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Spontaneous thrombosis of an unruptured large or giant saccular intracranial aneurysm is a well-known phenomenon and can cause ischemic stroke (IS), which is a rare event. The possible pathogenic mechanisms of IS include distal embolic occlusion secondary to migration of the intra-aneurysmal thrombus, occlusion of the parent artery lumen caused by the retrograde extension of the aneurysmal thrombosis, external compression of the parent artery due to the increased aneurysmal mass effect. Among these, IS due to simultaneous thromboses of the aneurysm and its parent artery is extremely rare, with only a few cases reported in the literature. Herein, we present a case of a 18-year-old woman who suffered an acute IS, attribute to spontaneous complete thrombosis of an unruptured large saccular aneurysm of the right middle cerebral artery with occlusion of the parent artery, and we review the literature simultaneously., (© 2024 The Authors. Published by Elsevier Inc. on behalf of University of Washington.)

Transparent heaters (THs) find widespread application in various indoor and outdoor settings, such as LCD panels and motorcycle helmet visors. Among the materials used for efficient TH performance, the AgNW network stands out due to its high conductivity, substantial transmittance, and minimal solution requirement. Extensive research has been directed towards enhancing AgNW characteristics, focusing on smaller diameters and longer wires. In TH applications, the primary considerations include a rapid response and elevated temperature. Consequently, this research delves into investigating the impact of parameters like diameter, length, and density on random AgNW networks under varying applied voltages. The finite element method is employed for analyzing temperature changes in response to voltage application, particularly in scenarios involving small-scale setups with high-density and high-percolation AgNW networks. The results reveal a significant increase in the thermal transition rate, ranging from 28% to 36%, with varying densities in the random network. Within the same density, the AgNW network with larger diameters and lengths demonstrates the highest temperatures, aligning with previous calculations. Furthermore, a trade-off exists between optical properties in smaller diameters and electrical properties in larger diameters within a relatively narrow temperature range., (© 2024 IOP Publishing Ltd.)

Background: Chitin, the main form of aminated polysaccharide in nature, is a biocompatible, polycationic, and antimicrobial biopolymer used extensively in industrial processes. Despite the abundance of chitin, applications thereof are hampered by difficulties in feedstock harvesting and limited structural versatility. To address these problems, we proposed a two-step cascade employing carbohydrate oxidoreductases and amine transaminases for plant polysaccharide aminations via one-pot reactions. Using a galactose oxidase from Fusarium graminearum for oxidation, this study compared the performance of CvATA (from Chromobacterium violaceum) and SpATA (from Silicibacter pomeroyi) on a range of oxidized carbohydrates with various structures and sizes. Using a rational enzyme engineering approach, four point mutations were introduced on the SpATA surface, and their effects on enzyme activity were evaluated., Results: Herein, a quantitative colorimetric assay was developed to enable simple and accurate time-course measurement of the yield of transamination reactions. With higher operational stability, SpATA produced higher product yields in 36 h reactions despite its lower initial activity. Successful amination of oxidized galactomannan by SpATA was confirmed using a deuterium labeling method; higher aminated carbohydrate yields achieved with SpATA compared to CvATA were verified using HPLC and XPS. By balancing the oxidase and transaminase loadings, improved operating conditions were identified where the side product formation was largely suppressed without negatively impacting the product yield. SpATA mutants with multiple alanine substitutions besides E407A showed improved product yield. The E407A mutation reduced SpATA activity substantially, supporting its predicted role in maintaining the dimeric enzyme structure., Conclusions: Using oxidase-amine transaminase cascades, the study demonstrated a fully enzymatic route to polysaccharide amination. Although the activity of SpATA may be further improved via enzyme engineering, the low operational stability of characterized amine transaminases, as a result of low retention of PMP cofactors, was identified as a key factor limiting the yield of the designed cascade. To increase the process feasibility, future efforts to engineer improved SpATA variants should focus on improving the cofactor affinity, and thus the operational stability of the enzyme., (© 2024. The Author(s).)

Introduction: Coronary microvascular dysfunction (CMD) plays a major role in hypertrophic cardiomyopathy (HCM) physiopathology but its assessment in clinical practice remains a challenge. Nowadays, innovations in invasive and noninvasive coronary evaluation using multimodal imaging provide options for the diagnosis of CMD. The objective of the present study was to investigate if new multimodal imaging diagnosis of CMD could detect HCM patients with more impaired cardiac function by left atrioventricular coupling index (LACI)., Methods and Results: A total of 32 consecutive patients with a confirmed diagnosis of HCM (62 ± 13 years, 62% men) were prospectively screened for CMD using a multimodal imaging method. LACI was assessed by cardiovascular magnetic resonance imaging. Fifteen (47%) patients had CMD by multimodal imaging method. Patients with CMD presented a significantly higher LACI (48.5 ± 25.4 vs. 32.5 ± 10.6, p = .03). A multivariate logistic regression analysis demonstrated that CMD was independently associated with LACI (OR = 1.069, 95% CI 1.00-1.135, p = .03)., Conclusion: Multimodal imaging diagnosis of CMD is applicable to HCM patients and is associated with more impaired cardiac function., (© 2024 John Wiley & Sons Ltd.)

Laccase-like multicopper oxidases are recognized for their potential to alter the reactivity of lignins for application in value-added products. Typically, model compounds are employed to discover such enzymes; however, they do not represent the complexity of industrial lignin substrates. In this work, a screening pipeline was developed to test enzymes simultaneously on model compounds and industrial lignins. A total of 12 lignin-active fungal multicopper oxidases were discovered, including 9 enzymes active under alkaline conditions (pH 11.0). Principal component analysis revealed the poor ability of model compounds to predict enzyme performance on industrial lignins. Additionally, sequence similarity analyses grouped these enzymes with Auxiliary Activity-1 sub-families with few previously characterized members, underscoring their taxonomic novelty. Correlation between the lignin-activity of these enzymes and their taxonomic origin, however, was not observed. These are critical insights to bridge the gap between enzyme discovery and application for industrial lignin valorization., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: This study aims to reveal the relationship between personality characteristics and verbal or physical aggression in Vietnamese adolescents., Patients and Methods: We recruited 3003 participants [1498 boys (49.9%) and 1505 girls (50.1%); mean age ± SD = 13.50 ± 0.936] who we tested with the Eysenck Personality Questionnaire - Brief version (EPQ-BV), and Vietnamese Aggression Scale (VAS). A multivariate analysis of variance test, Pearson Correlation, and analyzing mediating variable interaction is used to analyze data., Results: The findings suggested a significant interaction between personality traits, specifically extraversion and neuroticism, and physical aggression, verbal aggression, and anger. Students with higher levels of personality had higher levels of verbal aggression, and students with higher levels of physical aggression and anger had stronger personality traits than others and lower levels of physical aggression and anger. Personality traits, specifically extraversion, and neuroticism, differed significantly by gender and school years in adolescence. Mediation analysis revealed a positive and statistically significant indirect correlation between personality traits and physically aggressive behavior, with anger as a mediator. Similarly, a positive and statistically significant indirect correlation between personality traits and verbally aggressive behavior through anger was found. The relationship between personality traits and physical aggression was also significant via verbal aggression and anger., Conclusion: This study improved our understanding of personality traits and verbal or physical aggression. Most crucially, physical and verbal aggression mediate personality traits and aggressive conduct. In secondary school, gender and school year affected extraversion and neuroticism. This discovery illuminates personality-based aggressiveness intervention., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Le et al.)

Fouling at liquid-solid interfaces is a pernicious problem for a wide range of applications, including those that are implemented by digital microfluidics (DMF). There are several strategies that have been used to combat surface fouling in DMF, the most common being inclusion of amphiphilic surfactant additives in the droplets to be manipulated. Initial studies relied on Pluronic additives, and more recently, Tetronic additives have been used, which has allowed manipulation of complex samples like serum and whole blood. Here, we report our evaluation of 19 different Pluronic and Tetronic additives, with attempts to determine (1) the difference in antifouling performance between the two families, (2) the structural similarities that predict exceptional antifouling performance, and (3) the mechanism of the antifouling behavior. Our analysis shows that both Pluronic and Tetronic additives with modest molar mass, poly(propylene oxide) (PPO) ≥50 units, poly(ethylene oxide) (PEO) mass percentage ≤50%, and hydrophilic-lipophilic balance (HLB) ca. 13-15 allow for exceptional antifouling performance in DMF. The most promising candidates, P104, P105, and T904, were able to support continuous movement of droplets of serum for more than 2 h, a result (for devices operating in air) previously thought to be out of reach for this technique. Additional results generated using device longevity assays, intrinsic fluorescence measurements, dynamic light scattering, asymmetric flow field flow fractionation, supercritical angle fluorescence microscopy, atomic force microscopy, and quartz crystal microbalance measurements suggest that the best-performing surfactants are more likely to operate by forming a protective layer at the liquid-solid interface than by complexation with proteins. We propose that these results and their implications are an important step forward for the growing community of users of this technique, which may provide guidance in selecting surfactants for manipulating biological matrices for a wide range of applications.

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

In this study, a UV-curable acrylate epoxy nanocomposite coating has been prepared by incorporation of ZnO-Ag hybrid nanoparticles. For this purpose, firstly ZnO-Ag hybrid nanoparticles were fabricated by a seed-mediated growth method. Then, these ZnO-Ag hybrid nanoparticles (2 wt%) were added into the UV-curable acrylate resin matrices. The photocuring process of nanocomposite was evaluated by various factors, such as the conversion of acrylate double bonds, pendulum hardness and gel fraction. Under the 4.8 s UV-exposure time for full crosslinking, the obtained data indicated that incorporation of ZnO-Ag nanohybrids into the coating matrix changed the crosslinking process of coating significantly. A mechanical teat indicated that the presence of nanohybrids in photocurable coating matrix enhanced its abrasion resistance from 98.7 to 131.6 L per mil (33.3%). The antibacterial test against E. coli over 7 h indicated that E. coli bacteria were killed totally by nanocomposite coating, whereas it was 2.6 × 10 4 CFU mL -1 for the neat coating without nanoparticles., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Common periodontal disease treatment procedures often fail to restore the structural integrity of the junctional epithelium (JE), the epithelial attachment of the gum to the tooth, leaving the tooth-gum interface prone to bacterial colonization. To address this issue, we introduced a novel bio-inspired protein complex comprised of a proline-rich enamel protein, SCPPPQ1, and laminin 332 (LAM332) to enhance the JE attachment. Using quartz crystal microbalance with dissipation monitoring (QCM-D), we showed that SCPPPQ1 and LAM332 interacted and assembled into a protein complex with high-affinity adsorption of 5.9e -8 [M] for hydroxyapatite (HA), the main component of the mineralized tooth surfaces. We then designed a unique shear device to study the adhesion strength of the oral epithelial cells to HA. The SCPPPQ1/LAM332 complex resulted in a twofold enhancement in adhesion strength of the cells to HA compared to LAM332 (from 31 dyn/cm 2 to 63 dyn/cm 2 ). In addition, using a modified wound-healing assay, we showed that gingival epithelial cells demonstrated a significantly high migration rate of 2.7 ± 0.24 µm/min over SCPPPQ1/LAM332-coated surfaces. Our collective data show that this protein complex has the potential to be further developed in designing a bioadhesive to enhance the JE attachment and protect the underlying connective tissue from bacterial invasion. However, its efficacy for wound healing requires further testing in vivo. STATEMENT OF SIGNIFICANCE: This work is the first functional study towards understanding the combined role of the enamel protein SCPPPQ1 and laminin 332 (LAM332) in the epithelial attachment of the gum, the junctional epithelium (JE), to the tooth hydroxyapatite surfaces. Such studies are essential for developing therapeutic approaches to restore the integrity of the JE in the destructive form of gum infection. We have developed a model system that provided the first evidence of the strong interaction between SCPPPQ1 and LAM332 on hydroxyapatite surfaces that favored protein adsorption and subsequently oral epithelial cell attachment and migration. Our collective data strongly suggested using the SCPPPQ1/LAM332 complex to accelerate the reestablishment of the JE after surgical gum removal to facilitate gum regeneration., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Eddy-current sensors are widely used for precise displacement sensing and non-destructive testing. Application of printed-circuit board (PCB) technology for manufacturing sensor coils may reduce the cost of the sensor and enhance the performance by ensuring consistency. However, these prospects depend on the uniformness of the sensor coil. Inductance measurements of sample coils reveal rather considerable variations. In this paper, we investigate the sources of these variations. Through image analysis of cut-away cross-sections of sensor coils, four factors that contribute to the inductance variations are identified: the distance between layers, the distance between tracings, cross-sectional areas, and misalignment among layers. By using and extending existing method of calculating inductance of spiral coils, the inductance distributions are obtained when these factors are randomly varied. A sensitivity analysis shows that the inductance uncertainty is most affected by the uniformness of the spacings between coil traces and the distances between layers. Improvements in PCB manufacturing process can help to reduce the uncertainty in inductance.

Softwood kraft lignin is a major bioresource relevant to the production of sustainable bio-based products. Continued challenges to lignin valorization, however, include poor solubility in organic solvents and in aqueous solutions at neutral pH. Herein, an alkaline tolerant laccase was used to graft acrylate functionalities onto softwood kraft lignin, which is expected to enhance the reactivity of lignin with isocyanate when producing bio-based polyurethanes. Proton nuclear magnetic resonance, Fourier-transform infrared spectroscopy, and high-performance liquid chromatography were used to confirm successful grafting of the acrylate monomer onto lignin and verify the importance of including tert-butyl hydroperoxide as an initiator in the grafting reaction. Laccase-mediated grafting of softwood kraft lignin under alkaline conditions produced lignin products with approximately 30% higher hydroxyl value and higher reactivity toward isocyanate. The reported enzymatic and aqueous process presents an opportunity for the sustainable valorization of softwood kraft lignin. KEY POINTS: • Softwood kraft lignin displayed high phenolic hydroxyl content, polydispersity index and average molecular weight • Grafting hydroxyethyl acrylate (HEA) monomer onto kraft lignin by laccase was successful at 60 °C and alkaline conditions • Lignin-HEA grafted copolymer showed an increase in total OH value and an increase in average molecular weight., (© 2022. The Author(s).)

A frost filter (FRF) was developed as a humidity pretreatment device (HPD) to improve the measurement of ambient ozone (O 3 ). The FRF was produced in a tube, which was supercooled by a thermoelectric cooling device based on the Peltier effect. The relative humidity (RH) of the air samples varied from 30% to 80% at 25 °C, and the O 3 concentration was set as 100 ppbv. Besides O 3 , SO 2 at 150 ppbv was used for comparison. The density of the FRF was evaluated. Comparison studies on the humidity removal efficiencies and loss ratios of analytes among a FRF HPD, a short Nafion™ tube (NS), and a long Nafion™ tube (NL) HPDs were conducted. As results, the density of the FRF was dependent on the temperature at a fixed sampling flow rate. The outlet humidity of both the FRF and the NL HPDs were less than 8% RH at 25 °C. The mean concentrations of O 3 and SO 2 after the FRF HPD were similar to the initial concentrations at all humidity levels, whereas they were significantly different for both the NS and NL HPDs at higher humidity. This suggests that the FRF HPD is a reliable humidity pretreatment for O 3 measurements., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Xylan is one of the most abundant, natural polysaccharides, and much recent interest focuses on upgrading heteroxylan to make use of its unique structures and chemistries. Significant progress has been made in the discovery and application of novel enzymes for debranching and modifying heteroxylans. Debranching enzymes include acetylxylan esterases, α-l-arabinofuranosidases and α-d-glucuronidases that release side groups from the xylan backbone to recover both biochemicals and less substituted xylans for polymer applications in food packaging or drug delivery systems. Besides esterases and hydrolases, many oxidoreductases including carbohydrate oxidases, lytic polysaccharide monooxygenases, laccases and peroxidases have been also applied to alter different types of xylans for improved physical and chemical properties. This review will highlight the recent discovery and application of enzymes for upgrading xylans for use as added-value chemicals and in functional polymers., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The relative ability of the small laccase (sLac) and dye-decoloring peroxidase (DyP2) from Amycolatopsis sp. 75iv2 to transform a variety of lignins was investigated using time-of-flight secondary ion mass spectrometry (ToF-SIMS). The enzymes modified organosolv hardwood lignin to different extents even in the absence of an added mediator. More particularly, sLac decreased the lignin modification metric S (S-lignin)/Ar (total aromatics) by 58% over 16h, while DyP2 lowered this ratio by 31% in the absence of exogenous H 2 O 2 . When used on their own, both sLac and DyP2 also modified native lignin present in aspen wood powder, albeit to lesser extents than in the organosolv lignin. The addition of ABTS for sLac and Mn 2+ as well as H 2 O 2 for DyP2 led to increased lignin modification in aspen wood powder as reflected by a decrease in the G/Ar metric by up to a further 13%. This highlights the importance of exogenous mediators for transforming lignin within its native matrix. Furthermore, the addition of ABTS reduced the selectivity of sLac for S-lignin over G-lignin, indicating that the mediator also altered the product profiles. Finally, when sLac was included in reactions containing DyP2, in part to generate H 2 O 2 in situ , the relative abundance of lignin products differed from individual enzymatic treatments. Overall, these results identify possible routes to tuning lignin modification or delignification through choice of enzyme and mediator. Moreover, the current study expands the application of ToF-SIMS to evaluating enzyme action on technical lignins, which can accelerate the discovery and engineering of industrially relevant enzymes for lignin valorization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vuong, Singh, Eltis and Master.)

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species—aspen, pine, and spruce—under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

The coordinated action of carbohydrate-active enzymes has mainly been evaluated for the purpose of complete saccharification of plant biomass (lignocellulose) to sugars. By contrast, the coordinated action of accessory hemicellulases on xylan debranching and recovery is less well characterized. Here, the activity of two family GH115 α-glucuronidases (SdeAgu115A from Saccharophagus degradans, and AxyAgu115A from Amphibacillus xylanus) on spruce arabinoglucuronoxylan (AGX) was evaluated in combination with an α-arabinofuranosidase from families GH51 (AniAbf51A, aka E-AFASE from Aspergillus niger) and GH62 (SthAbf62A from Streptomyces thermoviolaceus). The α-arabinofuranosidases boosted (methyl)-glucuronic acid release by SdeAgu115A by approximately 50 % and 30 %, respectively. The impact of the α-arabinofuranosidases on AxyAgu115A activity was comparatively low, motivating its structural characterization. The crystal structure of AxyAgu115A revealed increased length and flexibility of the active site loop compared to SdeAgu115A. This structural difference could explain the ability of AxyAgu115A to accommodate more highly substituted arabinoglucuronoxylan, and inform enzyme selections for improved AGX recovery and use., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

This work aims to explore how ZnO nanoparticles enhance the mechanical, photoaging, and self‑cleaning properties of water‑borne acrylic coating. Micro/nano‑ZnO particles (at 2 wt.% of total solid resin) were dispersed into the acrylic polymer matrices using ultrasonication to understand the effect of the size of the coating properties. The effect of ZnO particles on the properties of composite coatings (25 µm of thick) have been evaluated through various tests, such as abrasion measurement, ultraviolet/condensation (UV/CON) weathering aging, and methylene blue self‑cleaning. Experimental data indicated that the incorporation of ZnO particles enhanced both abrasion resistance and methylene blue removal efficiency of the water‑borne acrylic coatings, with nano‑ZnO particles being the best. However, the weathering degradation of nanocomposite coatings was more severe as compared to the coating with micro‑ZnO (at the same ZnO content).

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate., (© 2020 Federation of European Biochemical Societies.)

Interference is a pivotal issue of a non-dispersive infrared (NDIR) sensor and analyzer. Therefore, the main contribution of this study is to introduce a potential method to compensate for the interference of the NDIR analysis. A potential method to compensate for the interference of a nitric oxide (NO) NDIR analyzer was developed. Double bandpass filters (BPFs) with HITRAN (high-resolution transmission molecular absorption database)-based wavelengths were used to create an ultranarrow bandwidth, where there were least-interfering effects with respect to the coal-fired power plant emission gas compositions. Key emission gases from a coal-fired power plant, comprising carbon monoxide (CO), NO, sulfur dioxide (SO 2 ), nitrogen dioxide (NO 2 ), carbon dioxide (CO 2 ), and water (H 2 O) (in the form of vapor), were used to investigate the gas interference. The mixtures of those gases were also used to investigate the performance of the double BPFs. We found that CO, CO 2 , SO 2 , and H 2 O significantly affected the detection of NO when a commercial, single narrow BPF was used. In contrast, the double BPFs could remove the interference of CO, NO 2 , SO 2 , and CO 2 in terms of their concentrations. In the case of H 2 O, the filter performed well until a level of 50% relative humidity at 25 °C. Moreover, the signal-to-noise ratio of the analyzer was approximately 10 when the double BPFs were applied. In addition, the limit of detection of the analyzer with the double BPFs was approximately 4 ppm, whereas that with the commercial one was 1.3 ppm. Therefore, double BPFs could be used for an NO NDIR analyzer instead of a gas filter correlation to improve the selectivity of the analyzer under the condition of a known gas composition, such as a coal-fired power plant. However, the sensitivity of the analyzer would be decreased.

In this manuscript, biological durability, cytotoxicity and MRI image contrast effect of chitosan modified magnetic nanoparticles were investigated. The result of durability study shows that the asprepared sample with average size of about 30 nm had a high stability under pH conditions in range of from 2 to 12 and at salt concentration in range of from 0 to 300 mM. The cytotoxicity testing indicates that the obtained Fe₃O₄@CS ferrofluid revealed a low cytotoxicity. After 48 h of test on the line of prostate tumor cells of Sarcoma 180, collected IC 50 value was 178.5±22 ( μ g/ml), 7.5 to 27.9 times less cytotoxicity than that of reported ferromagnetic fluids. MRI data shows that the transverse relaxation rate ( r 2) of the ferrite nanoparticles was 130.32 (mM -1 s -1 ), 2 and 1.44 times larger than that of the commercial products of Sinerem (AMI-227) and Ferumoxytol products, respectively. Invivo test in rabbit shows that the picture of body parts was clearly observed after the injection of the Fe₃O₄@CS ferrofluid. With these outstanding properties, this magnetic fluid based on the chitosan modified Fe₃O₄ nanoparticles had great potential for enhancing the image contrast in image diagnosis by MRI magnetic resonance imaging technique.

Conjugated polymers are promising materials for various cutting-edge technologies, especially for organic conducting materials and in the energy field. In this work, we have synthesized a new conjugated polymer and investigated the effect of distance between bond layers, side-chain functional groups (H, Br, OH, OCH 3 and OC 2 H 5 ) on structural characteristics, phase transition temperature (T), and electrical structure of C 13 H 8 OS using Density Functional Theory (DFT). The structural characteristics were determined by the shape, network constant (a, b and c), bond length (C-C, C-H, C-O, C-S, C-Br and O-H), phase transition temperatures, and the total energy (E tot ) on a base cell. Our finding shows that the increase of layer thickness (h) of C 13 H 8 OS-H has a negligible effect on the transition temperature, while the energy bandgap (E g ) increases from 1.646 eV to 1.675 eV. The calculation of bond length with different side chain groups was carried out for which C 13 H 8 OS-H has C-H = 1.09 Å; C 13 H 8 OS-Br has C-Br = 1.93 Å; C 13 H 8 OS-OH has C-O = 1.36 Å, O-H = 0.78 Å; C 13 H 8 OS-OCH 3 has C-O = 1.44 Å, O-H =1.10 Å; C 13 H 8 OS-OC 2 H 5 has C-O = 1.45 Å, C-C = 1.51Å, C-H = 1.10 Å. The transition temperature (T) for C 13 H 8 OS-H was 500 K < T < 562 K; C 13 H 8 OS-Br was 442 K < T < 512 K; C 13 H 8 OS-OH was 487 K < T < 543 K; C 13 H 8 OS-OCH 3 was 492 K < T < 558 K; and C 13 H 8 OS-OC 2 H 5 was 492 K < T < 572 K. The energy bandgap (E g ) of Br is of E g = 1.621 eV, the doping of side chain groups H, OH, OCH 3, and OC 2 H 5 , leads to an increase of E g from 1.621 eV to 1.646, 1.697, 1.920, and 2.04 eV, respectively.

Background: Dicarboxylic acids offer several applications in detergent builder and biopolymer fields. One of these acids, 4- O -methyl d-glucaric acid, could potentially be produced from glucuronoxylans, which are a comparatively underused fraction of wood and agricultural biorefineries., Results: Accordingly, an enzymatic pathway was developed that combines AxyAgu115A, a GH115 α-glucuronidase from Amphibacillus xylanus , and GOOX, an AA7 gluco-oligosaccharide oxidase from Sarocladium strictum , to produce this bio-based chemical from glucuronoxylan. AxyAgu115A was able to release almost all 4- O -methyl d-glucuronic acid from glucuronoxylan while a GOOX variant, GOOX-Y300A, could convert 4- O -methyl d-glucuronic acid to the corresponding glucaric acid at a yield of 62%. Both enzymes worked effectively at alkaline conditions that increase xylan solubility. Given the sensitivity of AxyAgu115A to hydrogen peroxide and optimal performance of GOOX-Y300A at substrate concentrations above 20 mM, the two-step enzyme pathway was demonstrated as a sequential, one-pot reaction. Additionally, the resulting xylan was easily recovered from the one-pot reaction, and it was enzymatically hydrolysable., Conclusions: The pathway in this study requires only two enzymes while avoiding a supplementation of costly cofactors. This cell-free approach provides a new strategy to make use of the underutilized hemicellulose stream from wood and agricultural biorefineries., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Water vapor is a pivotal obstacle when measuring ambient air pollutants. The effects of water vapor removal devices which are called KPASS (Key-compound PASSer) and Cooler. On the measurement of O 3 , SO 2 , and CO at ambient levels were investigated. Concentrations of O 3 , SO 2 , and CO were 100 ppb, 150 ppb, and 25 ppm, respectively. The amount of water vapor varied at different relative humidity levels of 30%, 50%, and 80% when the temperature was 25 °C and the pressure was 1 atm. Water vapor removal efficiencies and recovery rates of target gases were also determined. The KPASS showed a better performance than the Cooler device, removing 93.6% of water vapor and the Cooler removing 59.2%. In terms of recovery, the KPASS showed a better recovery of target gases than the Cooler. Consequently, it is suggested that the KPASS should be an alternative way to remove water vapor when measuring O 3 , SO 2 , and CO.

This study investigated the reduction of particulate matter (PM) in a subway platform using self-developed magnetic hybrid filters (magnet-magnet (MM) and magnet-cascade (MC) filter). The magnetic hybrid filter systems were installed and operated in Jegi-dong subway station (J station) platform. The removal efficiency of PM 10 (particular matter with aerodynamic diameter less than 10 μm) was evaluated according to various influencing factors such as the combination of filters, linear velocity, and operating conditions of trains. As a result, the average removal efficiency of the MC filter (40.5%) was higher than that of the MM one (27.0%). The maximum PM 10 removal efficiencies by MM (34.1%) and MC (47.2%) filters were observed at 20 (linear velocity: 2.41 m/s) and 30 jog (8 m/s) dials, respectively. We additionally found that the removal efficiency of PM 10 using MM and MC filters suddenly decreased when the concentration of background PM 10 in the platform increased. Based on the results of this study, hybrid technology using two or more capture principles can remove PM more efficiently than technology using a single such principle., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A chemo-enzymatic pathway was developed to transform 4-O-methylglucuronic acid (MeGlcpA) containing xylo-oligosaccharides from beechwood into clickable monomers capable of polymerizing at room temperature and in aqueous conditions to form unique polytriazoles. While the gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum was used to oxidize C6-propargylated oligosaccharides, the acid-amine coupling reagents 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDAC) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) were employed and compared for their ability to append click functionalities to carboxylic acid groups of enzyme-treated oligosaccharides. While DMT-MM was a superior coupling reagent for this application, a triazine side product was observed during C-1 amidation. Resulting bifunctional xylo-oligosaccharide monomers were polymerized using a Cu(I) catalyst, forming a soft gel which was characterized by 1 H NMR, confirming the triazole product.

Glucuronic acid and/or 4-O-methyl-glucuronic acid (GlcA/MeGlcA) are substituents of the main xylans present in hardwoods, conifers, and many cereal grains. α-Glucuronidases from glycoside hydrolase family GH115 can target GlcA/MeGlcA from both internally and terminally substituted regions of xylans. The current study describes the first GH115 α-glucuronidase, AxyAgu115A, from the alkaliphilic organism Amphilbacillus xylanus. AxyAgu115A was active in a wide pH range, and demonstrated better performance in alkaline condition compared to other characterized GH115 α-glucuronidases, which generally show optimal activity in acidic conditions. Specifically, its relative activity between pH 5.0 and pH 8.5 was above 80%, and was 35% of maximum at pH 10.5; although the enzyme lost 30% and 80% relative residual activity after 24-h pre-incubation at pH 9 and pH 10, respectively. AxyAgu115A was also similarly active towards glucuronoxylan as well as comparatively complex xylans such as spruce arabinoglucurunoxylan. Accommodation of complex xylans was supported by docking analyses that predicted accessibility of AxyAgu115A to branched xylo-oligosaccharides. MeGlcA release by AxyAgu115A from each xylan sample was increased by up to 30% by performing the reaction at pH 11.0 rather than pH 4.0, revealing applied benefits of AxyAgu115A for xylan recovery and processing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Most existing methods for screening the activity of lytic polysaccharide mono-oxygenases (LPMOs) on polysaccharides are based on the detection of soluble oxidized sugars. This approach might underestimate the total performance of LPMOs since oxidation events that do not lead to oligosaccharide release are not detected. Using PcLPMO9D as a model enzyme, a microplate-based method has been developed to detect C1-oxidizing LPMO activity by covalently linking a water-soluble fluorophore to oxidized positions within the cellulose fiber. This fluorescence method was validated using X-ray photoelectron spectroscopy and then combined with high-performance anion-exchange chromatography to track total PcLPMO9D activity.

Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A&#95;Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.

Glucuronic acid (GlcAp) and/or methylglucuronic acid (MeGlcAp) decorate the major forms of xylan in hardwood and coniferous softwoods as well as many cereal grains. Accordingly, the complete utilization of glucuronoxylans or conversion to sugar precursors requires the action of main chain xylanases as well as α-glucuronidases that release the α- (1→2)-linked (Me)GlcAp side groups. Herein, a family GH115 enzymefrom the marine bacterium Saccharophagus degradans 2-40(T), SdeAgu115A, demonstrated activity toward glucuronoxylan and oligomers thereof with preference toward MeGlcAp linked to internal xylopyranosyl residues. Unique biochemical characteristics of NaCl activation were also observed. The crystal structure of SdeAgu115A revealed a five-domain architecture, with an additional insertion C(+) domain that had significant impact on the domain arrangement of SdeAgu115A monomer and its dimerization. The participation of domain C(+) in substrate binding was supported by reduced substrate inhibition upon introducing W773A, W689A, and F696A substitutions within this domain. In addition to Asp-335, the catalytic essentiality of Glu-216 was revealed by site-specific mutagenesis. A primary sequence analysis suggested that the SdeAgu115A architecture is shared by more than half of GH115 members, thus defining a distinct archetype for GH115 enzymes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

A sampling system was designed to determine the composition ratios of VOCs emitted from 31 surface coating materials (SCMs). Representative architectural, automotive, and marine SCMs in Korea were investigated. Toluene, ethylbenzene, and xylene were the predominant VOCs. The VOC levels (wt%) from automotive SCMs were significantly higher than those from architectural and marine paints. It was found that target SCMs comprised mainly VOCs with 6-10 carbon atoms in molecules, which could be adsorbed by activated carbon. The saturated activated carbon which had already adsorbed toluene, ethylbenzene, and m-xylene was combusted. The saturated activated carbon was more combustible than new activated carbon because it comprised inflammable VOCs. Therefore, it could be an alternative fuel when using in a "fuelization system". To use the activated carbon as a fuel, a control technology of VOCs from a coating process was also designed and introduced., (Copyright © 2015. Published by Elsevier Ltd.)

Background: Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO., Results: Both GaO-CBM29 and CBM29-GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, kcat/Km values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO-CBM29., Major Conclusions: Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO-CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs., General Significance: This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The characteristics of volatile organic compounds (VOCs) emitted from several consumer and commercial products (body wash, dishwashing detergent, air freshener, windshield washer fluid, lubricant, hair spray, and insecticide) were studied and compared. The spray products were found to emit the highest amount of VOCs (~96 wt%). In contrast, the body wash products showed the lowest VOC contents (~1.6 wt%). In the spray products, 21.6-96.4 % of the VOCs were propane, iso-butane, and n-butane, which are the components of liquefied petroleum gas. Monoterpene (C10H16) was the dominant component of the VOCs in the non-spray products (e.g., body wash, 53-88 %). In particular, methanol was present with the highest amount of VOCs in windshield washer fluid products. In terms of the number of carbon, the windshield washer fluids, lubricants, insecticides, and hair sprays comprised >95 % of the VOCs in the range C2-C5. The VOCs in the range C6-C10 were predominantly found in the body wash products. The dishwashing detergents and air fresheners contained diverse VOCs from C2 to C11. Besides comprising hazardous VOCs, VOCs from consumer products were also ozone precursors. The ozone formation potential of the consumer and commercial spray products was estimated to be higher than those of liquid and gel materials. In particular, the hair sprays showed the highest ozone formation potential.

The gluco-oligosaccharide oxidase from Sarocladium strictum CBS 346.70 (GOOX) is a single domain flavoenzyme that favourably oxidizes gluco- and xylo- oligosaccharides. In the present study, GOOX was shown to also oxidize plant polysaccharides, including cellulose, glucomannan, β-(1→3,1→4)-glucan, and xyloglucan, albeit to a lesser extent than oligomeric substrates. To improve GOOX activity on polymeric substrates, three carbohydrate binding modules (CBMs) from Clostridium thermocellum, namely CtCBM3 (type A), CtCBM11 (type B), and CtCBM44 (type B), were separately appended to the amino and carboxy termini of the enzyme, generating six fusion proteins. With the exception of GOOX-CtCBM3 and GOOX-CtCBM44, fusion of the selected CBMs increased the catalytic activity of the enzyme (kcat) on cellotetraose by up to 50%. All CBM fusions selectively enhanced GOOX binding to soluble and insoluble polysaccharides, and the immobilized enzyme on a solid cellulose surface remained stable and active. In addition, the CBM fusions increased the activity of GOOX on soluble glucomannan by up to 30% and on insoluble crystalline as well as amorphous cellulose by over 50%.

Expansin and expansin-related proteins loosen plant cell wall architectures and are widely distributed in several types of organisms, including plants, fungi and bacteria. Here we describe sequence diversity and unique gene expression profiles of multiple expansin-related proteins identified in the basidiomycete, Phanerochaete carnosa. The protein sequences were homologous to loosenin, an expansin-related protein reported in the basidiomycete, Bjerkandera adusta. We identified homologous sequences of each of those P. carnosa proteins in many basidiomycete species. Twelve P. carnosa loosenin-like proteins (LOOLs) were classified into two subgroups according to sequence homology. Conservation of polysaccharide-binding amino acid residues was stricter in subgroup A. Subgroup A sequences included a conserved 8-9 amino acid insertion in a polysaccharide-binding groove whereas subgroup B contained a 12-18 amino acid insertion next to the binding groove. The P. carnosa genome also encodes the expansin-related protein, DREX1, which adopts a loosenin-like structure but has lower sequence homology to other LOOLs. The gene expression analysis of those proteins showed distinct patterns that were not significantly related to subgroupings. The variation in the protein sequences and gene expression patterns, and wide distribution among the basidiomycota, suggest that the diverse cell wall loosening proteins contribute to effective plant cell wall association and utilization by basidiomycetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA), salicylic acid, β-glucan (GLU), and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

The xylan-binding module Clostridium thermocellum CBM22A was successfully fused to a gluco-oligosaccharide oxidase, GOOX-VN, from Sarocladium strictum via a short TP linker, allowing the fused protein to effectively bind different xylans. The presence of the CtCBM22A at the N-terminal of GOOX-VN increased catalytic activity on mono- and oligo-saccharides by 2-3 fold while not affecting binding affinity to these substrates. Notably, both GOOX-VN and its CBM fusion also showed oxidation of xylo-oligosaccharides with degrees of polymerization greater than six. Whereas fusion to CtCBM22A did not alter the thermostability of GOOX-VN or reduce substrate inhibition, CtCBM22A&#95;GOOX-VN could be immobilized to insoluble oat spelt xylan while retaining wild-type activity. QCM-D analysis showed that the fused enzyme remained bound during oxidation. These features could be harnessed to generate hemicellulose-based biosensors that detect and quantify the presence of different oligosaccharides.

We removed particulate matter (PM) emitted from a subway tunnel using magnetic filters. A magnetic filter system was installed on the top of a ventilation opening. Magnetic field density was increased by increasing the number of permanent magnet layers to determine PM removal characteristics. Moreover, the fan's frequency was adjusted from 30 to 60 Hz to investigate the effect of wind velocity on PM removal efficiency. As a result, PM removal efficiency increased as the number of magnetic filters or fan frequency increased. We obtained maximum removal efficiency of PM10 (52%), PM2.5 (46%), and PM1 (38%) at a 60 Hz fan frequency using double magnetic filters. We also found that the stability of the PM removal efficiency by the double filter (RSD, 3.2-5.8%) was higher than that by a single filter (10.9-24.5%) at all fan operating conditions.

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family.

Background: The oxidation of carbohydrates from lignocellulose can facilitate the synthesis of new biopolymers and biochemicals, and also reduce sugar metabolism by lignocellulolytic microorganisms, reserving aldonates for fermentation to biofuels. Although oxidoreductases that oxidize cellulosic hydrolysates have been well characterized, none have been reported to oxidize substituted or branched xylo-oligosaccharides. Moreover, this is the first report that identifies amino acid substitutions leading to GOOX variants with reduced substrate inhibition., Results: The recombinant wild type gluco-oligosaccharide oxidase (GOOX) from the fungus Sarocladium strictum, along with variants that were generated by site-directed mutagenesis, retained the FAD cofactor, and showed high activity on cello-oligosaccharide and xylo-oligosaccharides, including substituted and branched xylo-oligosaccharides. Mass spectrometric analyses confirmed that GOOX introduces one oxygen atom to oxidized products, and 1H NMR and tandem mass spectrometry analysis confirmed that oxidation was restricted to the anomeric carbon. The A38V mutation, which is close to a predicted divalent ion-binding site in the FAD-binding domain of GOOX but 30 Å away from the active site, significantly increased the kcat and catalytic efficiency of the enzyme on all oligosaccharides. Eight amino acid substitutions were separately introduced to the substrate-binding domain of GOOX-VN (at positions Y72, E247, W351, Q353 and Q384). In all cases, the Km of the enzyme variant was higher than that of GOOX, supporting the role of corresponding residues in substrate binding. Most notably, W351A increased Km values by up to two orders of magnitude while also increasing kcat up to 3-fold on cello- and xylo-oligosaccharides and showing no substrate inhibition., Conclusions: This study provides further evidence that S. strictum GOOX has broader substrate specificity than the enzyme name implies, and that substrate inhibition can be reduced by removing aromatic side chains in the -2 binding subsite. Of the enzyme variants, W351A might be particularly advantageous when oxidizing oligosaccharides present at high substrate concentrations often experienced in industrial processes.

A gluco-oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX-VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX-VN oxidized xylose, galactose, and N-acetylglucosamine. Besides monosaccharides, GOOX-VN oxidized xylo-oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello-oligosaccharides. Of three mutant enzymes that were created in GOOX-VN to improve substrate specificity, Y300A and Y300N doubled kcat values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX-VN and its variants are particularly valuable for oxidative modification of cello- and xylo-oligosaccharides., (Copyright © 2011 Wiley Periodicals, Inc.)

Recent studies have shown that a number of glycoside hydrolase families do not follow the classical catalytic mechanisms, as they lack a typical catalytic base/nucleophile. A variety of mechanisms are used to replace this function, including substrate-assisted catalysis, a network of several residues, and the use of non-carboxylate residues or exogenous nucleophiles. Removal of the catalytic base/nucleophile by mutation can have a profound impact on substrate specificity, producing enzymes with completely new functions.

Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time. High radiological doses and the associated costs have been a major obstacle to the routine (and early) use of (14)C despite the recognized advantages of a (14)C-tracer for quantifying drug metabolism and disposition. Accelerator mass spectrometry eliminates this long-standing problem by reducing radioactivity levels while delivering matrix-independent quantitation to attomole levels of sensitivity in small samples or fractionated isolates. Accelerator mass spectrometry and trace (14)C-labeled drugs are now used to obtain early insights into the human metabolism of a drug candidate in ways that were not previously practical. With this article we describe some of our empirically based approaches for regualted bioanalysis and offer perspectives on current applications and opportunities for the future.

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.

Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family-6 glycoside hydrolases; however, a single catalytic base was not identified. Mutation of all putative catalytic base residues, within 6 A of the -1/+1 glucose subsites, including the highly conserved Asp226, Asp497 and Glu495, as well as Ser232 and Tyr220, did not reveal a catalytic base, although these residues are all important for activity. We propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function.

Thermobifida fuscaLam81A is a single domain family-81 beta-1,3-endoglucanase, but no structure is known for this family. Site-directed mutagenesis of 14 conserved residues chosen from sequence alignments was used to identify those with critical roles in catalysis, binding or substrate specificity. Mutant enzymes were assayed for their ability to bind and hydrolyze substrates with various glycosyl linkages. Residues D422, E499 and E503 were candidates for the catalytic acid or catalytic base, and E499 was shown to be the catalytic base by azide rescue. F425 was shown to have a major role in substrate binding possibly mediated by aromatic ring stacking with the sugar substrate. In addition, mutation of D424 to histidine altered the substrate specificity by increasing the rate of cleavage of mixed-linkage beta-glucan and carboxymethyl-cellulose, 60- and 16-fold, respectively, over the wild-type enzyme.