Quenching effect of cerium oxide nanoparticles on singlet oxygen: validation of the potential for reaction with multiple reactive oxygen species.

Here we studied cerium oxide nanoparticles (nanoceria) as an agent for the future treatment of oxidative damage by validating and evaluating its scavenging activity towards reactive oxygen species (ROS) in vitro. Nanoceria has been shown to mimic the activities of superoxide dismutase and catalase, degrading superoxide (O2•-) and hydrogen peroxide (H2O2). We examined the antioxidative activity of nanoceria, focusing on its ability to quench singlet oxygen (1O2) in an aqueous solution. Electron paramagnetic resonance (EPR) was used to determine the rates of second-order reactions between nanoceria and three ROS (1O2, O2•-, and H2O2) in aqueous solution, and its antioxidative abilities were demonstrated. Nanoceria shows a wide range of ultraviolet-light absorption bands and thus 1O2 was produced directly in a nanoceria suspension using high-frequency ultrasound. The quenching or scavenging abilities of nanoceria for 1O2 and hypoxanthine-xanthine oxidase reaction-derived O2•- were examined by EPR spin-trapping methods, and the consumption of H2O2 was estimated by the EPR oximetry method. Our results indicated that nanoceria interact not only with two previously reported ROS but also with 1O2. Nanoceria were shown to degrade O2•- and H2O2, and their ability to quench 1O2 may be one mechanism by which they protect against oxidative damage such as inflammation.

Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b.

Two methanol dehydrogenases (MDHs), MxaFI and XoxF, have been characterized in methylotrophic and methanotrophic bacteria. MxaFI contains a calcium ion in its active site, whereas XoxF contains a lanthanide ion. Importantly, the expression of MxaFI and XoxF is inversely regulated by lanthanide bioavailability, i.e., the "lanthanide switch." To reveal the genetic and environmental factors affecting the lanthanide switch, we focused on two Methylosinus trichosporium OB3b mutants isolated during routine cultivation. In these mutants, MxaF was constitutively expressed, but lanthanide-dependent XoxF1 was not, even in the presence of 25 µM cerium ions, which is sufficient for XoxF expression in the wild type. Genotyping showed that both mutants harbored a loss-of-function mutation in the CQW49_RS02145 gene, which encodes a TonB-dependent receptor. Gene disruption and complementation experiments demonstrated that CQW49_RS02145 was required for XoxF1 expression in the presence of 25 µM cerium ions. Phylogenetic analysis indicated that CQW49_RS02145 was homologous to the Methylorubrum extorquens AM1 lanthanide transporter gene (lutH). These findings suggest that CQW49_RS02145 is involved in lanthanide uptake across the outer membrane. Furthermore, we demonstrated that supplementation with cerium and glycerol caused severe growth arrest in the wild type. CQW49_RS02145 underwent adaptive laboratory evolution in the presence of cerium and glycerol ions, resulting in a mutation that partially mitigated the growth arrest. This finding implies that loss-of-function mutations in CQW49_RS02145 can be attributed to residual glycerol from the frozen stock. IMPORTANCE Lanthanides are widely used in many industrial applications, including catalysts, magnets, and polishing. Recently, lanthanide-dependent metabolism was characterized in methane-utilizing bacteria. Despite the global demand for lanthanides, few studies have investigated the mechanism of lanthanide uptake by these bacteria. In this study, we identify a lanthanide transporter in Methylosinus trichosporium OB3b and indicate the potential interaction between intracellular lanthanide and glycerol. Understanding the genetic and environmental factors affecting lanthanide uptake should not only help improve the use of lanthanides for the bioconversion of methane into valuable products like methanol but also be of value for developing biomining to extract lanthanides under neutral conditions.

N-Heterocyclic carbene-based C-centered Au(I)-Ag(I) clusters with intense phosphorescence and organelle-selective translocation in cells.

Photoluminescent gold clusters are functionally variable chemical modules by ligand design. Chemical modification of protective ligands and introduction of different metals into the gold clusters lead to discover unique chemical and physical properties based on their significantly perturbed electronic structures. Here we report the synthesis of carbon-centered Au(I)-Ag(I) clusters with high phosphorescence quantum yields using N-heterocyclic carbene ligands. Specifically, a heterometallic cluster [(C)(AuI-L)6AgI2]4+, where L denotes benzimidazolylidene-based carbene ligands featuring N-pyridyl substituents, shows a significantly high phosphorescence quantum yield (Φ = 0.88). Theoretical calculations suggest that the carbene ligands accelerate the radiative decay by affecting the spin-orbit coupling, and the benzimidazolylidene ligands further suppress the non-radiative pathway. Furthermore, these clusters with carbene ligands are taken up into cells, emit phosphorescence and translocate to a particular organelle. Such well-defined, highly phosphorescent C-centered Au(I)-Ag(I) clusters will enable ligand-specific, organelle-selective phosphorescence imaging and dynamic analysis of molecular distribution and translocation pathways in cells.

Intermolecular binding between bulk water and dissolved gases in earth's magnetic field.

Elucidation of the static states and dynamic behavior of oxygen and nitrogen dissolved in water is one of the most important issues in the life sciences. In the present study, experimental trials and theoretical calculations were performed based on the hypothesis that the dissolution of gas molecules in water is related to excitation by the Earth's magnetic field. Using quantum theories such as those used to describe electro magnetic resonance and nuclear magnetic resonance, this study investigated the states of oxygen, nitrogen and hydrogen dissolved in water. The results indicate that the Earth's magnetic field is involved in the bonding and dissociation of molecules at the gas-liquid interface. These calculations assessed the effect of a field strength of 1.0 x 10-4 T and reproduced the influences of temperature changes on dissolved gas concentrations. Molecular interactions caused by electromagnetic properties and the external geomagnetic field were found to affect intermolar bonding associated with water cluster structures. It is concluded that the binding between molecules typically attributed to Coulomb coupling by magnetic charge and van der Waals forces results from excitation in the Earth's magnetic field.

Identifying the chloroperoxyl radical in acidified sodium chlorite solution.

The present study identified the active radical species in acidic sodium chlorite and investigated the feasibility of quantifying these species with the diethylphenylenediamine (DPD) method. Electron spin resonance (ESR) spectroscopy was used to identify the active species generated in solutions containing sodium chlorite (NaClO2). The ESR signal was directly observed in an acidified sodium chlorite (ASC) aqueous solution at room temperature. This ESR signal was very long-lived, indicating that the radical was thermodynamically stable. The ESR parameters of this signal did not coincide with previously reported values of the chlorine radical (Cl●) or chlorine dioxide radical (O = Cl●-O and O = Cl-O●). We refer to this signal as being from the chloroperoxyl radical (Cl-O-O●). Quantum chemical calculations revealed that the optimal structure of the chloroperoxyl radical is much more thermodynamically stable than that of the chlorine dioxide radical. The UV-visible spectrum of the chloroperoxyl radical showed maximum absorbance at 354 nm. This absorbance had a linear relationship with the chloroperoxyl radical ESR signal intensity. Quantifying the free chlorine concentration by the DPD method also revealed a linear relationship with the maximum absorbance at 354 nm, which in turn showed a linear relationship with the chloroperoxyl radical ESR signal intensity. These linear relationships suggest that the DPD method can quantify chloroperoxyl radicals, which this study considers to be the active species in ASC aqueous solution.

Switching Between Methanol Accumulation and Cell Growth by Expression Control of Methanol Dehydrogenase in Methylosinus trichosporium OB3b Mutant.

Methanotrophs have been used to convert methane to methanol at ambient temperature and pressure. In order to accumulate methanol using methanotrophs, methanol dehydrogenase (MDH) must be downregulated as it consumes methanol. Here, we describe a methanol production system wherein MDH expression is controlled by using methanotroph mutants. We used the MxaF knockout mutant of Methylosinus trichosporium OB3b. It could only grow with MDH (XoxF) which has a cerium ion in its active site and is only expressed by bacteria in media containing cerium ions. In the presence of 0 µM copper ion and 25 µM cerium ion, the mutant grew normally. Under conditions conducive to methanol production (10 µM copper ion and 0 µM cerium ion), cell growth was inhibited and methanol accumulated (2.6 µmol·mg-1 dry cell weight·h-1). The conversion efficiency of the accumulated methanol to the total amount of methane added to the reaction system was ~0.3%. The aforementioned conditions were repeatedly alternated by modulating the metal ion composition of the bacterial growth medium.

Dendrimer porphyrins as the oxygen sensor for intracellular imaging to suppress interaction towards biological molecules.

Optical methods using phosphorescence quenching by oxygen are suitable for the measurement of oxygen concentration within cells. In cells, however, the dyes such as Pt-porphyrins interact with biological components so that their optical properties are changed. Therefore, the absolute oxygen concentration determination in cells is difficult. To suppress this interaction, we focussed on porphyrin-cored dendrimers (dendrimer-porphyrins) and synthesized 2nd-4th generation dendrimer-porphyrins with various surface functional groups (G2-G4, ARG, αGLU and γGLU). These dendrimer-porphyrins showed oxygen sensing property and the change of their spectroscopic properties by biomolecules was supressed. Additionally, the dendrimer-porphyrins were accumulated in cells even in the presence of serum, so oxygen concentration imaging without the effect of serum starvation was also achieved.

Methane Hydroxylation with Water as an Electron Donor under Light Irradiation in the Presence of Reconstituted Membranes Containing both Photosystem†II and a Methane Monooxygenase.

Methane/methanol conversion is one of the most important chemical reactions. Methane monooxygenases from methanotrophs are enzymes that catalyze methane/methanol conversion under mild conditions. Here we report the reconstitution of purified photosystem II (PSII) from Thermosynechococcus elongatus BP-1 into the membrane fraction containing particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b. Photoinduced hydroxylation of methane to methanol was successfully achieved by using the PSII-reconstituted membrane containing pMMO under light irradiation. This result indicates that the sequential redox chain from PSII through the quinone pool to pMMO can be constructed and that water can serve as the electron donor for methane hydroxylation under irradiation with light. pMMO in the membrane fraction produced hydrogen peroxide as a byproduct when an electron donor was added for methane hydroxylation, whereas under light irradiation conditions the PSII-reconstituted membrane containing pMMO did not generate hydrogen peroxide. Optimization of the electron-transfer rate can easily be achieved with this system by tuning the light intensity.

Real-time monitoring of intracellular redox changes in Methylococcus capsulatus (Bath) for efficient bioconversion of methane to methanol.

This study aimed to develop a novel method for real-time monitoring of the intracellular redox states in a methanotroph Methylococcus capsulatus, using Peredox as a genetically encoded fluorescent sensor of the NADH:NAD+ ratio. As expected, the fluorescence derived from the Peredox-expressing M. capsulatus transformant increased by supplementation of electron donor compounds (methane and formate), while it decreased by specifically inhibiting the methanol oxidation reaction. Electrochemical measurements confirmed that the Peredox fluorescence reliably represents the intracellular redox changes. This study is the first to construct a reliable redox-monitoring method for methanotrophs, which will facilitate to develop more efficient methane-to-methanol bioconversion processes.

Specific Interaction between Redox Phospholipid Polymers and Plastoquinone in Photosynthetic Electron Transport Chain.

Redox phospholipid polymers added in culture media are known to be capable of extracting electrons from living photosynthetic cells across bacterial cell membranes with high cytocompatibility. In the present study, we identify the intracellular redox species that transfers electrons to the polymers. The open-circuit electrochemical potential of an electrolyte containing the redox polymer and extracted thylakoid membranes shift to positive (or negative) under light irradiation, when an electron transport inhibitor specific to plastoquinone is added upstream (or downstream) in the photosynthetic electron transport chain. The same trend is also observed for a medium containing living photosynthetic cells of Synechococcus elongatus PCC7942. These results clearly indicate that the phospholipid redox polymers extract photosynthetic electrons mainly from plastoquinone.

Microbicidal action of photoirradiated aqueous extracts from wine lees.

Wine lees, a major waste product of winemaking, is a rich source of polyphenolic compounds. LED-light irradiation at 400-nm elicited microbicidal activity of aqueous extract from wine lees (WLE) against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, in addition to reactive oxygen species (ROS) formation, including hydroxyl radical (·OH) and hydrogen peroxide (H2O2). Although treatment for 20 min of photoirradiation alone exerted bactericidal activity with a 2- to 3-log reduction, photoirradiated WLE for 20 min achieved a 5-log or greater reduction in viable S. aureus and P. aeruginosa cells. Regarding C. albicans, a 1-log reduction (90 % reduction) of viable cells was achieved by photoirradiated WLE for 40 min, whereas photoirradiation alone did not show any fungicidal effect. ROS analyses revealed that approximately 170 µM ·OH and 600 µM H2O2 were generated in photoirradiated WLE for 20 min. Because the bactericidal activity of photoirradiated WLE was abolished by ·OH scavengers, ROS, especially highly oxidative ·OH, may be responsible for the microbicidal activity of photoirradiated WLE. In addition to its microbicidal activity, WLE may act as an antioxidant as it exerted radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl, a stable free radical.

Correction: Prooxidative Potential of Photo-Irradiated Aqueous Extracts of Grape Pomace, a Recyclable Resource from Winemaking Process.

[This corrects the article DOI: 10.1371/journal.pone.0158197.].

Prooxidative Potential of Photo-Irradiated Aqueous Extracts of Grape Pomace, a Recyclable Resource from Winemaking Process.

Our previous study revealed that aqueous extract of grape pomace obtained from a winemaking process could exert bactericidal action upon photo-irradiation via reactive oxygen species (ROS) formation. In the present study, we focused on chemical composition and prooxidative profile of the extract. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis showed that polyphenolic compounds including catechin monomers, dimers, trimers, and polyphenolic glucosides were contained. The polyphenol rich fraction used for the LC-ESI-MS analysis generated hydrogen peroxide (H2O2) upon photo-irradiation possibly initiated by photo-oxidation of phenolic hydroxyl group. That is, reduction of dissolved oxygen by proton-coupled electron transferred from the photo-oxidized phenolic hydroxyl group would form H2O2. The resultant H2O2 was then photolyzed to generate hydroxyl radical (•OH). The prooxidative profile of the extract in terms of •OH generation pattern upon photo-irradiation was similar to that of grape seed extract (GSE) as an authentic polyphenol product and (+)-catechin as a pure polyphenolic compound, and in all the three samples •OH generation could be retained during photo-irradiation for at least a couple of hours. The prooxidant activity of the photo-irradiated extract indicated by •OH yield was more potent than that of the photo-irradiated GSE and (+)-catechin, and this was well reflected in their bactericidal activity in which the photo-irradiated extract could kill the bacteria more efficiently than did the photo-irradiated GSE and (+)-catechin.

Bactericidal Action of Photo-Irradiated Aqueous Extracts from the Residue of Crushed Grapes from Winemaking.

Our previous studies revealed that photo-irradiation of polyphenols could exert bactericidal action via reactive oxygen species (ROS). In the present study, the photo-irradiation-induced bactericidal activity of the aqueous extract from the residue of crushed grapes from winemaking was investigated in relation to ROS formation. Staphylococcus aureus suspended in the extract was irradiated with LED light at 400 nm. This solution killed the bacteria, and a 3-4 log and a >5-log reduction of the viable counts were observed within 10 and 20 min, respectively. LED light irradiation alone also killed the bacteria, but the viable counts were 2-4 log higher than those of the photo-irradiated extract. In contrast, almost no change occurred in the suspension without LED irradiation. When hydroxyl radical scavengers were added to the suspension, the bactericidal effect of the photo-irradiated extract was attenuated. Furthermore, electron spin resonance analysis demonstrated that hydroxyl radicals were generated by the photo-irradiation of the extract. The present study suggests that polyphenolic compounds in the extract exert bactericidal activity via hydroxyl radical formation upon photo-irradiation.

High resolution imaging of intracellular oxygen concentration by phosphorescence lifetime.

Optical methods using phosphorescence quenching by oxygen are suitable for sequential monitoring and non-invasive measurements for oxygen concentration (OC) imaging within cells. Phosphorescence intensity measurement is widely used with phosphorescent dyes. These dyes are ubiquitously but heterogeneously distributed inside the whole cell. The distribution of phosphorescent dye is a major disadvantage in phosphorescence intensity measurement. We established OC imaging system for a single cell using phosphorescence lifetime and a laser scanning confocal microscope. This system had improved spatial resolution and reduced the measurement time with the high repetition rate of the laser. By the combination of ubiquitously distributed phosphorescent dye with this lifetime imaging microscope, we can visualize the OC inside the whole cell and spheroid. This system uses reversible phosphorescence quenching by oxygen, so it can measure successive OC changes from normoxia to anoxia. Lower regions of OC inside the cell colocalized with mitochondria. The time-dependent OC change in an insulin-producing cell line MIN6 by the glucose stimulation was successfully visualized. Assessing the detailed distribution and dynamics of OC inside cells achieved by the presented system will be useful to understanding a physiological and pathological oxygen metabolism.

Characterization of a radical S-adenosyl-L-methionine epimerase, NeoN, in the last step of neomycin B biosynthesis.

The last step of neomycin biosynthesis is the epimerization at C-5‴ of neomycin C to give neomycin B. A candidate enzyme responsible for the epimerization was a putative radical S-adenosyl-L-methionine (SAM) enzyme, NeoN, which is uniquely encoded in the neomycin biosynthetic gene cluster and remained an unassigned protein in the neomycin biosynthesis. The reconstituted and reduced NeoN showed the expected epimerization activity in the presence of SAM. In the epimerization, 1 equiv of SAM was consumed to convert neomycin C into neomycin B. The site of neomycin C reactive toward epimerization was clearly confirmed to be C-5‴ by detecting the incorporation of a deuterium atom from the deuterium oxide-based buffer solution. Further, alanine scanning of the NeoN cysteine residues revealed that C249 is a critical amino acid residue that provides a hydrogen atom to complete the epimerization. Furthermore, electron paramagnetic resonance analysis of the C249A variant in the presence of SAM and neomycin C revealed that a radical intermediate is generated at the C-5‴ of neomycin C. Therefore, the present study clearly illustrates that the epimerization of neomycin C to neomycin B is catalyzed by a unique radical SAM epimerase NeoN with a radical reaction mechanism.

A metabolomics-based approach for predicting stages of chronic kidney disease.

Chronic kidney disease (CKD) is a major epidemiologic problem and a risk factor for cardiovascular events and cerebrovascular accidents. Because CKD shows irreversible progression, early diagnosis is desirable. Renal function can be evaluated by measuring creatinine-based estimated glomerular filtration rate (eGFR). This method, however, has low sensitivity during early phases of CKD. Cystatin C (CysC) may be a more sensitive predictor. Using a metabolomic method, we previously identified metabolites in CKD and hemodialysis patients. To develop a new index of renal hypofunction, plasma samples were collected from volunteers with and without CKD and metabolite concentrations were assayed by quantitative liquid chromatography/mass spectrometry. These results were used to construct a multivariate regression equation for an inverse of CysC-based eGFR, with eGFR and CKD stage calculated from concentrations of blood metabolites. This equation was able to predict CKD stages with 81.3% accuracy (range, 73.9-87.0% during 20 repeats). This procedure may become a novel method of identifying patients with early-stage CKD.

Exploration of novel predictive markers in rat plasma of the early stages of chronic renal failure.

To identify blood markers for early stages of chronic kidney disease (CKD), blood samples were collected from rats with adenine-induced CKD over 28 days. Plasma samples were subjected to metabolomic profiling by liquid chromatography-mass spectrometry, followed by multivariate analyses. In addition to already-identified uremic toxins, we found that plasma concentrations of N6-succinyl adenosine, lysophosphatidylethanolamine 20:4, and glycocholic acid were altered, and that these changes during early CKD were more sensitive markers than creatinine concentration, a universal indicator of renal dysfunction. Moreover, the increase in plasma indoxyl sulfate concentration occurred earlier than increases in phenyl sulfate and p-cresol sulfate. These novel metabolites may serve as biomarkers in identifying early stage CKD.

Measurement of soil bacterial colony temperatures and isolation of a high heat-producing bacterium.

BACKGROUND: The cellular temperatures of microorganisms are considered to be the same as those of their surroundings because the cellular volume is too small to maintain a cellular temperature that is different from the ambient temperature. However, by forming a colony or a biofilm, microorganisms may be able to maintain a cellular temperature that is different from the ambient temperature. In this study, we measured the temperatures of bacterial colonies isolated from soils using an infrared imager and investigated the thermogenesis by a bacterium that increases its colony temperature. RESULTS: The temperatures of some colonies were higher or lower than that of the surrounding medium. A bacterial isolate with the highest colony temperature was identified as Pseudomonas putida. This bacterial isolate had an increased colony temperature when it grew at a temperature suboptimal for its growth. Measurements of heat production using a microcalorimeter showed that the temperature of this extraordinary, microcalorimetrically determined thermogenesis corresponded with the thermographically observed increase in bacterial colony temperature. When investigating the effects of the energy source on this thermal behavior, we found that heat production by this bacterium increased without additional biomass production at a temperature suboptimal for its growth. CONCLUSIONS: We found that heat production by bacteria affected the bacterial colony temperature and that a bacterium identified as Pseudomonas putida could maintain a cellular temperature different from the ambient temperature, particularly at a sub-optimal growth temperature. The bacterial isolate P. putida KT1401 increased its colony temperature by an energy-spilling reaction when the incubation temperature limited its growth.

The reactivity of α-oxoaldehyde with reactive oxygen species in diabetes complications.

The reactions of three α-oxoaldehydes (methylglyoxal, glyoxal, and pyruvic acid) with hydroxyl radicals generated by sonolysis of water were investigated using an electron spin resonance (electron paramagnetic resonance) spin-trapping method, and their reaction kinetics were investigated. It is apparent from our experimental results that methylglyoxal exhibits the highest reactivity of the three α-oxoaldehydes. These α-oxoaldehydes can react with hydroxyl radicals faster than other well-known antioxidants can. The reactivity of hydroxyl radicals is higher than that of hydrogen peroxides.