Molecular mechanism of the metal-independent production of hydroxyl radicals by thiourea dioxide and H2O2.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Unusual enantioselective cytoplasm-to-nucleus translocation and photosensitization of the chiral Ru(II) cationic complex via simple ion-pairing with lipophilic weak acid counter-anions.

Targeted and enantioselective delivery of chiral diagnostic-probes and therapeutics into specific compartments inside cells is of utmost importance in the improvement of disease detection and treatment. The classical DNA 'light-switch' ruthenium(II)-polypyridyl complex, [Ru(DIP)2(dppz)]Cl2 (DIP = 4,7-diphenyl-1,10-phenanthroline, dppz = dipyridophenazine) has been shown to be accumulated only in the cytoplasm and membrane, but excluded from its intended nuclear DNA target. In this study, the cationic [Ru(DIP)2(dppz)]2+ is found to be redirected into live-cell nucleus in the presence of lipophilic 3,5-dichlorophenolate or flufenamate counter-anions via ion-pairing mechanism, while maintaining its original DNA recognition characteristics. Interestingly and unexpectedly, further studies show that only the Δ-enantiomer is selectively translocated into nucleus while the Λ-enantiomer remains trapped in cytoplasm, which is found to be mainly due to their differential enantioselective binding affinities with cytoplasmic proteins and nuclear DNA. More importantly, only the nucleus-relocalized Δ-enantiomer can induce obvious DNA damage and cell apoptosis upon prolonged visible-light irradiation. Thus, the use of Δ-enantiomer can significantly reduce the dosage needed for maximal treatment effect. This represents the first report of enantioselective targeting and photosensitization of classical Ru(II) complex via simple ion-pairing with suitable weak acid counter-anions, which opens new opportunities for more effective enantioselective cancer treatment.

Unprecedented enantio-selective live-cell mitochondrial DNA super-resolution imaging and photo-sensitizing by the chiral ruthenium polypyridyl DNA "light-switch".

Mitochondrial DNA (mtDNA) is known to play a critical role in cellular functions. However, the fluorescent probe enantio-selectively targeting live-cell mtDNA is rare. We recently found that the well-known DNA 'light-switch' [Ru(phen)2dppz]Cl2 can image nuclear DNA in live-cells with chlorophenolic counter-anions via forming lipophilic ion-pairing complex. Interestingly, after washing with fresh-medium, [Ru(phen)2dppz]Cl2 was found to re-localize from nucleus to mitochondria via ABC transporter proteins. Intriguingly, the two enantiomers of [Ru(phen)2dppz]Cl2 were found to bind enantio-selectively with mtDNA in live-cells not only by super-resolution optical microscopy techniques (SIM, STED), but also by biochemical methods (mitochondrial membrane staining with Tomo20-dronpa). Using [Ru(phen)2dppz]Cl2 as the new mtDNA probe, we further found that each mitochondrion containing 1-8 mtDNA molecules are distributed throughout the entire mitochondrial matrix, and there are more nucleoids near nucleus. More interestingly, we found enantio-selective apoptotic cell death was induced by the two enantiomers by prolonged visible light irradiation, and in-situ self-monitoring apoptosis process can be achieved by using the unique 'photo-triggered nuclear translocation' property of the Ru complex. This is the first report on enantio-selective targeting and super-resolution imaging of live-cell mtDNA by a chiral Ru complex via formation and dissociation of ion-pairing complex with suitable counter-anions.

circRIP: an accurate tool for identifying circRNA-RBP interactions.

Circular ribonucleic acids (RNAs) (circRNAs) are formed by covalently linking the downstream splice donor and the upstream splice acceptor. One of the most important functions of circRNAs is mainly exerted through binding RNA-binding proteins (RBPs). However, there is no efficient algorithm for identifying genome-wide circRNA-RBP interactions. Here, we developed a unique algorithm, circRIP, for identifying circRNA-RBP interactions from RNA immunoprecipitation sequencing (RIP-Seq) data. A simulation test demonstrated the sensitivity and specificity of circRIP. By applying circRIP, we identified 95 IGF2BP3-binding circRNAs based on the IGF2BP3 RIP-Seq dataset. We further identified 2823 and 1333 circRNAs binding to >100 RBPs in K562 and HepG2 cell lines, respectively, based on enhanced cross-linking immunoprecipitation (eCLIP) data, demonstrating the significance to survey the potential interactions between circRNAs and RBPs. In this study, we provide an accurate and sensitive tool, circRIP (https://github.com/bioinfolabwhu/circRIP), to systematically identify RBP and circRNA interactions from RIP-Seq and eCLIP data, which can significantly benefit the research community for the functional exploration of circRNAs.

Molecular mechanism for the unusual enhancement of the second-step chemiluminescence production from the carcinogenic tetrabromohydroquinone and H2O2.

We have found recently that two-step intrinsic hydroxyl radical (·OH)-dependent chemiluminescence (CL) could be produced by carcinogenic tetrahaloquinone and H2O2. However, the first-step CL was too fast to clearly detect the stepwise generation of ·OH and CL, and to distinguish the exact dividing point between the first-step and second-step CL. Here we found that, extremely clear two-step intrinsic CL could be produced by the relative slow reaction of tetrabromohydroquinone (TBHQ) with H2O2, which was directly dependent on the two-step ·OH generation. Interestingly, the second-step, but not the first-step CL production of TBHQ/H2O2 (CRET donor) was markedly enhanced by fluorescein (a typical xanthene dye, CRET acceptor) through a unique chemiluminescence resonance energy transfer (CRET) process. The novel CRET system of TBHQ/H2O2/fluorescein was successfully applied for the sensitive detection of TBHQ with the detection limit as low as 2.5 µmol/L. These findings will help to develop more sensitive and highly efficient CL or CRET systems and specific CL sensor to detect the carcinogenic haloquinones, which may have broad environmental applications.

Whole-genome resequencing reveals complex effects of geographical-palaeoclimatic interactions on diversification of moustache toads in East Asia.

Geographical features and palaeoclimatic fluctuations are two classical evolutionary forces that shape genetic diversification within species. Fine-grained analysis of the mechanisms involved through population demographic processes, however, remains limited. Taking advantage of two recently published reference genomes, we resequenced the genomes and examined the evolutionary history of the moustache toads, a group endemic to East Asia where complex topography and fluctuating palaeoclimate are known to have had profound impacts on organisms. Moustache toads probably originated in southeast Yunnan, China, and diversified towards the northwestern of Yunnan, as well as central and eastern China. Further exploration based on three widespread species (Leptobrachium ailaonicum, L. boringii and L. liui) using demographic modelling and species distribution models revealed that mountains and river valleys in East Asia not only functioned as geographical barriers, but also provided dispersal corridors and facilitated continuous migration or post-glacial secondary contact among moustache toad populations. Furthermore, periodic oscillation of effective population sizes accompanying fluctuations of historical temperature and population contraction at the Last Glacial Maximum support the widespread impact of climatic changes of the Pleistocene on species diversification in East Asia. This impact was moderate for populations of L. ailaonicum and L. boringii in the southwestern mountains but severe for populations of L. liui in the eastern lowland regions of continental East Asia, which is supported by different degrees of change of their effective population sizes. Our findings reveal mechanisms underlying genetic diversification among moustache toads, and highlight the power of genomic data and demographic modelling for examining complex historical population-level processes and for understanding how geographical and palaeoclimatic factors interactively shape current intraspecific diversity.

The Profiles and Functions of RNA Editing Sites Associated with High-Altitude Adaptation in Goats.

High-altitude environments dramatically influenced the genetic evolution of vertebrates. However, little is known about the role of RNA editing on high-altitude adaptation in non-model species. Here, we profiled the RNA editing sites (RESs) of heart, lung, kidney, and longissimus dorsi muscle from Tibetan cashmere goats (TBG, 4500 m) and Inner Mongolia cashmere goats (IMG, 1200 m) to reveal RNA editing-related functions of high-altitude adaptation in goats. We identified 84,132 high-quality RESs that were unevenly distributed across the autosomes in TBG and IMG, and more than half of the 10,842 non-redundant editing sites were clustered. The majority (62.61%) were adenosine-to-inosine (A-to-I) sites, followed by cytidine-to-uridine (C-to-U) sites (19.26%), and 32.5% of them had a significant correlation with the expression of catalytic genes. Moreover, A-to-I and C-to-U RNA editing sites had different flanking sequences, amino acid mutations, and alternative splicing activity. TBG had higher editing levels of A-to-I and C-to-U than IMG in the kidney, whereas a lower level was found in the longissimus dorsi muscle. Furthermore, we identified 29 IMG and 41 TBG population-specific editing sites (pSESs) and 53 population-differential editing sites (pDESs) that were functionally involved in altering RNA splicing or recoding protein products. It is worth noting that 73.3% population-differential, 73.2% TBG-specific, and 80% IMG-specific A-to-I sites were nonsynonymous sites. Moreover, the pSESs and pDESs editing-related genes play critical functions in energy metabolisms such as ATP binding molecular function, translation, and adaptive immune response, which may be linked to goat high-altitude adaptation. Our results provide valuable information for understanding the adaptive evolution of goats and studying plateau-related diseases.

Combination of artesunate and WNT974 induces KRAS protein degradation by upregulating E3 ligase ANACP2 and ß-TrCP in the ubiquitin-proteasome pathway.

BACKGROUND: KRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin. METHODS: The synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or ß-TrCP. Western blot assay was utilized for the protein levels of NRAS, HRAS, KRAS, ANAPC2, ß-TrCP, GSK-3ß, p-Akt (Ser473), t-Akt, p-PI3K (Tyr458), t-PI3K, p-mTOR (Ser2448), t-mTOR. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, ß-TrCP, GSK-3ß or ANAPC2 in tumor tissues. RESULTS: Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3ß and E3 ligase ß-TrCP that is known to degrade GSK-3ß-phosphorylated KRAS protein. Knockdown of ß-TrCP- and inhibition of GSK-3ß abolish the combination treatment-induce KRAS ubiquitination and reduction in expression. Last but not least, combination treatment suppresses PI3K/Akt/m-TOR signaling pathway. CONCLUSIONS: Our data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin-proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment. Video Abstract.

Mechanistic Study on Oxidative DNA Damage and Modifications by Haloquinoid Carcinogenic Intermediates and Disinfection Byproducts.

Haloquinones (XQs) are a group of carcinogenic intermediates of the haloaromatic environmental pollutants and newly identified chlorination disinfection byproducts (DBPs) in drinking water. The highly reactive hydroxyl radicals/alkoxyl radicals and quinone enoxy/ketoxy radicals were found to arise in XQs and H2O2 or organic hydroperoxides system, independent of transition-metal ions. However, it was not clear whether these haloquinoid carcinogens and hydroperoxides can cause oxidative DNA damage and modifications, and if so, what are the underlying molecular mechanisms. We found that 8-oxodeoxyguanosine (8-oxodG), DNA strand breaks, and three methyl oxidation products could arise when DNA was treated with tetrachloro-1,4-benzoquinone and H2O2 via a metal-independent and intercalation-enhanced oxidation mechanism. Similar effects were observed with other XQs, which are generally more efficient than the typical Fenton system. We further extended our studies from isolated DNA to genomic DNA in living cells. We also found that potent oxidation of DNA to the more mutagenic imidazolone dIz could be induced by XQs and organic hydroperoxides such as t-butylhydroperoxide or the physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid via an unprecedented quinone-enoxy radical-mediated mechanism. These findings should provide new perspectives to explain the potential genotoxicity, mutagenesis, and carcinogenicity for the ubiquitous haloquinoid carcinogenic intermediates and DBPs.

Structure-Activity Relationship Investigation on Reaction Mechanism between Chlorinated Quinoid Carcinogens and Clinically-Used Aldoxime Nerve-Agent Antidote under Physiological Condition.

Pyridinium aldoximes are best-known therapeutic antidotes used for clinical treatment of poisonings by organophosphorus nerve-agents and pesticides. Recently, we found that pralidoxime (2-PAM, a currently clinically used nerve-agent antidote) could also detoxify tetrachloro-1,4-benzoquinone (TCBQ), which is a carcinogenic quinoid metabolite of the widely used wood preservative pentachlorophenol under normal physiological conditions, via an unusually mild and facile Beckmann fragmentation mechanism accompanied by radical homolysis. However, it is not clear whether the less-chlorinated benzoquinones (CnBQs, n ≤ 3) act similarly; if so, what is the structure-activity relationship? In this study, we found that (1) The stability of reaction intermediates produced by different CnBQs and 2-PAM was dependent not only on the position but also the degree of Cl-substitution on CnBQs, which can be divided into TCBQ- and DCBQ (dichloro-1,4-benzoquinone)-subgroup; (2) The pKa value of hydroxlated quinones (Cn-1BQ-OHs, the hydrolysis products of CnBQs), determined the stability of corresponding intermediates, that is, the decomposition rate of the intermediates depended on the acidity of Cn-1BQ-OHs; (3) The pKa value of the corresponding Cn-1BQ-OHs could also determine the reaction ratio of Beckmann fragmentation to radical homolysis in CnBQs/2-PAM. These new findings on the structure-activity relationship of the halogenated quinoid carcinogens detoxified by pyridinium aldoxime therapeutic agents via Beckmann fragmentation and radical homolysis reaction may have broad implications on future biomedical and environmental research.

Targeted live-cell nuclear delivery of the DNA 'light-switching' Ru(II) complex via ion-pairing with chlorophenolate counter-anions: the critical role of binding stability and lipophilicity of the ion-pairing complexes.

We have found recently that nuclear uptake of the cell-impermeable DNA light-switching Ru(II)-polypyridyl cationic complexes such as [Ru(bpy)2(dppz)]Cl2 was remarkably enhanced by pentachlorophenol (PCP), by forming ion-pairing complexes via a passive diffusion mechanism. However, it is not clear whether the enhanced nuclear uptake of [Ru(bpy)2(dppz)]2+ is only limited to PCP, or it is a general phenomenon for other highly chlorinated phenols (HCPs); and if so, what are the major physicochemical factors in determining nuclear uptake? Here, we found that the nuclear uptake of [Ru(bpy)2(dppz)]2+ can also be facilitated by other two groups of HCPs including three tetrachlorophenol (TeCP) and six trichlorophenol (TCP) isomers. Interestingly and unexpectedly, 2,3,4,5-TeCP was found to be the most effective one for nuclear delivery of [Ru(bpy)2(dppz)]2+, which is even better than the most-highly chlorinated PCP, and much better than its two other TeCP isomers. Further studies showed that the nuclear uptake of [Ru(bpy)2(dppz)]2+ was positively correlated with the binding stability, but to our surprise, inversely correlated with the lipophilicity of the ion-pairing complexes formed between [Ru(bpy)2(dppz)]Cl2 and HCPs. These findings should provide new perspectives for future investigations on using ion-pairing as an effective method for delivering other bio-active metal complexes into their intended cellular targets.

Sulfur-centered hemi-bond radicals as active intermediates in S-DNA phosphorothioate oxidation.

Phosphorothioate (PS) modifications naturally appear in bacteria and archaea genome and are widely used as antisense strategy in gene therapy. But the chemical effects of PS introduction as a redox active site into DNA (S-DNA) is still poorly understood. Herein, we perform time-resolved spectroscopy to examine the underlying mechanisms and dynamics of the PS oxidation by potent radicals in free model, in dinucleotide, and in S-oligomer. The crucial sulphur-centered hemi-bonded intermediates -P-S∴S-P- were observed and found to play critical roles leading to the stable adducts of -P-S-S-P-, which are backbone DNA lesion products. Moreover, the oxidation of the PS moiety in dinucleotides d[GPSG], d[APSA], d[GPSA], d[APSG] and in S-oligomers was monitored in real-time, showing that PS oxidation can compete with adenine but not with guanine. Significantly, hole transfer process from A+• to PS and concomitant -P-S∴S-P- formation was observed, demonstrating the base-to-backbone hole transfer unique to S-DNA, which is different from the normally adopted backbone-to-base hole transfer in native DNA. These findings reveal the distinct backbone lesion pathway brought by the PS modification and also imply an alternative -P-S∴S-P-/-P-S-S-P- pathway accounting for the interesting protective role of PS as an oxidation sacrifice in bacterial genome.

Detecting and Quantifying Polyhaloaromatic Environmental Pollutants by Chemiluminescence-Based Analytical Method.

Polyhaloaromatic compounds (XAr) are ubiquitous and recalcitrant in the environment. They are potentially carcinogenic to organisms and may induce serious risks to the ecosystem, raising increasing public concern. Therefore, it is important to detect and quantify these ubiquitous XAr in the environment, and to monitor their degradation kinetics during the treatment of these recalcitrant pollutants. We have previously found that unprecedented intrinsic chemiluminescence (CL) can be produced by a haloquinones/H2O2 system, a newly-found ●OH-generating system different from the classic Fenton system. Recently, we found that the degradation of priority pollutant pentachlorophenol by the classic Fe(II)-Fenton system could produce intrinsic CL, which was mainly dependent on the generation of chloroquinone intermediates. Analogous effects were observed for all nineteen chlorophenols, other halophenols and several classes of XAr, and a novel, rapid and sensitive CL-based analytical method was developed to detect these XAr and monitor their degradation kinetics. Interestingly, for those XAr with halohydroxyl quinoid structure, a Co(II)-mediated Fenton-like system could induce a stronger CL emission and higher degradation, probably due to site-specific generation of highly-effective ●OH. These findings may have broad chemical and environmental implications for future studies, which would be helpful for developing new analytical methods and technologies to investigate those ubiquitous XAr.

MicroRNA-96 is a potential tumor repressor by inhibiting NPTX2 in renal cell carcinoma.

MicroRNA-96 (miR-96) is a vertebrate conserved microRNA which plays important roles in various cancers including renal cell carcinoma (RCC). However, its function and mechanism in RCC are still unclear. In this study, miR-96 was found to be downregulated in RCC based on The Cancer Genome Atlas datasets analyses, and its target genes, which predicted by TargetScan, were investigated. Among these target genes, neuronal pentraxin 2 (NPTX2) was upregulated more than 15-fold in RCC, and moreover, closely related to patient survival. To validate its targeting of NPTX2 experimentally, reverse transcription polymerase chain reaction, Western blot analysis, and dual-luciferase assays were performed, and results of these assays demonstrated that miR-96 inhibited expression of NPTX2 through a single 3'-untranslated region targeting site. Furthermore, transfection assays in RenCa and 786-O cells showed miR-96 and small interfering RNA of NPTX2 inhibited cell proliferation, migration, and invasion and overexpression of NPTX2 recovered the inhibition of miR-96. In conclusion, the present study reveals a novel regulatory mechanism of miR-96 on NPTX2 expression in RCC, and the potential of miR-96 as a RCC tumor repressor deserves further investigation.

Apigenin inhibits STAT3/CD36 signaling axis and reduces visceral obesity.

Visceral obesity is the excess deposition of visceral fat within the abdominal cavity that surrounds vital organs. Visceral obesity is directly associated with metabolic syndrome, breast cancer and endometrial cancer. In visceral obese subjects, signal transducer and activator of the transcription 3 (STAT3) in adipocytes is constitutively active. In this study, we aimed to screen for dietary herbal compounds that possess anti-visceral obesity effect. Apigenin is abundant in fruits and vegetables. Our data show that apigenin significantly reduces body weight and visceral adipose tissue (VAT), but not subcutaneous (SAT) and epididymal adipose tissues (EAT), of the high fat diet (HFD)-induced obese mice. Mechanistic studies show that HFD increases STAT3 phosphorylation in VAT, but not in SAT and EAT. Further studies suggest that apigenin binds to non-phosphorylated STAT3, reduces STAT3 phosphorylation and transcriptional activity in VAT, and consequently reduces the expression of STAT3 target gene cluster of differentiation 36 (CD36). The reduced CD36 expression in adipocytes reduces the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ) which is the critical nuclear factor in adipogenesis. Our data show that apigenin reduces CD36 and PPAR-γ expressions and inhibits adipocyte differentiation; overexpression of constitutive active STAT3 reverses the apigenin-inhibited adipogenesis. Taken together, our data suggest that apigenin inhibits adipogenesis via the STAT3/CD36 axis. Our study has delineated the mechanism of action underlying the anti-visceral obesity effect of apigenin, and provide scientific evidence to support the development of apigenin as anti-visceral obesity therapeutic agent.

Unusual Two-Step Claisen-type Rearrangement Reaction under Physiological Conditions.

N-aryl hydroxamic acids, which are best known for their metal-chelating properties in chemical and biomedical research, have been found to markedly detoxify carcinogenic halogenated quinones. However, the exact chemical mechanism underlying such detoxication remains unclear. Here, we show that a very fast reaction took place between N-phenylbenzohydroxamic acid (N-PhBHA) and 2,5-dichloro-1,4-benzoquinone (DCBQ), forming an unexpected new carbon-carbon bonding phenyl-quinone product with high yield. In contrast, no reaction was observed with O-benzoyl N-PhBHA. Analogous results were observed for other N-aryl hydroxamic acids and halogenated quinones, which have an ortho-hydrogen adjacent to the reaction site (DCBQ-type). Interestingly, no free radical intermediates could be detected by both ESR spin-trapping and radical-scavenging methods during the reaction process. Taken together, we proposed that nucleophilic substitution followed by an unusual two-step Claisen-type rearrangement reaction was responsible for the formation of a new C-C bonding compound and the detoxication reaction. This represents the first report of an unusually mild and facile two-step Claisen-type rearrangement, which could take place under normal physiological conditions.

First Direct and Unequivocal Electron Spin Resonance Spin-Trapping Evidence for pH-Dependent Production of Hydroxyl Radicals from Sulfate Radicals.

Recently, the sulfate radical (SO4•-) has been found to exhibit broad application prospects in various research fields such as chemical, biomedical, and environmental sciences. It has been suggested that SO4•- could be transformed into a more reactive hydroxyl radical (•OH); however, no direct and unequivocal experimental evidence has been reported yet. In this study, using an electron spin resonance (ESR) secondary radical spin-trapping method coupled with the classic spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and the typical •OH-scavenging agent dimethyl sulfoxide (DMSO), we found that •OH can be produced from three SO4•--generating systems from weakly acidic (pH = 5.5) to alkaline conditions (optimal at pH = 13.0), while SO4•- is the predominant radical species at pH < 5.5. A comparative study with three typical •OH-generating systems strongly supports the above conclusion. This is the first direct and unequivocal ESR spin-trapping evidence for •OH formation from SO4•- over a wide pH range, which is of great significance to understand and study the mechanism of many SO4•--related reactions and processes. This study also provides an effective and direct method for unequivocally distinguishing •OH from SO4•-.

Potent Oxidation of DNA by Haloquinoid Disinfection Byproducts to the More Mutagenic Imidazolone dIz via an Unprecedented Haloquinone-Enoxy Radical-Mediated Mechanism.

Halogenated quinones are a class of carcinogenic intermediates and newly identified chlorination disinfection byproducts in drinking water. We found recently that halogenated quinones could enhance the decomposition of hydroperoxides independent of transition-metal ions and formation of the novel quinone enoxy/ketoxy radicals. Here, we show that the major oxidation product was 2-amino-5-[(2-deoxy-ß-d-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz) when the nucleoside 2'-deoxyguanosine (dG) was treated with tetrachloro-1,4-benzoquinone (TCBQ) and t-butyl hydroperoxide (t-BuOOH). The formation of dIz was markedly inhibited by typical radical spin-trapping agents. Interestingly and unexpectedly, we found that the generated quinone enoxy radical played a critical role in dIz formation. Using [15N5]-8-oxodG, dIz was found to be produced either directly from dG or through the transient formation of 8-oxodG. Based on these data, we proposed that the production of dIz might be through an unusual haloquinone-enoxy radical-mediated mechanism. Analogous results were observed in the oxidation of ctDNA by TCBQ/t-BuOOH and when t-BuOOH was substituted by the endogenously generated physiologically relevant hydroperoxide 13S-hydroperoxy-9Z,11E-octadecadienoic acid. This is the first report that halogenated quinoid carcinogens and hydroperoxides can induce potent oxidation of dG to the more mutagenic product dIz via an unprecedented quinone-enoxy radical-mediated mechanism, which may partly explain their potential carcinogenicity.

Palmitic acid is an intracellular signaling molecule involved in disease development.

Emerging evidence shows that palmitic acid (PA), a common fatty acid in the human diet, serves as a signaling molecule regulating the progression and development of many diseases at the molecular level. In this review, we focus on its regulatory roles in the development of five pathological conditions, namely, metabolic syndrome, cardiovascular diseases, cancer, neurodegenerative diseases, and inflammation. We summarize the clinical and epidemiological studies; and also the mechanistic studies which have identified the molecular targets for PA in these pathological conditions. Activation or inactivation of these molecular targets by PA controls disease development. Therefore, identifying the specific targets and signaling pathways that are regulated by PA can give us a better understanding of how these diseases develop for the design of effective targeted therapeutics.

The effect of Jiedu Huoxue decoction on rat model of experimental nonbacterial prostatitis via regulation of miRNAs.

CONTEXT: The underlying mechanisms of Jiedu Huoxue decoction (JDHXD) in treating chronic prostatitis have not been fully explored. OBJECTIVE: This study investigates the miRNAs as potential biomarkers and the effect of JDHXD on the rat model of experimental nonbacterial prostatitis. MATERIALS AND METHODS: Fifty-four Sprague-Dawley male rats were randomly divided into normal control, model, JDHXD low dose (0.5 g/kg/day), medium dose (1 g/kg/day), high dose (2 g/kg/day) and western medicine (cernilton 0.094 g/kg/day) groups, and intragastrically administered once daily for 30 days. The control and model (upon successful establishment) groups received distilled water. Differential expression of miRNAs was analysed with high-throughput miRNA sequencing and validated with qRT-PCR and Northern blot. Prediction of specific target genes and functional enrichment analysis were performed with bioinformatics. RESULTS: LD50 test showed no sign of toxicity with maximum feasible dose 4 g/kg JDHXD. Compared with control, 495 miRNAs showed expression changes in CAP/CPPS rats, of which 211 were significantly different and 37 were prostatic-related. There were 181 differentially expressed miRNAs between the model and high dose JDHXD groups, of which 23 were identical with the control and model groups. Compared with control, miR-146a, miR-423 and miR-205 expression increased significantly in the model group, decreased dose-dependently in the JDHXD groups (p < 0.05), and vice-versa for miR-96 (p < 0.05). The effect of low dose JDHXD was comparable to cernilton (p > 0.05). DISCUSSION AND CONCLUSIONS: Future studies may explore the contributions of the active components in JDHXD. The study design is generalisable. The effect can be repeatedly verified in clinical trials.