Mechanisms Associated with Superoxide Radical Scavenging Reactions Involving Phenolic Compounds Deduced Based on the Correlation between Oxidation Peak Potentials and Second-Order Rate Constants Determined Using Flow-Injection Spin-Trapping EPR Methods.

Flow-injection spin-trapping electron paramagnetic resonance (FI-EPR) methods that involve the use of 5,5-dimethyl-pyrroline-N-oxide (DMPO) as a spin-trapping reagent have been developed for the kinetic study of the O2•- radical scavenging reactions occurring in the presence of various plant-derived and synthetic phenolic antioxidants (Aox), such as flavonoid, pyrogallol, catechol, hydroquinone, resorcinol, and phenol derivatives in aqueous media (pH 7.4 at 25 °C). The systematically estimated second-order rate constants (ks) of these phenolic compounds span a wide range (from 4.5 × 10 to 1.0 × 106 M-1 s-1). The semilogarithm plots presenting the relationship between ks values and oxidation peak potential (Ep) values of phenolic Aox are divided into three groups (A1, A2, and B). The ks-Ep plots of phenolic Aox bearing two or three OH moieties, such as pyrogallol, catechol, and hydroquinone derivatives, belonged to Groups A1 and A2. These molecules are potent O2•- radical scavengers with ks values above 3.8 × 104 (M-1 s-1). The ks-Ep plots of all phenol and resorcinol derivatives, and a few catechol and hydroquinone derivatives containing carboxyl groups adjacent to the OH groups, were categorized into the group poor scavengers (ks < 1.6 × 103 M-1 s-1). The ks values of each group correlated negatively with Ep values, supporting the hypothesis that the O2•- radical scavenging reaction proceeds via one-electron and two-proton processes. The processes were accompanied by the production of hydrogen peroxide at pH 7.4. Furthermore, the correlation between the plots of ks and the OH proton dissociation constant (pKa•) of the intermediate aroxyl radicals (ks-pKa• plots) revealed that the second proton transfer process could potentially be the rate-determining step of the O2•- radical scavenging reaction of phenolic compounds. The ks-Ep plots provide practical information to predict the O2•- radical scavenging activity of plant-derived phenolic compounds based on those molecular structures.

Crystal structure of L-2-keto-3-deoxyfuconate 4-dehydrogenase reveals a unique binding mode as a α-furanosyl hemiketal of substrates.

L-2-Keto-3-deoxyfuconate 4-dehydrogenase (L-KDFDH) catalyzes the NAD+-dependent oxidization of L-2-keto-3-deoxyfuconate (L-KDF) to L-2,4-diketo-3-deoxyfuconate (L-2,4-DKDF) in the non-phosphorylating L-fucose pathway from bacteria, and its substrate was previously considered to be the acyclic α-keto form of L-KDF. On the other hand, BDH2, a mammalian homolog with L-KDFDH, functions as a dehydrogenase for cis-4-hydroxy-L-proline (C4LHyp) with the cyclic structure. We found that L-KDFDH and BDH2 utilize C4LHyp and L-KDF, respectively. Therefore, to elucidate unique substrate specificity at the atomic level, we herein investigated for the first time the crystal structures of L-KDFDH from Herbaspirillum huttiense in the ligand-free, L-KDF and L-2,4-DKDF, D-KDP (D-2-keto-3-deoxypentonate; additional substrate), or L-2,4-DKDF and NADH bound forms. In complexed structures, L-KDF, L-2,4-DKDF, and D-KDP commonly bound as a α-furanosyl hemiketal. Furthermore, L-KDFDH showed no activity for L-KDF and D-KDP analogs without the C5 hydroxyl group, which form only the acyclic α-keto form. The C1 carboxyl and α-anomeric C2 hydroxyl groups and O5 oxygen atom of the substrate (and product) were specifically recognized by Arg148, Arg192, and Arg214. The side chain of Trp252 was important for hydrophobically recognizing the C6 methyl group of L-KDF. This is the first example showing the physiological role of the hemiketal of 2-keto-3-deoxysugar acid.

Minimally Invasive Trans-sacral Canal Plasty for Lumbar Canal Stenosis.

Objective The elderly population is increasing in Japan. Along with the increase in the elderly population, the number of patients with lumbar degenerative diseases is also on the rise. In general, elderly patients tend to have more complications and are at higher risk for surgery. Many elderly people suffer from lumbar degenerative disease. We reviewed our initial experience with trans-sacral canal plasty (TSCP) for patients with lumbar spinal canal stenosis and examined the pertinent literature for this report. Methods An analytical observational study was performed on 120 patients with lumbar spinal canal stenosis who underwent TSCP at our single institution from March 2019 to October 2021. These patients had leg pain and/or lower back pain due to degenerative lumbar disease. Patients who had coagulation abnormality, pregnancy, contrast allergy, pyogenic spondylitis, or spinal metastasis were excluded. Results Immediately after TSCP, the average Visual Analog Scale (VAS) score for back pain improved from 58.2 to 29.3, and for leg pain from 72.0 to 31.3. Two years after TSCP, the average VAS score for back pain increased slightly and the average score for leg pain remained almost the same. Additional surgery was performed in 37 of 120 (31%) patients who underwent TSCP. The additional surgery group had significantly worse back pain at one and three months postoperatively than the conservative treatment group. The additional surgery group had significantly worse leg pain immediately after TSCP and at one and three months postoperatively than the conservative treatment group. Logistic regression analysis demonstrated that a decreased spinal canal area (OR 0.986, p = 0.039) was associated with additional surgery. Conclusions We reviewed the outcomes of TSCP at our hospital. The average VAS score for back pain and leg pain improved. However, 31% of patients who underwent TSCP required additional surgery. It was found that the spinal canal area was a major factor in the need for additional surgery.

Gluconeogenesis during development of the grass puffer (Takifugu niphobles).

During the development of teleost fish, the sole nutrient source is the egg yolk. The yolk consists mostly of proteins and lipids, with only trace amounts of carbohydrates such as glycogen and glucose. However, past evidence in some fishes showed transient increase in glucose during development, which may have supported the development of the embryos. Recently, we found in zebrafish that the yolk syncytial layer (YSL), an extraembryonic tissue surrounding the yolk, undergoes gluconeogenesis. However, in other teleost species, the knowledge on such gluconeogenic functions during early development is lacking. In this study, we used a marine fish, the grass puffer (Takifugu niphobles) and assessed possible gluconeogenic functions of their YSL, to understand the difference or shared features of gluconeogenesis between these species. A liquid chromatography (LC) / mass spectrometry (MS) analysis revealed that glucose and glycogen content significantly increased in the grass puffer during development. Subsequent real-time PCR results showed that most of the genes involved in gluconeogenesis increased in segmentation stages and/or during hatching. Among these genes, many were expressed in the YSL and liver, as shown by in situ hybridization analysis. In addition, glycogen immunostaining revealed that this carbohydrate source was accumulated in many tissues at segmentation stage but exclusively in the liver in hatched individuals. Taken together, these results suggest that developing grass puffer undergoes gluconeogenesis and glycogen synthesis during development, and that gluconeogenic activity is shared in YSL of zebrafish and grass puffer.

Investigation of long lateral mass screw insertion torque.

Background: Here, we assessed a new trajectory and insertion torque for the placement of a long lateral mass screw (LLMS) that offers stronger posterior fixation versus a shorter lateral mass screw (LMS) in the posterior cervical spine. We report a short technical note of the insertion torque of LLMS. Methods: The insertion trajectory/torque was evaluated in 30 patients (10 males and 20 females) undergoing posterior cervical LLMS fusions (2021-2023). Patients averaged 65 years of age. Pathology included eight cervical spine injuries, ten cord injuries, four dislocations/fractures, and eight other entities. Variables studied included the length of the LLMS inserted from C3-7, screw deviation rates, insertion torque, and adverse events. Results: A total of 146 screws were inserted: 11 pedicle screws (PSs) and 135 LLMS. The average insertion torque was 105.9 cNm for PS and 64.9 cNm for LLMS. As the screw lengthened by 1 mm, the insertion torque increased by approximately 4.4 cNm. Conclusion: Here, we documented that the insertion torque of LLMS was 66.1 cNm, greater than the 51.0 cNm for LMS, which should provide stronger posterior cervical fixation.

Cellulolytic enzymes in Microbulbifer sp. Strain GL-2, a marine fish intestinal bacterium, with emphasis on endo-1,4-ß-glucanases Cel5A and Cel8.

Cellulose is an abundant biomass on the planet. Various cellulases from environmental microbes have been explored for industrial use of cellulose. Marine fish intestine is of interest as one source of new enzymes. Here, we report the discovery of genes encoding two ß-glucosidases (Bgl3A and Bgl3B) and four endo-1,4-ß-glucanases (Cel5A, Cel8, Cel5B, and Cel9) as part of the genome sequence of a cellulolytic marine bacterium, Microbulbifer sp. Strain GL-2. Five of these six enzymes (excepting Cel5B) are presumed to localize to the periplasm or outer membrane. Transcriptional analysis demonstrated that all six genes were highly expressed in stationary phase. The transcription was induced by cello-oligosaccharides rather than by glucose, suggesting that the cellulases are produced primarily for nutrient acquisition following initial growth, facilitating the secondary growth phase. We cloned the genes encoding two of the endo-1,4-ß-glucanases, Cel5A and Cel8, and purified the corresponding recombinant enzymes following expression in Escherichia coli. The activity of Cel5A was observed across a wide range of temperatures (10-40 ˚C) and pHs (6-8). This pattern differed from those of Cel8 and the commercial cellulase Enthiron, both of which exhibit decreased activities below 30 ˚C and at alkaline pHs. These characteristics suggest that Cel5A might find use in industrial applications. Overall, our results reinforce the hypothesis that marine bacteria remain a possible source of novel cellulolytic activities.

Predicting Skeletal-related Events Using SINS.

STUDY DESIGN: Predictive study utilized retrospectively collected data. OBJECTIVE: The primary objective was to evaluate the predictive association between the Spine Instability Neoplastic Score (SINS) and Skeletal-related events (SREs). Secondary objectives included examining characteristics of cases with SINS < 6 among those who developed SRE, and evaluating the impact of additional predictors on prediction accuracy. SUMMARY OF BACKGROUND DATA: Advances in cancer treatment have prolonged the lives of cancer patients, emphasizing the importance of maintaining quality of life. Skeletal-related events from metastatic spinal tumors significantly impact quality of life. However, currently, there is no scientifically established method to predict the occurrence of SRE. SINS, developed by the Spine Oncology Study Group, assesses spinal instability using six categories. Therefore, the predictive performance of SINS for SRE occurrence is of considerable interest to clinicians. METHODS: This predictive study utilized retrospectively collected data from a single-center registry comprising over 1,000 patients with metastatic spinal tumors. SINS and clinical data were collected. Logistic regression was used to create a prediction equation for SRE using SINS. Additional analyses explored factors associated with SRE in patients with SINS < 6. RESULTS: The study included 1,041 patients with metastatic spinal tumors. SRE occurred in 121 cases (12%). The prediction model for SRE using SINS demonstrated an area under the curve (AUC) of 0.832. Characteristics associated with SRE included lower female prevalence, surgeries to primary sites, bone metastases to non-spinal sites, and metastases to other organs. A post hoc analysis incorporating additional predictors improved the AUC to 0.865. CONCLUSION: The SINS demonstrated reasonable predictive performance for SRE within one month of the initial visit. Incorporating additional factors improved prediction accuracy. The study emphasizes the need for a comprehensive clinical prediction model for SRE in metastatic spinal tumors.

Characterization of a novel L-fuconate dehydratase involved in the non-phosphorylated pathway of L-fucose metabolism from bacteria.

A sugar acid dehydratase from Paraburkholderia mimosarum, potentially involved in the non-phosphorylated L-fucose pathway, was functionally characterized. A biochemical analysis revealed its unique heterodimeric structure and higher specificity toward L-fuconate than D-arabinonate, D-altronate, and L-xylonate, which differed from homomeric homologs. This unique L-fuconate dehydratase has a poor phylogenetic relationship with other functional members of the D-altronate dehydratase/galactarate dehydratase protein family.

Molecular evolutionary insight of structural zinc atom in yeast xylitol dehydrogenases and its application in bioethanol production by lignocellulosic biomass.

Xylitol dehydrogenase (XDH) catalyzes the NAD+-dependent oxidization of xylitol into D-xylulose, and belongs to a zinc-dependent medium-chain dehydrogenase/reductase family. This protein family consists of enzymes with one or two zinc atoms per subunit, among which catalytic zinc is necessary for the activity. Among many XDHs from yeast and fungi, XDH from Pichia stipitis is one of the key enzymes for bioethanol production by lignocellulosic biomass, and possesses only a catalytic zinc atom. Despite its importance in bioindustry, a structural data of XDH has not yet been available, and little insight into the role of a second zinc atom in this protein family is known. We herein report the crystal structure of XDH from P. stipitis using a thermostabilized mutant. In the refined structure, a second zinc atom clearly coordinated with four artificially introduced cysteine ligands. Homologous mutations in XDH from Saccharomyces cerevisiae also stabilized and enhanced activity. The substitution of each of the four cysteine ligands with an aspartate in XDH from Schizosaccharomyces pombe contributed to the significantly better maintenance of activity and thermostability than their substitution with a serine, providing a novel hypothesis for how this zinc atom was eliminated.

Crystal Structure of l-2,4-Diketo-3-deoxyrhamnonate Hydrolase Involved in the Nonphosphorylated l-Rhamnose Pathway from Bacteria.

2,4-Diketo-3-deoxy-l-rhamnonate (L-DKDR) hydrolase (LRA6) catalyzes the hydrolysis reaction of L-DKDR to pyruvate and l-lactate in the nonphosphorylated l-rhamnose pathway from bacteria and belongs to the fumarylacetoacetate hydrolase (FAH) superfamily. Most of the members of the FAH superfamily are involved in the microbial degradation of aromatic substances and share low sequence similarities with LRA6, by which the underlying catalytic mechanism remains unknown at the atomic level. We herein elucidated for the first time the crystal structures of LRA6 from Sphingomonas sp. without a ligand and in complex with pyruvate, in which a magnesium ion was coordinated with three acidic residues in the catalytic center. Structural, biochemical, and phylogenetic analyses suggested that LRA6 is a close but distinct subfamily of the fumarylpyruvate hydrolase (FPH) subfamily, and amino acid residues at equivalent position to 84 in LRA6 are related to different substrate specificities between them (Leu84 and Arg86 in LRA6 and FPH, respectively). Structural transition induced upon the binding of pyruvate was observed within a lid-like region, by which a glutamate-histidine dyad that is critical for catalysis was arranged sufficiently close to the ligand. Among several hydroxylpyruvates (2,4-diketo-5-hydroxycarboxylates), L-DKDR with a C6 methyl group was the best substrate for LRA6, conforming to the physiological role. Significant activity was also detected in acylpyruvate including acetylpyruvate. The structural analysis presented herein provides a more detailed understanding of the molecular evolution and physiological role of the FAH superfamily enzymes (e.g., the FAH like-enzyme involved in the mammalian l-fucose pathway).

Efficiency of Long Lateral Mass Screws.

Introduction: Lateral mass screws (LMS) have been widely used for the posterior fusion of the cervical spine. Even though LMS are safe, the screws are short and postoperative fixation is uncertain. Therefore, we measured and reported a technique using long lateral mass screws (LLMS), a new method of screw insertion, using a Zed spine from LEXI (Tokyo, Japan). Materials and Methods: In this study, we evaluated the outcomes of 35 patients who underwent surgery using LLMS at our hospital from 2019 to 2021. Operative time, blood loss, complications, inserted screw length, screw length based on gender differences, and screw deviation rate were evaluated. The Mann−Whitney U test was used to determine the gender differences in screw length. Screw deviation was evaluated by postoperative CT and a Zed spine to determine the screw insertion angle. Results: The mean operative time was 185 ± 51 min (120−327 min), and the mean blood loss was 236 ± 316 g (10−1720 g). The total number of screws was 183. The screw length was 22.2 (16−28) mm for males and 20.8 (16−28) mm for females, with an average length of 21 ± 2.7 mm. No gender differences were observed in terms of screw length (p > 0.01 NS). The number of deviated screws above G3 was one in the third cervical vertebra, three in the fourth cervical vertebra, one in the fifth cervical vertebra, and one in the sixth cervical vertebra. The number of deviated screws was 6 out of 183, and the deviation rate was 3.2%. Conclusions: In this study, the LLMS deviation rate was 3.2%, and strong fixation was possible without any complications. We measured the screw length and screw deviation rate in cases in which LLMS were actually inserted.

Crystal structure of L-arabinose 1-dehydrogenase as a short-chain reductase/dehydrogenase protein.

l-Arabinose 1-dehydrogenase (AraDH) catalyzes the NAD(P)+-dependent oxidation of l-arabinose to L-arabinono-1,4-lactone in the non-phosphorylative l-arabinose pathway, and is classified into glucose-fructose oxidoreductase and short-chain dehydrogenase/reductase (SDR). We herein report the crystal structure of a SDR-type AraDH (from Herbaspirillum huttiense) for the first time. The interactions between Asp49 and the 2'- and 3'-hydroxyl groups of NAD+ were consistent with strict specificity for NAD+. In a binding model for the substrate, Ser155 and Tyr168, highly conserved in the SDR superfamily, interacted with the C1 and/or C2 hydroxyl(s) of l-arabinose, whereas interactions between Asp107, Arg109, and Gln206 and the C2 and/or C3 hydroxyl(s) were unique to AraDH. Trp200 significantly contributed to the selectivities of the C4 hydroxyl and C6 methyl of substrates.

Effect of Minimally Invasive Spine Stabilization in Metastatic Spinal Tumors.

Background and Objectives: There have been numerous advances in spine surgery for metastatic spinal tumors, and minimally invasive spine stabilization (MISt) is becoming increasingly popular in Japan. MISt is a minimally invasive fixation procedure that temporarily stabilizes the spine, thereby reducing pain, preventing pathological fractures, and improving activities of daily living at an early stage. MISt may be useful given the recent shift toward outpatient cancer treatment. Materials and Methods: This study enrolled 51 patients with metastatic spinal tumors who underwent surgery using MISt between December 2013 and October 2020. The Spinal Instability Neoplastic Score, an assessment of spinal instability, was used to determine the indication for surgery, and the Epidural Spinal Cord Compression scale was used for additional decompression. Results: The patients comprised 34 men and 17 women, and the mean age at surgery was 68.9 years. The mean postoperative follow-up period was 20.8 months, and 35 of 51 patients (67%) had died by the last survey. The mean operative time was 159.8 min, mean blood loss was 115.7 mL, and mean time to ambulation was 3.2 days. No perioperative complications were observed, although two patients required refixation surgery. Preoperatively, 37 patients (72.5%) were classified as Frankel grade E. There were no cases of postoperative exacerbation, and six patients showed improvement of one or more Frankel grades after surgery. The median duration of patient survival was about 22.0 months. Patients with breast, prostate, renal, and thyroid cancers had a good prognosis, whereas those with gastrointestinal and head and neck cancers had a poor prognosis. Conclusions: MISt can benefit patients who are ineligible for conventional, highly invasive surgery and is also suitable because cancer treatment is increasingly performed on an outpatient basis. Furthermore, choosing the right surgery for the right patient at the right time can significantly affect life expectancy.

Processes of molecular adsorption and ordering enhanced by mechanical stimuli under high contact pressure.

Adsorbed molecular films, referred to as boundary films in tribology, are widely used in various industrial products as a keyway for surface functionalisation, such as lubricity, wettability, and adhesion. Because boundary films are thin nanometre-scale molecular layers and can easily be removed, their formation process cannot be elucidated in detail. In this study, to analyse the growth dynamics of boundary films, the film thickness and molecular orientation of the boundary film of a fatty acid used as an additive in rolling contact as mechanical stimuli were measured in situ. The measurements were performed on simple test lubricants, which were composed of n-hexadecane and stearic acid, at rolling tribological condition between steel and glass (or sapphire) surfaces by ultrathin film interferometry combined with sum-frequency generation spectroscopy according to a unique protocol. The results quantitatively demonstrate shear-induced boundary film formation. The insight gained from these results is anticipated to enable the formulation of high-performance lubricant additives to further reduce friction loss and high-performance glues that can be freely designed for removability.

Prophylactic Effect of Liaison Treatment on the Occurrence of Skeletal-Related Events in Patients with Metastatic Spinal Tumours: An Exploratory Interrupted Time Series Study.

INTRODUCTION: There is a growing momentum for the collaboration between multiple disciplines for the prevention and treatment of skeletal-related events (SREs) in patients with metastatic spinal tumors. However, the effectiveness of multidisciplinary approaches remains unclear. Hence, we conducted an exploratory study to examine the impact of liaison treatment for metastatic spinal tumor (LMST) on the prevention of SREs among patients with a metastatic spinal tumor. METHODS: This study was an exploratory interrupted time series conducted in a single medical center. Overall, 1,043 patients with a metastatic spinal tumor diagnosed between January 2011 and December 2020 were included. The LMST was implemented in January 2014. The LMST team consisted of the orthopedic surgery, thoracic surgery, breast and thyroid surgery, clinical oncology, urology, and radiology departments. Monthly joint conferences were held for patients with spinal instability, and the incidence of SRE was measured at 6-month intervals. RESULTS: Throughout the study period, we identified 66 SRE incidences. After the implementation of the LMST, a level change of -5.2% (95% confidence interval [CI]: -11.7 to 1.3, p = 0.11) was observed. Subsequently, a post-implementation trend change of -0.3% (95% CI: -2.0 to 1.5, p = 0.75) beyond the baseline was noted. CONCLUSIONS: We suggest both immediate and gradual effects of the introduction of the LMST on deterring the development of SREs. Our results support the global trend of introducing a multidisciplinary approach for the treatment of metastatic spinal tumors.

Crystal structures of aconitase X enzymes from bacteria and archaea provide insights into the molecular evolution of the aconitase superfamily.

Aconitase superfamily members catalyze the homologous isomerization of specific substrates by sequential dehydration and hydration and contain a [4Fe-4S] cluster. However, monomeric and heterodimeric types of function unknown aconitase X (AcnX) have recently been characterized as a cis-3-hydroxy-L-proline dehydratase (AcnXType-I) and mevalonate 5-phosphate dehydratase (AcnXType-II), respectively. We herein elucidated the crystal structures of AcnXType-I from Agrobacterium tumefaciens (AtAcnX) and AcnXType-II from Thermococcus kodakarensis (TkAcnX) without a ligand and in complex with substrates. AtAcnX and TkAcnX contained the [2Fe-2S] and [3Fe-4S] clusters, respectively, conforming to UV and EPR spectroscopy analyses. The binding sites of the [Fe-S] cluster and substrate were clearlydifferent from those that were completely conserved in other aconitase enzymes; however, theoverall structural frameworks and locations of active sites were partially similar to each other.These results provide novel insights into the evolutionary scenario of the aconitase superfamilybased on the recruitment hypothesis.

Video-Assisted Thoracoscopic Surgery for Re-Collapse of Vertebrae after Percutaneous Vertebral Augmentation (PVA).

INTRODUCTION: Due to the increase in osteoporosis accompanying the aging society in Japan, osteoporotic vertebral fractures (OVFs) are increasing. Percutaneous vertebral augmentation (PVA) has been widely used for OVFs because it reduces pain immediately with less invasiveness. Re-collapse of vertebral body after PVA is a rare, but important, complication. Once the re-collapse has occurred, patients should undergo an additional invasive salvage surgery. METHODS: We treated 5 patients with re-collapse after PVA in our hospital. For re-collapse after PVA, we performed anterior column reconstruction with video-assisted thoracoscopic surgery (VATS), posterior fixation with percutaneous pedicle screws (PPSs) and minimally invasive spine stabilization (MISt). RESULTS: The mean postoperative follow-up was at 62.8 months. At the final follow-up, the patients were free of low back pain, and bony union was achieved in all cases. The postoperative correction loss was 6 degrees. Perioperative complications included aspiration pneumonia in one patient and bone fracture of an adjacent vertebral body in two patients. There were no reoperation cases. CONCLUSIONS: We perform minimally invasive combined anterior and posterior surgery with VATS for re-collapse after PVA. This procedure is useful in elderly patients with less reserve capacity.

Crystal structure of l-rhamnose 1-dehydrogenase involved in the nonphosphorylative pathway of l-rhamnose metabolism in bacteria.

Several microorganisms can utilize l-rhamnose as a carbon and energy source through the nonphosphorylative metabolic pathway, in which l-rhamnose 1-dehydrogenase (RhaDH) catalyzes the NAD(P)+ -dependent oxidization of l-rhamnose to l-rhamnono-1,4-lactone. We herein investigated the crystal structures of RhaDH from Azotobacter vinelandii in ligand-free, NAD+ -bound, NADP+ -bound, and l-rhamnose- and NAD+ -bound forms at 1.9, 2.1, 2.4, and 1.6 Å resolution, respectively. The significant interactions with the 2'-phosphate group of NADP+ , but not the 2'-hydroxyl group of NAD+ , were consistent with a preference for NADP+ over NAD+ . The C5-OH and C6-methyl groups of l-rhamnose were recognized by specific residues of RhaDH through hydrogen bonds and hydrophobic contact, respectively, which contribute to the different substrate specificities from other aldose 1-dehydrogenases in the short-chain dehydrogenase/reductase superfamily.

Isolation of cellulase-producing Microbulbifer sp. from marine teleost blackfish (Girella melanichthys) intestine and the enzyme characterization.

Most animals cannot digest cellulose but have symbiotic microbes that degrade the matrix polysaccharides of plant matter. Herbivorous and omnivorous marine fish are similarly expected to rely on symbiotic microbes, but reports to date on cellulase-producing bacteria in fish intestines are limited. Here, we report the isolation of new cellulase-producing bacteria from the marine omnivorous teleost, blackfish (Girella melanichthys), and the characterization of cellulase activity. Three strains of cellulase-producing bacteria sp. were isolated from the hindgut of wild G. melanichthys. The strains of cellulase-producing bacteria grew in medium with artificial seawater but not in NaCl alone. Growth was optimum at 20-35°C, but there was no growth at 40°C, suggesting adaptation in a marine environment at a low temperature. Isolates were identified to Microbulbifer sp., among which GL-2 strain produced a high enzyme activity. The GL-2 strain was further used for enzyme characterization with carboxymethyl cellulose (CMC) as the substrate. Maximum activity of the cellulase was observed at 60°C, and activity was more than 30% at 20°C, while commercial cellulase Enthiron showed an optimum activity at 50°C and 17% activity at 20°C. Hydrolytic products by GL-2 cellulase were cellobiose but not glucose, suggesting a deficiency of ß-glucosidase activity. Active gel electrophoresis containing CMC showed five bands, suggesting several cellulolytic enzymes. The GL-2 strain and its enzyme are potential probiotics for aquaculture fish and the industrial production of cellobiose.

Effect of water on the electroresponsive structuring and friction in dilute and concentrated ionic liquid lubricant mixtures.

The effect of water on the electroactive structuring of a tribologically relevant ionic liquid (IL) when dispersed in a polar solvent has been investigated at a gold electrode interface using neutron reflectivity (NR). For all solutions studied, the addition of small amounts of water led to clear changes in electroactive structuring of the IL at the electrode interface, which was largely determined by the bulk IL concentration. At a dilute IL concentration, the presence of water gave rise to a swollen interfacial structuring, which exhibited a greater degree of electroresponsivity with applied potential compared to an equivalent dry solution. Conversely, for a concentrated IL solution, the presence of water led to an overall thinning of the interfacial region and a crowding-like structuring, within which the composition of the inner layer IL layers varied systematically with applied potential. Complementary nanotribotronic atomic force microscopy (AFM) measurements performed for the same IL concentration, in dry and ambient conditions, show that the presence of water reduces the lubricity of the IL boundary layers. However, consistent with the observed changes in the IL layers observed by NR, reversible and systematic control of the friction coefficient with applied potential was still achievable. Combined, these measurements provide valuable insight into the implications of water on the interfacial properties of ILs at electrified interfaces, which inevitably will determine their applicability in tribotronic and electrochemical contexts.