Evaluation of Phosphorus Enrichment in Groundwater by Legacy Phosphorus in Orchard Soils with High Phosphorus Adsorption Capacity Using Phosphate Oxygen Isotope Analysis.

Long-term phosphorus (P) fertilization results in P accumulation in agricultural soil and increases the risk of P leaching into water bodies. However, evaluating P leaching into groundwater is challenging, especially in clay soil with a high P sorption capacity. This study examined whether the combination of PO4 oxygen isotope (δ18OPO4) analysis and the P saturation ratio (PSR) was useful to identify P enrichment mechanisms in groundwater. We investigated the groundwater and possible P sources in Kubi, western Japan, with intensive citrus cultivation. Shallow groundwater had oxic conditions with high PO4 concentrations, and orchard soil P accumulation was high compared with forest soil. Although the soil had a high P sorption capacity, the PSR was above the threshold, indicating a high risk of P leaching from the surface orchard soil. The shallow groundwater δ18OPO4 values were higher than the expected isotopic equilibrium with pyrophosphatase. The high PSR and δ18OPO4 orchard soil values indicated that P leaching from orchard soil was the major P enrichment mechanism. The Bayesian mixing model estimated that 76.6% of the P supplied from the orchard soil was recycled by microorganisms. This demonstrates the utility of δ18OPO4 and the PSR to evaluate the P source and biological recycling in groundwater.

Effects of plasmon coupling on circular dichroism of chiral nanoparticle arrays.

Arrays and ensembles of chiral nanostructures have potential applications in the field of enantioselective sensors, metamaterials, and metasurfaces. In particular, chiral nanostructures fabricated through chemical and bottom-up approaches have attracted much attention from the viewpoint of cost and scalability, but the heterogeneity of the unit nanostructure constituting the array or ensemble often deteriorates its chiroptical responses. Here, we report that their deteriorated responses can be recovered or even enhanced further by interparticle plasmon coupling. We employed chiral silver (Ag) hexamers as models for electromagnetic simulations and investigated the effect of their parameters, such as interparticle spacing, chiral purity, and enantiomeric excess, on their g-factor, which is an index for chiroptical responses. The maximum value of g-factor (gmax) of the Ag hexamer surpasses that of the chiral monomer and augments with decreasing interparticle spacing. This enhancement in g-factor is observed even when chiral purity and enantiomeric excess are less than 100%, showing the potent role of plasmon coupling in amplifying chiroptical responses. Furthermore, our research highlights the amplification of the effect of plasmon coupling on the gmax value of infinite periodic chiral nanostructures. These results corroborate the potential of plasmon coupling to improve chiroptical responses by precisely controlling the interparticle spacing of chiral plasmonic nanostructures, thus mitigating the loss of g-factor caused by low purity and enantiomeric excess of the nanostructures fabricated by chemical and bottom-up approaches.

Distinctive alterations in the mesocorticolimbic circuits in various psychiatric disorders.

AIM: Increasing evidence suggests that psychiatric disorders are linked to alterations in the mesocorticolimbic dopamine-related circuits. However, the common and disease-specific alterations remain to be examined in schizophrenia (SCZ), major depressive disorder (MDD), and autism spectrum disorder (ASD). Thus, this study aimed to examine common and disease-specific features related to mesocorticolimbic circuits. METHODS: This study included 555 participants from four institutes with five scanners: 140 individuals with SCZ (45.0% female), 127 individuals with MDD (44.9%), 119 individuals with ASD (15.1%), and 169 healthy controls (HC) (34.9%). All participants underwent resting-state functional magnetic resonance imaging. A parametric empirical Bayes approach was adopted to compare estimated effective connectivity among groups. Intrinsic effective connectivity focusing on the mesocorticolimbic dopamine-related circuits including the ventral tegmental area (VTA), shell and core parts of the nucleus accumbens (NAc), and medial prefrontal cortex (mPFC) were examined using a dynamic causal modeling analysis across these psychiatric disorders. RESULTS: The excitatory shell-to-core connectivity was greater in all patients than in the HC group. The inhibitory shell-to-VTA and shell-to-mPFC connectivities were greater in the ASD group than in the HC, MDD, and SCZ groups. Furthermore, the VTA-to-core and VTA-to-shell connectivities were excitatory in the ASD group, while those connections were inhibitory in the HC, MDD, and SCZ groups. CONCLUSION: Impaired signaling in the mesocorticolimbic dopamine-related circuits could be an underlying neuropathogenesis of various psychiatric disorders. These findings will improve the understanding of unique neural alternations of each disorder and will facilitate identification of effective therapeutic targets.

A new sampling method with zirconium-loaded resin for phosphate oxygen isotope analysis in oligotrophic freshwater systems.

RATIONALE: The phosphate oxygen isotope ratio ( δ 18 O PO 4 ) is a useful technique to trace the sources and biogeochemical cycles of phosphorus (P) in aquatic ecosystems. However, δ 18 O PO 4 has not been widely used in oligotrophic freshwater systems due to technical and methodological difficulties in collecting sufficient phosphate (PO4 ) for the δ 18 O PO 4 analysis, which sometimes requires hundreds of liters of the water sample. In this study, a new approach (PaS-Zir) was developed for the δ 18 O PO 4 analysis in oligotrophic freshwater systems using zirconium (Zr)-loaded (ZrIRC) resin, which has a high affinity for PO4 . METHODS: ZrClO2 was added to Amberlite IRC748 to obtain the ZrIRC resin. The adsorption/desorption experiment using KH2 PO4 with a known value of δ 18 O PO 4 was conducted to determine the adsorption/desorption properties of the resin and the likelihood of isotopic fractionation. By installing mesh bags filled with the resin, the PaS-Zir approach was used in two rivers with low PO4 concentrations (0.2 and 5.3 µmol/L). A conventional sampling method was also performed in the study river with a higher PO4 concentration to validate the efficacy of the PaS-Zir method. RESULTS: The adsorption/desorption experiment demonstrated that the ZrIRC resin possessed a sufficient adsorption capacity (153 µmol/resin-mL) and exhibited little isotopic fractionation during the adsorption/desorption processes. Using the PaS-Zir method, we were able to collect sufficient PO4 samples for the δ 18 O PO 4 analysis from the rivers within at least 4 days of mesh bag installation. The δ 18 O PO 4 values (14.2‰ ± 0.2‰) obtained using the PaS-Zir method were comparable to those obtained using the conventional method (14.0‰ ± 0.03‰). CONCLUSION: We proved that the PaS-Zir method is applicable to oligotrophic freshwater systems and is generally more efficient than the conventional method. In addition, our method is useful for improving the understanding of the P dynamics of oligotrophic ecosystems because of the extremely low concentration of PO4 commonly found in them, which are often prone to P pollution.

A new method for phosphate purification for oxygen isotope ratio analysis in freshwater and soil extracts using solid-phase extraction with zirconium-loaded resin.

RATIONALE: Phosphate (PO4 ) oxygen isotope (δ18 OPO4 ) analysis is increasingly applied to elucidate phosphorus cycling. Due to its usefulness, analytical methods continue to be developed and improved to increase processing efficiency and applicability to various sample types. A new pretreatment procedure to obtain clean Ag3 PO4 using solid-phase extraction (SPE) with zirconium-loaded resin (ZrME), which can selectively adsorb PO4 , is presented and evaluated here. METHODS: Our method comprises (1) PO4 concentration, (2) PO4 separation by SPE, (3) cation removal, (4) Cl- removal, and (5) formation of Ag3 PO4 . The method was tested by comparing the resulting δ18 OPO4 of KH2 PO4 reagent, soil extracts (NaHCO3 , NaOH, and HCl), freshwater, and seawater with data obtained using a conventional pretreatment method. RESULTS: PO4 recovery of our method ranged from 79.2% to 97.8% for KH2 PO4 , soil extracts, and freshwater. Although the recovery rate indicated incomplete desorption of PO4 from the ZrME columns, our method produced high-purity Ag3 PO4 and accurate δ18 OPO4 values (i.e., consistent with those obtained using conventional pretreatment methods). However, for seawater, the PO4 recovery was low (1.1%), probably due to the high concentrations of F- and SO4 2- which interfere with PO4 adsorption on the columns. Experiments indicate that the ZrME columns could be regenerated and used repeatedly at least three times. CONCLUSIONS: We demonstrated the utility of ZrME for purification of PO4 from freshwater and soil extracts for δ18 OPO4 analysis. Multiple samples could be processed in three days using this method, increasing sample throughput and potentially facilitating more widespread use of δ18 OPO4 analysis to deepen our understanding of phosphorus cycling in natural environments.

Crystal Structure of the Catalytic and Cytochrome b Domains in a Eukaryotic Pyrroloquinoline Quinone-Dependent Dehydrogenase.

Pyrroloquinoline quinone (PQQ) was discovered as a redox cofactor of prokaryotic glucose dehydrogenases in the 1960s, and subsequent studies have demonstrated its importance not only in bacterial systems but also in higher organisms. We have previously reported a novel eukaryotic quinohemoprotein that exhibited PQQ-dependent catalytic activity in a eukaryote. The enzyme, pyranose dehydrogenase (PDH), from the filamentous fungus Coprinopsis cinerea (CcPDH) of the Basidiomycete division, is composed of a catalytic PQQ-dependent domain classified as a member of the novel auxiliary activity family 12 (AA12), an AA8 cytochrome b domain, and a family 1 carbohydrate-binding module (CBM1), as defined by the Carbohydrate-Active Enzymes (CAZy) database. Here, we present the crystal structures of the AA12 domain in its apo- and holo-forms and the AA8 domain of this enzyme. The crystal structures of the holo-AA12 domain bound to PQQ provide direct evidence that eukaryotes have PQQ-dependent enzymes. The AA12 domain exhibits a six-blade ß-propeller fold that is also present in other known PQQ-dependent glucose dehydrogenases in bacteria. A loop structure around the active site and a calcium ion binding site are unique among the known structures of bacterial quinoproteins. The AA8 cytochrome domain has a positively charged area on its molecular surface, which is partly due to the propionate group of the heme interacting with Arg181; this feature differs from the characteristics of cytochrome b in the AA8 domain of the fungal cellobiose dehydrogenase and suggests that this difference may affect the pH dependence of electron transfer.IMPORTANCE Pyrroloquinoline quinone (PQQ) is known as the "third coenzyme" following nicotinamide and flavin. PQQ-dependent enzymes have previously been found only in prokaryotes, and the existence of a eukaryotic PQQ-dependent enzyme was in doubt. In 2014, we found an enzyme in mushrooms that catalyzes the oxidation of various sugars in a PQQ-dependent manner and that was a PQQ-dependent enzyme found in eukaryotes. This paper presents the X-ray crystal structures of this eukaryotic PQQ-dependent quinohemoprotein, which show the active site, and identifies the amino acid residues involved in the binding of the cofactor PQQ. The presented X-ray structures reveal that the AA12 domain is in a binary complex with the coenzyme, clearly proving that PQQ-dependent enzymes exist in eukaryotes as well as prokaryotes. Because no biosynthetic system for PQQ has been reported in eukaryotes, future research on the symbiotic systems is expected.

Identification of Phosphorus Sources in a Watershed Using a Phosphate Oxygen Isoscape Approach.

Identifying nonpoint phosphorus (P) sources in a watershed is essential for addressing cultural eutrophication and for proposing best-management solutions. The oxygen isotope ratio of phosphate (δ18OPO4) can shed light on P sources and P cycling in ecosystems. This is the first assessment of the δ18OPO4 distribution in a whole catchment, namely, the Yasu River Watershed in Japan. The observed δ18OPO4 values in the river water varied spatially from 10.3‰ to 17.6‰. To identify P sources in the watershed, we used an isoscape approach involving a multiple-linear-regression model based on land use and lithological types. We constructed two isoscape models, one using data only from the whole watershed and the other using data from the small tributaries. The model results explain 69% and 96% of the spatial variation in the river water δ18OPO4. The lower R2 value for the whole watershed model is attributed to the relatively large travel time for P in the main stream of the lower catchment that can result in cumulative biological P recycling. Isoscape maps and a correlation analysis reveal the relative importance of P loading from paddy fields and bedrock. This work demonstrates the utility of δ18OPO4 isoscape models for assessing nonpoint P sources in watershed ecosystems.

Crystal Structure and Substrate Specificity Modification of Acetyl Xylan Esterase from Aspergillus luchuensis.

Acetyl xylan esterase (AXE) catalyzes the hydrolysis of the acetyl bonds present in plant cell wall polysaccharides. Here, we determined the crystal structure of AXE from Aspergillus luchuensis (AlAXEA), providing the three-dimensional structure of an enzyme in the Esterase_phb family. AlAXEA shares its core α/ß-hydrolase fold structure with esterases in other families, but it has an extended central ß-sheet at both its ends and an extra loop. Structural comparison with a ferulic acid esterase (FAE) from Aspergillus niger indicated that AlAXEA has a conserved catalytic machinery: a catalytic triad (Ser119, His259, and Asp202) and an oxyanion hole (Cys40 and Ser120). Near the catalytic triad of AlAXEA, two aromatic residues (Tyr39 and Trp160) form small pockets at both sides. Homology models of fungal FAEs in the same Esterase_phb family have wide pockets at the corresponding sites because they have residues with smaller side chains (Pro, Ser, and Gly). Mutants with site-directed mutations at Tyr39 showed a substrate specificity similar to that of the wild-type enzyme, whereas those with mutations at Trp160 acquired an expanded substrate specificity. Interestingly, the Trp160 mutants acquired weak but significant type B-like FAE activity. Moreover, the engineered enzymes exhibited ferulic acid-releasing activity from wheat arabinoxylan.IMPORTANCE Hemicelluloses in the plant cell wall are often decorated by acetyl and ferulic acid groups. Therefore, complete and efficient degradation of plant polysaccharides requires the enzymes for cleaving the side chains of the polymer. Since the Esterase_phb family contains a wide array of fungal FAEs and AXEs from fungi and bacteria, our study will provide a structural basis for the molecular mechanism of these industrially relevant enzymes in biopolymer degradation. The structure of the Esterase_phb family also provides information for bacterial polyhydroxyalkanoate depolymerases that are involved in biodegradation of thermoplastic polymers.

Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood.

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

pH-dependent electron transfer reaction and direct bioelectrocatalysis of the quinohemoprotein pyranose dehydrogenase.

A pyranose dehydrogenase from Coprinopsis cinerea (CcPDH) is an extracellular quinohemoeprotein, which consists a b-type cytochrome domain, a pyrroloquinoline-quinone (PQQ) domain, and a family 1-type carbohydrate-binding module. The electron transfer reaction of CcPDH was studied using some electron acceptors and a carbon electrode at various pH levels. Phenazine methosulfate (PMS) reacted directly at the PQQ domain, whereas cytochrome c (cyt c) reacted via the cytochrome domain of intact CcPDH. Thus, electrons are transferred from reduced PQQ in the catalytic domain of CcPDH to heme b in the N-terminal cytochrome domain, which acts as a built-in mediator and transfers electron to a heterogenous electron transfer protein. The optimal pH values of the PMS reduction (pH 6.5) and the cyt c reduction (pH 8.5) differ. The catalytic currents for the oxidation of l-fucose were observed within a range of pH 4.5 to 11. Bioelectrocatalysis of CcPDH based on direct electron transfer demonstrated that the pH profile of the biocatalytic current was similar to the reduction activity of cyt c characters.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes.

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the ß-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE: Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a brown-rot fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of brown rot through degradation of the primary cell wall.

Relationships between oxycodone pharmacokinetics, central symptoms, and serum interleukin-6 in cachectic cancer patients.

PURPOSE: Elevated serum proinflammatory cytokines are associated with the reduction of cytochrome P450 enzyme (CYP) activity. This study aimed to evaluate the oxycodone pharmacokinetics, central symptoms, and serum proinflammatory cytokines based on cachexia stage in cancer patients. METHODS: Forty-seven cancer patients receiving extended-release oxycodone were enrolled. Predose plasma concentrations of oxycodone and its metabolites were normalized with the daily dose and body weight. The central symptoms and serum level of proinflammatory cytokines were investigated at each cachexia stage. RESULTS: The plasma concentrations of oxycodone in patients with cachexia and refractory cachexia were significantly higher than that in patients with precachexia. The metabolic ratio to noroxycodone in patients with cachexia was significantly lower than that in patients with precachexia. The patients with a higher cachexia stage had a higher serum level of interleukin-6 (IL-6), but not tumor necrosis factor-α and interleukin-1ß. The serum IL-6 level was correlated with the plasma concentration of oxycodone and inversely with the metabolic ratio to noroxycodone. The incidence of somnolence was not associated with the plasma oxycodone concentration. In contrast, the cachexia stage and its associated serum IL-6 level were correlated with the incidence of somnolence. CONCLUSIONS: Cancer cachexia raised the plasma exposure of oxycodone through the reduction of CYP3A metabolic pathway. The reduction of CYP3A in cachectic cancer patients was associated with an elevation of serum IL-6. Although cachectic cancer patients with higher serum IL-6 levels had the symptom of somnolence, the alterations in oxycodone pharmacokinetics were not related to the incidence of symptom.

A novel pyrroloquinoline quinone-dependent 2-keto-D-glucose dehydrogenase from Pseudomonas aureofaciens.

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from Pseudomonas aureofaciens. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an Escherichia coli expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl2, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-d-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl2 were added. (1)H nuclear magnetic resonance ((1)H-NMR) analysis of reaction products revealed 2-keto-d-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (Pa2KGDH). Considering the high substrate specificity, the physiological function of Pa2KGDH may be for production of 2KGA.

Structural and biochemical analyses of glycoside hydrolase family 26 ß-mannanase from a symbiotic protist of the termite Reticulitermes speratus.

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of ß-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites -5 and -2, respectively, in addition to accommodation of both glucose and mannose at subsites -3 and -4. RsMan26C has a long open cleft with a hydrophobic platform of Trp(94) at subsite -5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met(85) specifically interacts with the equatorial O-2 of glucose at subsite -3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.

Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of a lytic polysaccharide monooxygenase by X-ray photoreduction.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes that employ a copper-mediated, oxidative mechanism to cleave glycosidic bonds. The LPMO catalytic mechanism likely requires that molecular oxygen first binds to Cu(I), but the oxidation state in many reported LPMO structures is ambiguous, and the changes in the LPMO active site required to accommodate both oxidation states of copper have not been fully elucidated. Here, a diffraction data collection strategy minimizing the deposited x-ray dose was used to solve the crystal structure of a chitin-specific LPMO from Enterococcus faecalis (EfaCBM33A) in the Cu(II)-bound form. Subsequently, the crystalline protein was photoreduced in the x-ray beam, which revealed structural changes associated with the conversion from the initial Cu(II)-oxidized form with two coordinated water molecules, which adopts a trigonal bipyramidal geometry, to a reduced Cu(I) form in a T-shaped geometry with no coordinated water molecules. A comprehensive survey of Cu(II) and Cu(I) structures in the Cambridge Structural Database unambiguously shows that the geometries observed in the least and most reduced structures reflect binding of Cu(II) and Cu(I), respectively. Quantum mechanical calculations of the oxidized and reduced active sites reveal little change in the electronic structure of the active site measured by the active site partial charges. Together with a previous theoretical investigation of a fungal LPMO, this suggests significant functional plasticity in LPMO active sites. Overall, this study provides molecular snapshots along the reduction process to activate the LPMO catalytic machinery and provides a general method for solving LPMO structures in both copper oxidation states.

Simultaneous determination of erlotinib and its isomeric major metabolites in human plasma using isocratic liquid chromatography-tandem mass spectrometry and its clinical application.

This study developed a method for the simultaneous determination of erlotinib and its isomeric major metabolites, OSI-413 and OSI-420, in human plasma using an isocratic liquid chromatography-tandem mass spectrometry. Plasma specimens deproteinized with acetonitrile were separated using a 3-µm particle size octadecylsilyl column. The m/z values of the precursor and product ions for the analytes were as follows: erlotinib, 394.2/278.2; and OSI-413 and OSI-420, 380.2/278.2. The total run time was 21 min and no peaks interfering with the analytes and internal standard (d6 -erlotinib) in human plasma were observed. The calibration curves of erlotinib, OSI-413 and OSI-420 were linear over the concentration ranges of 10-3000, 2-500 and 2-100 ng/mL, respectively. The pretreatment recovery ratios were >86.1%. The intra- and inter-assay precisions and accuracies were <12.7 and 89.0-108.9% for all analytes. This validated method was applied to the determination of plasma samples in lung cancer patients receiving 150 mg of oral erlotinib. The plasma concentration ranges of erlotinib, OSI-413 and OSI-420 were 373-2354, 15.7-379 and 2.5-43.6 ng/mL, respectively. In conclusion, the present method can be helpful for evaluating the plasma exposures of erlotinib and its major isomeric metabolites in clinical settings.

Quantitative Reconstruction of Sulfur Deposition Using a Mixing Model Based on Sulfur Isotope Ratios in Tree Rings.

Quantification of sulfur (S) deposition is critical to deciphering the environmental archive of S in terrestrial ecosystems. Here we propose a mixing model that quantifies S deposition based on the S isotope ratio (δS) in tree rings. We collected samples from Japanese cedar ( D. Don) stumps from two sites: one near Yokkaichi City (YOK), which is well known for having the heaviest S air pollution in the world, and one at Inabu-cho (INA) in central Japan, which has been much less affected by air pollution. The δS profiles at both sites are consistent with S air pollution and contributions of anthropogenic S. The minimum value in YOK is lower than the δS values of anthropogenic S or any other possible source. Because the δS in the tree rings is affected by fractionation in the forest ecosystems, we used a mixing model to account for the isotope effects and to distinguish the sources of S. Based on the model results, we infer that the peak of S emissions at YOK occurred sometime between the late 1960s and early 1970s (489 mmol m yr). This estimated value is comparable with the highest reported values in Europe. This is the first quantitative estimate of anthropogenic input of S in forest systems based on δS in tree rings. Our results suggest that tree ring data can be used when monitoring stations of atmospheric S are lacking and that estimates of S deposition using δS in tree rings will advance our understanding of the local-scale S dynamics and the effect of human activities on it.

Multidimensional QoE of Multiview Video and Selectable Audio IP Transmission.

We evaluate QoE of multiview video and selectable audio (MVV-SA), in which users can switch not only video but also audio according to a viewpoint change request, transmitted over IP networks by a subjective experiment. The evaluation is performed by the semantic differential (SD) method with 13 adjective pairs. In the subjective experiment, we ask assessors to evaluate 40 stimuli which consist of two kinds of UDP load traffic, two kinds of fixed additional delay, five kinds of playout buffering time, and selectable or unselectable audio (i.e., MVV-SA or the previous MVV-A). As a result, MVV-SA gives higher presence to the user than MVV-A and then enhances QoE. In addition, we employ factor analysis for subjective assessment results to clarify the component factors of QoE. We then find that three major factors affect QoE in MVV-SA.

Characterization of sulfur deposition over the period of industrialization in Japan using sulfur isotope ratio in Japanese cedar tree rings taken from stumps.

We characterized the sulfur deposition history over the period of industrialization in Japan based on the sulfur isotope ratio (δ(34)S) in tree rings of Japanese cedar (Cryptomeria japonica D. Don) stumps. We analyzed and compared δ(34)S values in the rings from two types of disk samples from 170-year-old stumps that had been cut 5 years earlier (older forest stand) and from 40-year-old living trees (younger forest stand) in order to confirm the validity of using stump disks for δ(34)S analysis. No differences in δ(34)S values by age were found between the sample types, indicating that stump disks can be used for δ(34)S analysis. The δ(34)S profile in tree rings was significantly correlated with anthropogenic SO2 emissions in Japan (r = -0.76, p < 0.05) and, thus, tree rings serve as a record of anthropogenic sulfur emissions. In addition, the values did not change largely from pre-industrialization to the 1940s (+4.2 to +6.1‰). The values before the 1940s are expected to reflect the background sulfur conditions in Japan and, thus, disks containing rings formed before the 1940s contain information about the natural environmental sulfur, which is useful for biogeochemical studies.

Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium.

Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain.