylated ß-cyclodextrin (perm ß-CD)-protonated L-Lysine non-covalent complex as a prototypical system, we present the infrared multiple photon dissociation (IRMPD) spectrum of the gas phase complex produced by electrospray ionization technique. In order to elucidate the structure of perm ß-CD)/LysH+ complex in the gas phase, we carry out quantum chemical calculations to assign the two strong peaks at 3,340 and 3,560 cm-1 in the IRMPD spectrum, finding that the carboxyl forms loose hydrogen bonding with the perm ß-CD, whereas the ammonium group of L-Lysine is away from the perm ß-CD unit. By simulating the structures of perm ß-CD/H+/L-Lysine complex in solution using the supramolecule/continuum model, we find that the extremely unstable gas phase structure corresponds to the most stable conformer in solution.

Brain somatic mutations as RNA therapeutic targets in neurological disorders.

Research into the genetic etiology of a neurological disorder can provide directions for genetic diagnosis and targeted therapy. In the past, germline mutations, which are transmitted from parents or newly arise from parental germ cells, were considered as major genetic causes of neurological disorders. However, recent evidence has shown that somatic mutations in the brain, which can arise from neural stem cells during development or over aging, account for a significant number of brain disorders, ranging from neurodevelopmental, neurodegenerative, and neuropsychiatric to neoplastic disease. Moreover, the identification of disease-causing somatic mutations or mutated genes has provided new insights into molecular pathogenesis and unveiled potential therapeutic targets for treating neurological disorders that have few, or no, therapeutic options. RNA therapeutics, including antisense oligonucleotide (ASO) and small interfering RNA (siRNA), are emerging as promising therapeutic tools for treating genetic neurological disorders. As the number of approved and investigational ASO and siRNA drugs for neurological disorders associated with germline mutations increases, they may also prove to be attractive modalities for treating neurologic disorders resulting from somatic mutations. In this perspective, we highlight several neurological diseases caused by brain somatic mutations and discuss the potential role of RNA therapeutics in these conditions.

Nucleophilic Radiofluorination Using Tri-tert-Butanol Ammonium as a Bifunctional Organocatalyst: Mechanism and Energetics.

We present a quantum chemical analysis of the 18F-fluorination of 1,3-ditosylpropane, promoted by a quaternary ammonium salt (tri-(tert-butanol)-methylammonium iodide (TBMA-I) with moderate to good radiochemical yields (RCYs), experimentally observed by Shinde et al. We obtained the mechanism of the SN2 process, focusing on the role of the -OH functional groups facilitating the reactions. We found that the counter-cation TBMA+ acts as a bifunctional promoter: the -OH groups function as a bidentate 'anchor' bridging the nucleophile [18F]F- and the -OTs leaving group or the third -OH. These electrostatic interactions cooperate for the formation of the transition states of a very compact configuration for facile SN2 18F-fluorination.

Origin of Salt Effects in SN2 Fluorination Using KF Promoted by Ionic Liquids: Quantum Chemical Analysis.

Quantum chemical analysis is presented, motivated by Grée and co-workers' observation of salt effects [Adv. Synth. Catal. 2006, 348, 1149-1153] for SN2 fluorination of KF in ionic liquids (ILs). We examine the relative promoting capacity of KF in [bmim]PF6 vs. [bmim]Cl by comparing the activation barriers of the reaction in the two ILs. We also elucidate the origin of the experimentally observed additional rate acceleration in IL [bmim]PF6 achieved by adding KPF6. We find that the anion PF6- in the added salt acts as an extra Lewis base binding to the counter-cation K+ to alleviate the strong Coulomb attractive force on the nucleophile F-, decreasing the Gibbs free energy of activation as compared with that in its absence, which is in good agreement with experimental observations of rate enhancement. We also predict that using 2 eq. KF together with an eq. KPF6 would further activate SN2 fluorination.

Inter- and Intra-Molecular Organocatalysis of SN2 Fluorination by Crown Ether: Kinetics and Quantum Chemical Analysis.

We present the intra- and inter-molecular organocatalysis of SN2 fluorination using CsF by crown ether to estimate the efficacy of the promoter and to elucidate the reaction mechanism. The yields of intramolecular SN2 fluorination of the veratrole substrates are measured to be very small (<1% in 12 h) in the absence of crown ether promoters, whereas the SN2 fluorination of the substrate possessing a crown ether unit proceeds to near completion (~99%) in 12 h. We also studied the efficacy of intermolecular rate acceleration by an independent promoter 18-crown-6 for comparison. We find that the fluorinating yield of a veratrole substrate (leaving group = -OMs) in the presence of 18-crown-6 follows the almost identical kinetic course as that of intramolecular SN2 fluorination, indicating the mechanistic similarity of intra- and inter-molecular organocatalysis of the crown ether for SN2 fluorination. The calculated relative Gibbs free energies of activation for these reactions, in which the crown ether units act as Lewis base promoters for SN2 fluorination, are in excellent agreement with the experimentally measured yields of fluorination. The role of the metal salt CsF is briefly discussed in terms of whether it reacts as a contact ion pair or as a "free" nucleophile F-.

The SARS-CoV-2 RNA interactome.

SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.

Mechanistic study of nucleophilic fluorination for the synthesis of fluorine-18 labeled fluoroform with high molar activity from N-difluoromethyltriazolium triflate.

The synthesis of fluorine-18 labeled fluoroform with high molar activity has grown in importance for the development of fluorine-18 labeled aryl-CF3 radiopharmaceuticals that are useful as diagnostic radiotracers for the powerful technique of positron emission tomography (PET). We designed a strategy of synthesizing fluorine-18 labeled fluoroform from N1-difluoromethyl-N3-methyltriazolium triflate (1) via SN2 fluorination without stable fluorine isotope scrambling. Fluoroform was generated at rt in 10 min by fluorination of the triazolium precursor with TBAF (6 equiv.). We propose three routes (a), (b), and (c) for this fluorination. Quantum chemical calculations have been carried out to elucidate the mechanism of experimentally observed nucleophilic attack of fluoride at difluoromethyl group via route (a), not N3-methyl via route (b). 1H and 19F NMR studies using deuterium source have been performed to examine the competition between SN2 fluorination (route (a)) and the formation of difluorocarbene (route (c)). The observed superiority of SN2 pathway to formation of difluorocarbene in the reaction of the precursor using CsF in (CD3CN/(CD3)3COD (17.8 : 1)) gives the possibility of preparing the fluorine-18 labeled fluoroform in high molar activity.

Noncovalent Complexes of Cyclodextrin with Small Organic Molecules: Applications and Insights into Host-Guest Interactions in the Gas Phase and Condensed Phase.

Cyclodextrins (CDs) have drawn a lot of attention from the scientific communities as a model system for host-guest chemistry and also due to its variety of applications in the pharmaceutical, cosmetic, food, textile, separation science, and essential oil industries. The formation of the inclusion complexes enables these applications in the condensed phases, which have been confirmed by nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and other methodologies. The advent of soft ionization techniques that can transfer the solution-phase noncovalent complexes to the gas phase has allowed for extensive examination of these complexes and provides valuable insight into the principles governing the formation of gaseous noncovalent complexes. As for the CDs' host-guest chemistry in the gas phase, there has been a controversial issue as to whether noncovalent complexes are inclusion conformers reflecting the solution-phase structure of the complex or not. In this review, the basic principles governing CD's host-guest complex formation will be described. Applications and structures of CDs in the condensed phases will also be presented. More importantly, the experimental and theoretical evidence supporting the two opposing views for the CD-guest structures in the gas phase will be intensively reviewed. These include data obtained via mass spectrometry, ion mobility measurements, infrared multiphoton dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations.

Harnessing Ionic Interactions and Hydrogen Bonding for Nucleophilic Fluorination.

We review recent works for nucleophilic fluorination of organic compounds in which the Coulombic interactions between ionic species and/or hydrogen bonding affect the outcome of the reaction. SN2 fluorination of aliphatic compounds promoted by ionic liquids is first discussed, focusing on the mechanistic features for reaction using alkali metal fluorides. The influence of the interplay of ionic liquid cation, anion, nucleophile and counter-cation is treated in detail. The role of ionic liquid as bifunctional (both electrophilic and nucleophilic) activator is envisaged. We also review the SNAr fluorination of diaryliodonium salts from the same perspective. Nucleophilic fluorination of guanidine-containing of diaryliodonium salts, which are capable of forming hydrogen bonds with the nucleophile, is exemplified as an excellent case where ionic interactions and hydrogen bonding significantly affect the efficiency of reaction. The origin of experimental observation for the strong dependence of fluorination yields on the positions of -Boc protection is understood in terms of the location of the nucleophile with respect to the reaction center, being either close to far from it. Recent advances in the synthesis of [18F]F-dopa are also cited in relation to SNAr fluorination of diaryliodonium salts. Discussions are made with a focus on tailor-making promoters and solvent engineering based on ionic interactions and hydrogen bonding.

PUMILIO hyperactivity drives premature aging of Norad-deficient mice.

Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.

Chiral differentiation of d- and l-isoleucine using permethylated ß-cyclodextrin: infrared multiple photon dissociation spectroscopy, ion-mobility mass spectrometry, and DFT calculations.

Chiral differentiation of protonated isoleucine (Ile) using permethylated ß-cyclodextrin (perCD) in the gas-phase was studied using infrared multiple photon dissociation (IRMPD) spectroscopy, ion-mobility, and density functional theory (DFT) calculations. The gaseous protonated non-covalent complexes of perCD and d-Ile or l-Ile produced by electrospray ionization were interrogated by laser pulses in the wavenumber region of 2650 to 3800 cm-1. The IRMPD spectra showed remarkably different IR spectral features for the d-Ile or l-Ile and perCD non-covalent complexes. However, drift-tube ion-mobility experiments provided only a small difference in their collision cross-sections, and thus a limited separation of the d- and l-Ile complexes. DFT calculations revealed that the chiral distinction of the d- and l-complexes by IRMPD spectroscopy resulted from local interactions of the protonated Ile with perCD. Furthermore, the theoretical results showed that the IR absorption spectra of higher energy conformers (by ∼13.7 kcal mol-1) matched best with the experimentally observed IRMPD spectra. These conformers are speculated to be formed from kinetic-trapping of the solution-phase conformers. This study demonstrated that IRMPD spectroscopy provides an excellent platform for differentiating the subtle chiral difference of a small amino acid in a cyclodextrin-complexation environment; however, drift-tube ion-mobility did not have sufficient resolution to distinguish the chiral difference.

Chiral differentiation of d- and l-alanine by permethylated ß-cyclodextrin: IRMPD spectroscopy and DFT methods.

The gaseous chiral differentiation of alanine by permethylated ß-cyclodextrin was studied using IRMPD spectroscopy and density functional theory calculations. The protonated non-covalent complexes of permethylated ß-cyclodextrin and d- or l-alanine were mass-selected and investigated by IR laser pulses in the wavelength region of 2650-3800 cm-1. The remarkably different features of the IRMPD spectra for d- and l-alanine are described, and their origin is elucidated by quantum chemical calculations. We show that the differentiation of the experimentally observed spectral features is the result of different local interactions of d- and l-alanine with permethylated ß-cyclodextrin. We also assign the extremely high-frequency (>3700 cm-1) bands in the observed spectra to the stretch motions of completely isolated alanine -OH groups.

Hydrogen-bond promoted nucleophilic fluorination: concept, mechanism and applications in positron emission tomography.

Due to the tremendous interest in carbon-fluorine bond-forming reactions, research efforts in this area have been dedicated to the development of facile processes to synthesize small fluorine-containing organic molecules. Among others, PET (Positron Emission Tomography) is one of the most important applications of fluorine chemistry. Recognizing the specific requirements of PET processes, some groups have focused on fluorination reactions using alkali metal fluorides, particularly through SN2-type reactions. However, a common "misconception" about the role of protic solvents and hydrogen bonding interactions in this class of reactions has hampered the employment of these excellent promoters. Herein, we would like to review recent discoveries in this context, showing straightforward nucleophilic fluorination reactions using alkali metal fluorides promoted by protic solvents. Simultaneous dual activation of reacting partners by intermolecular hydrogen bonding and the enhancement of the "effective fluoride nucleophilicity", which is Nature's biocatalytic approach with the fluorinase enzyme, are the key to this unprecedentedly successful nucleophilic fluorination.

Stereo-epitaxial growth of single-crystal Ni nanowires and nanoplates from aligned seed crystals.

Epitaxially grown anisotropic Ni nanostructures are promising building blocks for the development of miniaturized and stereo-integrated data storage kits because they can store multiple magnetic domain walls (DWs). Here, we report stereo-epitaxially grown single-crystalline Ni nanowires (NWs) and nanoplates, and their magnetic properties. Vertical and inclined Ni NWs were grown at the center and edge regions of c-cut sapphire substrates, respectively. Vertical Ni nanoplates were grown on r-cut sapphire substrates. The morphology and growth direction of Ni nanostructures can be steered by seed crystals. Cubic Ni seeds grow into vertical Ni NWs, tetrahedral Ni seeds grow into inclined Ni NWs, and triangular Ni seeds grow into vertical Ni nanoplates. The shapes of the Ni seeds are determined by the interfacial energy between the bottom plane of the seeds and the substrates. The as-synthesized Ni NWs and nanoplates have blocking temperature values greater than 300 K at 500 Oe, verifying that these Ni nanostructures can form large magnetic DWs with high magnetic anisotropy properties. We anticipate that epitaxially grown Ni NWs and nanoplates will be used in various types of 3-dimensional magnetic devices.