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The best-fit method is frequently used to evaluate the accuracy of different implant impression techniques. However, the method includes inherent superimposition errors, which may accumulate and become more exaggerated in complete-arch impressions.The purpose of this in vitro study was to evaluate and compare the trueness and precision of conventional open-tray impressions and intraoral digital scans at the implant level in an edentulous maxillary model with 6 implant replicas without superimposition.A master model was fabricated using epoxy resin by duplicating a maxillary edentulous cast that had 6 implant replicas in the right first molar, right first premolar, right lateral incisor, left lateral incisor, left first premolar, and left first molar positions. The conventional open-tray, splinted-coping impression technique was used to fabricate 10 definitive casts (group CI). Intraoral digital scans were performed, after which scan bodies were connected to each implant replica to fabricate 10 digital models (group IOS). For the master model and group CI, a computerized coordinate-measuring machine was used to determine the 3D spatial orientation of the implant replicas. For group IOS, the scan bodies were converted to implant replicas using a digital library, and an inspection software program was used to measure the implant replicas. To compare the accuracies of different impression techniques, a 3D part coordinate system was set to compute the centroid and projection angles of each implant replica. The changes in the centroid coordinates (linear displacement: Δx, Δy, Δz, and ΔD; ΔD=ΔxGroup CI gave more accurate trueness values than group IOS for overall Δx (P.001), Δy (P =.029), Δz (P.001), and ΔD (P.001). Furthermore, group CI had more accurate precision values for Δx, Δy, and Δz. Group IOS exhibited a statistically greater angular displacement in the ZX plane (P=.002), but the difference was only 0.24 degrees. No differences were found between the 2 groups for the angular displacement in the XY plane (P=.529).Conventional open-tray impressions produced significantly smaller linear displacements than the digital scan obtained using an intraoral scanner at the implant level in a complete-arch model.

Thiophenol is the simplest aromatic thiol that is utilized for various applications in industry and agriculture. However, it should be used with care because thiophenol is readily absorbed into the human body by inhalation and ingestion, which leads to serious internal injuries. Thus, there is an urgent need for real-time and accurate monitoring of thiophenol. Despite remarkable advantages of electrogenerated chemiluminescence (ECL) analysis, ECL thiophenol probes have never been reported. Herein, a new strategy for the rapid detection of thiophenol by use of an ECL turn-on chemodosimeter based on a cyclometalated Ir(III) complex is described. This analytical system showed superior sensitivity [limit of detection (LOD) value, 3.8 nM] in comparison to the conventional fluorescence method. In addition, our system exhibited remarkable selectivity and reaction rate toward thiophenol over other analytes. Moreover, it was successfully applied to quantify thiophenol in real water samples, providing a new proof-of-concept for field monitoring based on ECL.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB5, HXD3, HXE3, TrXB5, TrXD3 and TrXE3. We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB3, HXB4, HXD3, TrXB3 and TrXD3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators., Hepoxilins (HXs) and trioxilins (TrXs) are lipid metabolites with roles in inflammation and insulin secretion. Here, the authors discover a prokaryotic source of HXs and TrXs, identify the biosynthetic enzymes and heterologously express HXs and TrXs in E. coli.

To produce tagatose from fructose with a high conversion rate and to establish a high-yield purification method of tagatose from the reaction mixture. Fructose at 1 M (180 g l−1) was converted to 0.8 M (144 g l−1) tagatose by a three-step enzymatic cascade reaction, involving hexokinase, plus ATP, fructose-1,6-biphosphate aldolase, phytase, over 16 h with a productivity of 9 g l−1 h−1. No byproducts were detected. Tagatose was recrystallized from ethanol to a purity of 99.9% and a yield of 96.3%. Overall, tagatose at 99.9% purity was obtained from fructose with a yield of 77%. This is the first biotechnological production of tagatose from fructose and the first application of solvent recrystallization for the purification of rare sugars.

A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus . The amino acid sequence of ALDH from B. cereus ( Bc ALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all- trans -retinal to all- trans -retinoic acid with NAD(P) + . We newly found that Bc ALDH and human ALDH (ALDH1A1) could reduce all- trans -retinal to all- trans -retinol with NADPH. The catalytic residues in Bc ALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency ( k cat / K m ) for the reduction of all- trans -retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD + converted 400 μM all- trans -retinal to 210 μM all- trans -retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 μM all- trans -retinal to 20, 90, and 40 μM all- trans -retinol, respectively. These results indicate that Bc ALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, Bc ALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. IMPORTANCE Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P) + as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all- trans -retinal to all- trans -retinoic acid by Bc ALDH and human ALDH was altered to the biotransformation to all- trans -retinol by a cofactor switch using NADPH. Moreover, the production of all- trans -retinal to all- trans -retinol was changed by mutations at positions 194 and 457 in Bc ALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Recombinant Escherichia coli cells expressing linoleate 13-hydratase from Lactobacillus acidophilus were permeabilized by treating with 0.2 M NaCl. The optimal conditions for the production of 13-hydroxy-9,15(Z,Z)-octadecadienoic acid (13-HODE) from α-linolenic acid by permeabilized recombinant cells were pH 6.0, 40 °C, 7.5 % (v/v) methanol, 60 g/l permeabilized cells, and 15 g/l α-linolenic acid. Under these conditions, permeabilized cells produced 7.5 g/l 13-HODE after 6 h, with a conversion yield of 50 % (w/w) and a volumetric productivity of 1.25 g/l/h. These values were 161 and 160 % of those obtained by nonpermeabilized cells, respectively. To the best of our knowledge, this is the first report on the process optimization for the biotechnological production of 13-HODE.

Electrogenerated chemiluminescence (ECL) is a luminescent process which generates light through sequential electron transfer reactons on the electrode surface. ECL-based molecular sensors have several advantages over the conventional analytical techniques such as high sensitivity and low background signal. Additionally, the ECL provides the possibility of potential point-of-care-testing and field-monitoring with the simplicity of equipment and the method. However, ECL detection of small molecules is still a great challenge because most of ECL detection methods have been developed via specific biomacromolecular recognition such as antibody-antigen and aptamer-protein interactions. Herein, we report ECL molecular sensors for qualitative and quantitative detection of biologically important and environmentally toxic molecules. In the first part, a chemodosimetric approach will be introduced for one-step analysis of Hcy levels based on the ECL. A rationally designed cyclometalated iridium(III) complex possessing a phenylisoquinoline main ligand underwent a selective ring-formation reaction with Hcy to generate a binding adduct, which produced highly luminescent excited states, and yielded strong ECL signals on the surface of an electrode. The level of Hcy was successfully monitored by the ECL increment with a linear correlation between 0–40 µM in 99.9% aqueous media. The approach required neither sample preparation nor bulky instrument, suggesting the point-of-care testing of Hcy levels, and is potentially useful for routine, cost-effective, and precautionary diagnosis of various cardiovascular diseases. ECL sensors for other biologically important analytes (pyrophosphate, hydrogen sulfide, cysteine, H2O2) and environmentally toxic molecules (thiophenol, glyphosate, Hg(II)) will also be presented. In the second part, we report a totally different approach to discriminate a target among various interferences. This is called potential-dependent electrochemiluminescence. The iridium complex having a dicyanovinyl group on the main ligand cannot perfectly distinguish cyanide from sulfide and cysteine because not only cyanide but also sulfide and cysteine can attack the beta position of the dicyanovinyl group to increase the PL intensity. However, cyanide adduct is electrochemically oxidized at much less positive potential (~ 1.0 V) than those of sulfide and cysteine adducts, and this enables the discrimination of cyanide via ECL approach. This method enables complete removal of the interfering signals from sulfide and other thiols by controlling the potential range (0 – 1.4 V). References [1] H. J. Kim, K.-S. Lee, Y.-J. Jeon, I.-S. Shin, J.-I. Hong, Electrochemiluminescent chemodosimeter based on iridium(III) complex for point-of-care detection of homocysteine levels, Biosens. Bioelectron. 91 (2017) 497–503. doi: 10.1016/j.bios.2017.01.010. [2] S.-Y. Kim, H. J. Kim, J.-I. Hong, Electrochemiluminescent chemodosimetric probes for sulfide based on cyclometalated Ir(III) complexes, RSC Adv. 7 (2017) 10865–10868. doi: 10.1039/C6RA26826A. [3] I.-S. Shin, S. W. Bae, H. Kim, J.-I. Hong, Electrogenerated Chemiluminescent Anion Sensing: Selective Recognition and Sensing of Pyrophosphate, Anal. Chem. 82 (2010) 8259-8265. doi: 10.1021/ac1017293. [4] H. Rhee, T. Kim, J.-I. Hong, Ir(III) complex–based phosphorescence and electrochemiluminescence chemodosimetric probes for Hg(II) ion with high selectivity and sensitivity, Dalton Trans. 47 (2018) 3803-3810. doi: 10.1039/c8dt00309b. [5] K.-R. Kim, H. J. Kim, J.-I. Hong, Electrogenerated chemiluminescent chemodosimeter based on a cyclometalated-iridium(III) complex for rapid and sensitive detection of thiophenol, Anal. Chem. 91 (2019) 1353-1359. doi: 10.1021/acs.analchem.8b03445. [6] J. Park, T. Kim, H. J. Kim, J.-I. Hong, Iridium(III) complex-based electrochemiluminescent probe for H2S, Dalton Trans. 48 (2019) 4565-4573. doi: 10.1039/c8dt04901g. [7] T. Kim, J.-I. Hong, Photoluminescence and Electrochemiluminescence Dual-signaling Sensors for Selective Detection of Cysteine Based on Iridium(III) Complexes, ACS Omega. 4 (2019) 12616-12625. doi: 10.1021/acsomega.9b01501.

Linoleate 13-hydratase from Lactobacillus acidophilus LMG 11470 converted linoleic acid to hydroxyl fatty acid, which was identified as 13S-hydroxy-9(Z)-octadecenoic acid (13-HOD) by GC-MS and NMR. The expression of linoleate 13-hydratase gene in Escherichia coli was maximized by using pACYC plasmid and super optimal broth with catabolite repression (SOC) medium containing 40mM Mg(2+). To optimize induction conditions, recombinant cells were cultivated at 37°C, 1mM isopropyl-β-d-thiogalactopyranoside was added at 2h, and the culture was further incubated at 16°C for 18h. Recombinant cells expressing linoleate 13-hydratase from L. acidophilus were obtained under the optimized expression conditions and used for 13-HOD production from linoleic acid. The optimal reaction conditions were pH 6.0, 40°C, 0.25% (v/v) Tween 40, 25gl(-1) cells, and 100gl(-1) linoleic acid, and under these conditions, whole recombinant cells produced 79gl(-1) 13-HOD for 3h with a conversion yield of 79% (w/w), a volumetric productivity of 26.3gl(-1)h(-1), and a specific productivity of 1.05g g-cells(-1)h(-1). To the best of our knowledge, the recombinant cells produced hydroxy fatty acid with the highest concentration and productivity reported so far.

The aim of this study is the first time demonstration of cis-12 regio-selective linoleate double-bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis-9 and cis-12 double-bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio-selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13-dihydroxyoctadecanoic acid biosynthesis. Linoleate 13-hydratase (LHT-13) selectively converted 10 mM linoleic acid to 13S-hydroxy-9(Z)-octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid-hydroxylation enzymes and facilitate the designed production of the regio-selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks. Biotechnol. Bioeng. 2015;112: 2206–2213. © 2015 Wiley Periodicals, Inc.

Hydroxy fatty acids are used as starting materials for the production of secondary metabolites and signalling molecules as well as in the manufacture of industrial fine chemicals. However, these compounds are usually difficult to produce from renewable biomass by chemical means. In this study, linoleate double bond hydratases of Lactobacillus acidophilus NBRC 13951 were cloned for the first time. These enzymes were highly specific for the hydration of the C-9 or the C-12 double bond of unsaturated fatty acids (e.g., linoleic acid). Thereby, the enzymes allowed the selective production of hydroxy fatty acids such as 13-hydroxy-cis-9-octadecenoic acid and 10-hydroxy-cis-12-octadecenoic acid from linoleic acid. In addition, the hydroxy fatty acids were further converted into industrially relevant carboxylic acids (e.g., 12-hydroxy-cis-9-dodecenoic acid, α,ω-tridec-9-enedioic acid) and lactones (i.e., δ-decalactone, γ-dodecelactone) via whole-cell biocatalysis using an enzyme cascade. This study thus contributes to the preparation of hydroxy fatty acids, unsaturated carboxylic acids, and lactones from renewable unsaturated fatty acids.

Sugar 4-epimerization reactions are important for the production of rare sugars and their derivatives, which have various potential industrial applications. For example, the production of tagatose, a functional sweetener, from fructose by sugar 4-epimerization is currently constrained because a fructose 4-epimerase does not exist in nature. We found that class II d-fructose-1,6-bisphosphate aldolase (FbaA) catalyzed the 4-epimerization of d-fructose-6-phosphate (F6P) to d-tagatose-6-phosphate (T6P) based on the prediction via structural comparisons with epimerase and molecular docking and the identification of the condensed products of C3 sugars. In vivo, the 4-epimerization activity of FbaA is normally repressed. This can be explained by our results showing the catalytic efficiency of d-fructose-6-phosphate kinase for F6P phosphorylation was significantly higher than that of FbaA for F6P epimerization. Here, we identified the epimerization reactions and the responsible catalytic residues through observation of the reactions of FbaA and l-rhamnulose-1-phosphate aldolases (RhaD) variants with substituted catalytic residues using different substrates. Moreover, we obtained detailed potential epimerization reaction mechanism of FbaA and a general epimerization mechanism of the class II aldolases l-fuculose-1-phosphate aldolase, RhaD, and FbaA. Thus, class II aldolases can be used as 4-epimerases for the stereo-selective synthesis of valuable carbohydrates.

Purpose: Penile circular fasciocutaneous flap urethroplasty is a useful technique for a long anterior urethral stricture due to the flap’s hairless nature and ample length. We investigated the surgical outcomes of urethroplasty for a complex anterior urethral stricture, performed using a penile circular fasciocutaneous flap. Materials and Methods: Between 2008 and 2013, we performed a retrospective review of 29 patients who underwent urethroplasty using a penile circular fasciocutaneous flap and had at least 6 months of follow-up. A total of 20 cases utilized only a fasciocutaneous flap, while 9 cases combined a fasciocutaneous flap with other surgery. Success was defined as no requirement of additional urethral instrumentation. Results: The overall success rate was 68.9% (20 out of 29 cases) at a median follow-up of 19 months. Furthermore, fasciocutaneous flap urethroplasty rendered the actual stricture-free rate of 79.3%. The location of recurrence was mostly at the junction of the flap. Among 9 surgical failures, 5 cases were treated successfully by using an additional surgical procedure. Fistula repair was needed in 1 case 4 months later. Further, periodic urethral dilation was performed in the remaining 3 cases. The failure rate was significantly higher in patients with suprapubic cystostomy than in patients without suprapubic cystostomy. The most common complication was post-micturition dribbling. Conclusions: Penile circular fasciocutaneous flap urethroplasty is a useful method for the reconstruction of a long anterior urethral stricture. A sufficient healthy margin should be acquired for better surgical results due to the fact that most recurrence occurs at the junction of the flap.

One of the most significant properties of lipoxygenase (LOX) as a biocatalyst is its stereo-selective oxygenation. In this study, the stereo-specific production of 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from linoleic acid was achieved using whole recombinant Escherichia coli cells expressing LOX from Nostoc sp. SAG 25.82. The optimal conditions for the production of 9R-HODE were pH 7.5, 25 °C, 40 g l−1 cells, 15 g l−1 linoleic acid, 2% (v/v) methanol, 1 working volume/oxygen volume/min (vvm) oxygenation rate, and 250 rpm agitation speed in 500 ml-baffled flask containing a working volume of 50 ml. Under these optimized conditions, whole recombinant cells expressing 9R-LOX protein produced 14.3 g l−1 9R-HODE for 1 h, with a conversion yield of 95% (w/w) and a productivity of 14.3 g l−1 h−1. The oxygen supply method significantly influenced stereo- and regio-selectivity of the oxygenation of linoleic acid. Among the oxygen supply methods tested, oxygenation (1 vvm) with agitation (250 rpm) resulted in the highest 9R/13S-HODE ratio of the products at 96:4. This is the first application using whole recombinant cells harboring R-specific LOX for the stereo-selective production of an R-specific hydroxy fatty acid.

A recombinant aldo-keto reductase (AKR) from Marivirga tractuosa was purified with a specific activity of 0.32 unit ml −1 for all- trans -retinal with a 72 kDa dimer. The enzyme had substrate specificity for aldehydes but not for alcohols, carbonyls, or monosaccharides. The enzyme turnover was the highest for benzaldehyde ( k cat = 446 min −1 ), whereas the affinity and catalytic efficiency were the highest for all- trans -retinal ( K m = 48 μM, k cat / K m = 427 mM −1 min −1 ) among the tested substrates. The optimal reaction conditions for the production of all- trans -retinol from all- trans -retinal by M. tractuosa AKR were pH 7.5, 30 °C, 5% (v/v) methanol, 1% (w/v) hydroquinone, 10 mM NADPH, 1710 mg l −1 all- trans -retinal, and 3 unit ml −1 enzyme. Under these optimized conditions, the enzyme produced 1090 mg ml −1 all- trans -retinol, with a conversion yield of 64% (w/w) and a volumetric productivity of 818 mg l −1 h −1 . AKR from M. tractuosa showed no activity for all- trans -retinol using NADP + as a cofactor, whereas human AKR exhibited activity. When the cofactor-binding residues (Ala158, Lys212, and Gln270) of M. tractuosa AKR were changed to the corresponding residues of human AKR (Ser160, Pro212, and Glu272), the A158S and Q270E variants exhibited activity for all- trans -retinol. Thus, amino acids at positions 158 and 270 of M. tractuosa AKR are determinant residues of the activity for all- trans -retinol.

Amorphous InGaZnOx (a-IGZO) thin-film transistors (TFTs) are currently used in flat-panel displays due to their beneficial properties. However, the mobility of ∼10 cm2/(V s) for the a-IGZO TFTs used in commercial organic light-emitting diode TVs is not satisfactory for high-resolution display applications such as virtual and augmented reality applications. In general, the electrical properties of amorphous oxide semiconductors are strongly dependent on their chemical composition; the indium (In)-rich IGZO achieves a high mobility of 50 cm2/(V s). However, the In-rich IGZO TFTs possess another issue of negative threshold voltage owing to intrinsically high carrier density. Therefore, the development of an effective way of carrier density suppression in In-rich IGZO will be a key strategy to the realization of practical high-mobility a-IGZO TFTs. In this study, we report that In-rich IGZO TFTs with vertically stacked InOx, ZnOx, and GaOx atomic layers exhibit excellent performances such as saturation mobilities of ∼74 cm2/(V s), threshold voltage of -1.3 V, on/off ratio of 8.9 × 108, subthreshold swing of 0.26 V/decade, and hysteresis of 0.2 V, while keeping a reasonable carrier density of ∼1017 cm-3. We found that the vertical dimension control of IGZO active layers is critical to TFT performance parameters such as mobility and threshold voltage. This study illustrates the potential advantages of atomic layer deposition processes for fabricating ultrahigh-mobility oxide TFTs.

Hydroxy fatty acids are widely used in chemical, food, and cosmetic industries as starting materials for the synthesis of polymers and as additives for the manufacture of lubricants, emulsifiers, and stabilizers. They have antibiotic, anti-inflammatory, and anticancer activities and therefore can be applied for medicinal uses. Microbial fatty acid-hydroxylation enzymes, including P450, lipoxygenase, hydratase, 12-hydroxylase, and diol synthase, synthesize regio-specific hydroxy fatty acids. In this article, microbial fatty acid-hydroxylation enzymes, with a focus on region-specificity and diversity, are summarized and the production of mono-, di-, and tri-hydroxy fatty acids is introduced. Finally, the production methods of regio-specific and diverse hydroxy fatty acids, such as gene screening, protein engineering, metabolic engineering, and combinatory biosynthesis, are suggested.

A novel β-glucosidase from Penicillium aculeatum was purified as a single 110.5-kDa band on SDS-PAGE with a specific activity of 75.4 U mg⁻¹ by salt precipitation and Hi-Trap Q HP and Resource Q ion exchange chromatographies. The purified enzyme was identified as a member of the glycoside hydrolase 3 family based on its amino acid sequence. The hydrolysis activity for p-nitrophenyl-β-D-glucopyranoside was optimal at pH 4.5 and 70 °C with a half-life of 55 h. The enzyme hydrolyzed exo-, 3-O-, and 6-O-β-glucosides but not 20-O-β-glucoside and other glycosides of ginsenosides. Because of the novel specificity, this enzyme had the transformation pathways for ginsenosides: Rb₁ → Rd → F₂ → compound K, Rb₂ → compound O → compound Y, Rc → compound Mc₁ → compound Mc, Rg₃ → Rh₂ → aglycone protopanaxadiol (APPD), Rg₁ → F₁, and Rf → Rh₁ → aglycone protopanaxatriol (APPT). Under the optimum conditions, the enzyme converted 0.5 mM Rb₂, Rc, Rd, Rg₃, Rg₁, and Rf to 0.49 mM compound Y, 0.49 mM compound Mc, 0.47 mM compound K, 0.23 mM APPD, 0.49 mM F₁, and 0.44 mM APPT after 6 h, respectively.

Hydroxy fatty acids (HFAs) such as 10-hydroxystearic acid (10-HSA) and 10-hydroxy-12(Z)-octadecenoic acid (10-HOD), which are similar to ricinoleic acid, are important starting materials and intermediates for the industrial manufacture of many commodities. Stenotrophomonas sp. KACC 91585, which was isolated from lake sediment, is an efficient bacterium for unsaturated fatty acid hydration that produces 10-HSA and 10-HOD from oleic acid and linoleic acid, respectively, with high conversion rates. The complete genome of this strain is 4,541,729bp with 63.83% GC content and devoid of plasmids. Sets of genes involved in the fatty acid biosynthesis and modification as well as modified lipids were identified in the genome, and these genes were concerned with HFA production. This genome sequence provides molecular information and elucidation for HFA production, and will be used as an efficient biocatalyst source for the biotechnological production of HFA.

The optimal temperature and pH for retinal production using metabolically engineered Escherichia coli in a 7-l fermentor were found to be 30°C and 7.0, respectively. The agitation speed was a critical factor for retinal production. The optimal agitation speed was 400 rpm (oxygen transfer coefficient, k(L)a, = 92 1/h) in batch culture and 600 rpm (k(L)a=148 1/h) in a fed-batch culture of glycerol. Span 80 was selected as a surfactant for retinal production in metabolically engineered E. coli because Span 80 had proven the most effective for increased retinal production among the tested surfactants. Under the optimal conditions in the fed-batch culture with 5 g/l Span 80, the cell mass and the concentration, content, and productivity of retinal were 24.7 g/l, 600 mg/l, 24.3mg/g-cells, and 18 mg l(-1)h(-1) after 33 h, respectively. They were 1.2-, 2.7-, 2.3-, and 2.7-fold higher than those in the fed-batch culture without Span 80, respectively. The concentration and productivity of retinal in this study were the highest ever reported. The hydrophilic portion of Span 80 (sorbitan) did not affect cell growth and retinal production, but the hydrophobic portion (oleic acid) stimulated cell growth. However, oleic acid plus sorbitan did not stimulate retinal production. Thus, Span 80, as a linked compound of oleic acid and sorbitan produced by esterification, proved to be an effective surfactant for the enhancement of retinal production.

A putative fatty acid hydratase from Stenotrophomonas maltophilia was cloned and expressed in Escherichia coli. The recombinant enzyme showed the highest hydration activity for oleic acid among the fatty acids tested, indicating that the enzyme is an oleate hydratase. The optimal conditions for the production of 10-hydroxystearic acid from oleic acid using whole cells of recombinant E. coli containing the oleate hydratase were pH 6.5, 35°C, 0.05% (w/v) Tween 40, 10 g l(-1) cells, and 50 g l(-1) oleic acid. Under these conditions, whole recombinant cells produced 49 g l(-1) 10-hydroxystearic acid for 4 h, with a conversion yield of 98% (w/w), a volumetric productivity of 12.3 g l(-1) h(-1), and a specific productivity of 1.23 g g-cells(-1) h(-1), which were 18%, 2.5-, and 2.5-fold higher than those of whole wild-type S. maltophilia cells, respectively. This is the first report of 10-hydroxystearic acid production using recombinant cells and the concentration and productivity are the highest reported thus far among cells.

A recombinant enzyme from Lysinibacillus fusiformis was expressed, purified, and identified as an oleate hydratase because the hydration activity of the enzyme was the highest for oleic acid (with a k (cat) of 850 min(-1) and a K (m) of 540 μM), followed by palmitoleic acid, γ-linolenic acid, linoleic acid, myristoleic acid, and α-linolenic acid. The optimal reaction conditions for the enzymatic production of 10-hydroxystearic acid were pH 6.5, 35 °C, 4% (v/v) ethanol, 2,500 U ml(-1) (8.3 mg ml(-1)) of enzyme, and 40 g l(-1) oleic acid. Under these conditions, 40 g l(-1) (142 mM) oleic acid was converted into 40 g l(-1) (133 mM) 10-hydroxystearic acid for 150 min, with a molar yield of 94% and a productivity of 16 g l(-1) h(-1), and olive oil hydrolyzate containing 40 g l(-1) oleic acid was converted into 40 g l(-1) 10-hydroxystearic acid for 300 min, with a productivity of 8 g l(-1) h(-1).

Fusarium moniliforme var. subglutinans was selected from among 100 strains of fungi for producing ginsenoside F(1) from ginsenoside Rg(1). The enzyme responsible was purified as a single 85 kDa band with a specific activity of 136 U mg(-1). It hydrolysed glucose-linked ginsenosides Rb(1), Rd and Rg(1) but not for other monosaccharide-linked ginsenosides, Rb(2), Rc, R(1), and Re. Under the optimum conditions of pH 6.0, 50 °C, 30 U l(-1) of enzyme, and 5 mg Rg(1) ml(-1), 4 mg F(1) ml(-1) was produced after 4 h, with a molar yield of 100% and a productivity of 1 g l(-1) h(-1). This represents the highest productivity and conversion yield of F(1) yet reported.

Biological hydrogen production by the green alga, Chlamydomonas reinhardtii can be induced in conditions of sulfur deprivation. In this study, we investigated the repeated and enhanced hydrogen production afforded by the re-addition of sulfate with monitoring of pH and concentration of chlorophyll and sulfate. Without adjustment of the pH, the optimal concentration of re-added sulfate was 30 μM for the hydrogen production. By the re-addition of 30 μM of sulfate and the adjustment of the pH during 4 cycles of repeated production, we obtained the maximum amount of 789 ml H2 l−1 culture, which is 3.4 times higher than that of one batch production without adjustment of pH, 236 ml H2 l−1 culture. This means that the enhancement of the hydrogen production can be achieved by the careful control of the sulfate re-addition and pH adjustment in the sulfur deprived culture.

Biomass of the green algae has been recently an attractive feedstock source for bio-fuel production because the algal carbohydrates can be derived from atmospheric CO2 and their harvesting methods are simple. We utilized the accumulated starch in the green alga Chlamydomonas reinhardtii as the sole substrate for fermentative hydrogen (H2) production by the hyperthermophilic eubacterium Thermotoga neapolitana. Because of possessing amylase activity, the bacterium could directly ferment H2 from algal starch with H2 yield of 1.8–2.2 mol H2/mol glucose and the total accumulated H2 level from 43 to 49% (v/v) of the gas headspace in the closed culture bottle depending on various algal cell-wall disruption methods concluding sonication or methanol exposure. Attempting to enhance the H2 production, two pretreatment methods using the heat-HCl treatment and enzymatic hydrolysis were applied on algal biomass before using it as substrate for H2 fermentation. Cultivation with starch pretreated by 1.5% HCl at 121 °C for 20 min showed the total accumulative H2 yield of 58% (v/v). In other approach, enzymatic digestion of starch by thermostable α-amylase (Termamyl) applied in the SHF process significantly enhanced the H2 productivity of the bacterium to 64% (v/v) of total accumulated H2 level and a H2 yield of 2.5 mol H2/mol glucose. Our results demonstrated that direct H2 fermentation from algal biomass is more desirably potential because one bacterial cultivation step was required that meets the cost-savings, environmental friendly and simplicity of H2 production.

The gene cluster for spectinomycin biosynthesis from Streptomyces spectabilis was analyzed completely and registered under the accession number EU255259 at the National Center for Biotechnology Information. Based on sequence analysis, spcM of the S. spectabilis cluster is the only methyltransferase candidate required for methylation in spectinomycin biosynthesis. It has high similarity with the conserved domain of DNA methylase, which contains both N-4 cytosine–specific DNA methylases and N-6 adenine–specific DNA methylases. Nucleotide methylation can provide antibiotic resistance, such as 16S rRNA methyltransferase, to Enterobacteriaceae. We therefore tested a hypothesis that SpcM offers aminoglycoside resistance to bacteria. The heterologous expression of spcM in Escherichia coli and S. lividans enhanced resistance against spectinomycin and its relative aminoglycoside antibiotics. We therefore propose that one of the functions of SpcM may be conferring aminoglycoside antibiotic resistance to cells.

A mobile robot localization problem can be classified into following three sub-problems as an observation likelihood model, a motion model and a filtering technique. So far, we have developed the range sensor based, integrated localization scheme, which can be used in human-coexisting real environment such as a science museum and office buildings. From those experiences, we found out that there are several significant issues to be solved. In this paper, we focus on three key issues, and then illustrate our solutions to the presented problems. Three issues are listed as follows: (1) Investigation of design requirements of a desirable observation likelihood model, and performance analysis of our design (2) Performance evaluation of the localization result by computing the matching error (3) The semi-global localization scheme to deal with localization failure due to abrupt wheel slippage In this paper, we show the significance of each concept, developed solutions and the experimental results. Experiments were carried out in a typical modern building environment, and the results clearly show that the proposed solutions are useful to develop practical and integrated localization schemes.

Retinoids are a class of compounds that are forms of vitamin A and include retinal, retinol, retinoic acid, and retinyl ester. Retinal is involved in visual cycle, retinol has anti-infective, anticancer, antioxidant, and anti-wrinkle functions, and retinoic acid is used to treat acne and cancer. Retinol, retinoic acid, and retinyl ester are used in cosmetic and pharmaceutical industries. In this article, the biochemical properties and active sites and reaction mechanisms of retinoid-converting enzymes in animals and bacteria, including retinol dehydrogenase, alcohol dehydrogenase, aldo-keto reductase, and aldehyde dehydrogenase, are reviewed. The production of retinoids, using retinoid-producing enzymes and metabolically engineered cells, is also described. Uncharacterized bacterial proteins are suggested as retinoid-converting enzymes, and the production of retinoids using metabolically engineered cells is proposed as a feasible method.

Secondary metabolites of plants are often difficult to synthesize in high yields because of the large complexity of the biosynthetic pathways and challenges encountered in the functional expression of the required biosynthetic enzymes in microbial cells. In this study, the biosynthesis of plant oxylipins--a family of oxygenated unsaturated carboxylic acids--was explored to enable a high-yield production through a designed microbial synthetic system harboring a set of microbial enzymes (i.e., fatty acid double-bond hydratases, alcohol dehydrogenases, Baeyer-Villiger monooxygenases, and esterases) to produce a variety of unsaturated carboxylic acids from γ-linolenic acid. The whole cell system of the recombinant Escherichia coli efficiently produced (6Z,9Z)-12-hydroxydodeca-6,9-dienoic acid (7), (Z)-9-hydroxynon-6-enoic acid (15), (Z)-dec-4-enedioic acid (17), and (6Z,9Z)-13-hydroxyoctadeca-6,9-dienoic acid (2). This study demonstrated that various secondary metabolites of plants can be produced by implementing artificial biosynthetic pathways into whole-cell biocatalysis.

The aim of this study is the first time demonstration of cis-12 regio-selective linoleate double-bond hydratase. Hydroxylation of fatty acids, abundant feedstock in nature, is an emerging alternative route for many petroleum replaceable products thorough hydroxy fatty acids, carboxylic acids, and lactones. However, chemical route for selective hydroxylation is still quite challenging owing to low selectivity and many environmental concerns. Hydroxylation of fatty acids by hydroxy fatty acid forming enzymes is an important route for selective biocatalytic oxyfunctionalization of fatty acids. Therefore, novel fatty acid hydroxylation enzymes should be discovered. The two hydratase genes of Lactobacillus acidophilus were identified by genomic analysis, and the expressed two recombinant hydratases were identified as cis-9 and cis-12 double-bond selective linoleate hydratases by in vitro functional validation, including the identification of products and the determination of regio-selectivity, substrate specificity, and kinetic parameters. The two different linoleate hydratases were the involved enzymes in the 10,13-dihydroxyoctadecanoic acid biosynthesis. Linoleate 13-hydratase (LHT-13) selectively converted 10 mM linoleic acid to 13S-hydroxy-9(Z)-octadecenoic acid with high titer (8.1 mM) and yield (81%). Our study will expand knowledge for microbial fatty acid-hydroxylation enzymes and facilitate the designed production of the regio-selective hydroxy fatty acids for useful chemicals from polyunsaturated fatty acid feedstocks.

The validamycin biosynthetic gene cluster was isolated from Streptomyces hygroscopicus var. limoneus KTCC 1715 (IFO 12704) using a pair of degenerated PCR primers designed from the sequence of AcbC, 2-epi-5-epi-valiolone synthase in the acarbose biosynthesis. The nucleotide sequence analysis of the 37-kb DNA region revealed 22 complete ORFs including vldA, the acbC ortholog. Located around vldA, vldB to K were predicted to encode adenyltransferase, kinase, ketoreductase (or epimerase/dehydratase), glycosyltransferase, aminotransferase, dehydrogenase, phosphatase/phosphomutase, glycosyl hydrolase, transport protein, and glycosyltransferase, respectively. Apparently absent were any regulatory components within the sequenced region. The disruption of vldA abolished the validamycin biosynthesis and the plasmid-based complementation with vldABC restored production to the vldA-mutant; this substantiated that vldABC are essential to validamycin biosynthesis. This finding enabled us to discover the complete validamycin biosynthetic cluster. The cosmid clone of pJWS3001 harboring the 37-kb DNA region conferred validamycin-accumulation to Streptomyces lividans, indicating that the entire gene cluster of validamycin biosynthesis had been isolated. Additionally, Streptomyces albus, transformed with pJWS3001, produced a high level of α-glucosidase inhibitory activity in a R2YE liquid culture, which highlights the portability of the cluster within Streptomyces. The product of vldI was characterized as a glucoamylase (kcat, 32 s− 1; Km, 5 mg/ml of starch) that does not play any apparent role in the validamycin biosynthesis. In order to characterize the upstream region, a vldW knockout was achieved via gene-replacement. A phenotypic study of the resulting mutant revealed that vldW is not essential for the host's ability to control Pellicularia filamentosa growth. The current information suggests that vldA to vldH is the genetic region essential to validamycin biosynthesis. This promises excellent opportunities to elucidate biosynthetic route(s) to the validamycin complex and to engineer the pathway for industrial application.

Aspergillus oryzae KACC 40247 was selected from among 60 fungal strains as an effective 7,8,4'-trihydroxyisoflavone (8-hydroxydaidzein)-producing fungus. The optimal culture conditions for production by this strain in a 7-L fermentor were found to be 30 °C, pH 6, and 300 rpm. Under these conditions, A. oryzae KACC 40247 produced 62 mg/L of 8-hydroxydaidzein from soybean extract in 30 h, with a productivity of 2.1 mg/L/h. These are the highest production and productivity for 8-hydroxydaidzein ever reported. To increase production, several concentrations of daidzin and of daidzein as precursor were added at several culture times. The optimal addition time and concentration for daidzin were 12 h and 1,248 mg/L, and those for daidzein were 12 h and 254 mg/L respectively. Maximum production and productivity for 8-hydroxydaidzein with the addition of daidzein were 95 mg/L and 3.2 mg/L/h respectively, and those with the addition of daidzin were 160 mg/L and 4.4 mg/L/h respectively.

A D-allulose 3-epimerase from Flavonifractor plautii was cloned and expressed in Escherichia coli and Corynebacterium glutamicum. The maximum activity of the enzyme purified from recombinant E. coli cells was observed at pH 7.0, 65 degrees C, and 1 mM Co2+ with a half-life of 40 min at 65 degrees C, K-m of 162 mM, and k(cat) of 25280 1/s. For increased D-allulose production, recombinant C. glutamicum cells were permeabilized via combined treatments with 20 mg/L penicillin and 10% (v/v) toluene. Under optimized conditions, 10 g/L permeabilized cells produced 235 g/L D-allulose from 750 g/L D-fructose after 40 min, with a conversion rate of 31% (w/w) and volumetric productivity of 353 g/L/h, which were 1.4- and 2.1-fold higher than those obtained for nonpermeabilized cells, respectively.

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH 6.5, 30 °C, 15 g l(-1) ricinoleic acid, 9 mg ml(-1) of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g l(-1) 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g l(-1) h(-1). The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.

L-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the L-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l L-ribose from 300 g/l L-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co(2+), with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h(-1) produced an average of 100 g/l L-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for L-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from L-arabinose as the substrate.

Optimal hydrolytic activity of β-glucosidase from Dictyoglomus turgidum for the ginsenoside Rd was at pH 5.5 and 80 °C, with a half-life of ~11 h. The enzyme hydrolysed β-linked, but not α-linked, sugar moieties of ginsenosides. It produced the rare ginsenosides, aglycon protopanaxadiol (APPD), compounds Y, and Mc, via three unique transformation pathways: Rb(1) → Rd → F(2) → compound K → APPD, Rb(2) → compound Y, and Rc → compound Mc. The enzyme converted 0.5 mM Rb(2) and 0.5 mM Rc to 0.5 mM compound Y and 0.5 mM compound Mc after 3 h, respectively, with molar conversion yields of 100 %.

A putative D-lyxose isomerase from Dictyoglomus turgidum was purified with a specific activity of 19 U/mg for D-lyxose isomerization by heat treatment and affinity chromatography. The native enzyme was estimated as a 42 kDa dimer by gel-filtration chromatography. The activity of the enzyme was highest for D-lyxose, suggesting that it is a D-lyxose isomerase. The maximum activity of the enzyme was at pH 7.5 and 75°C in the presence of 0.5 mM Co(2+), with a half-life of 108 min, K(m) of 39 mM, and k(cat) of 3,570 1/min. The enzyme is the most thermostable D-lyxose isomerase among those characterized to date. It converted 500 g D-xylulose/l to 380 g D-lyxose/l after 2 h. This is the highest concentration and productivity of D-lyxose reported thus far.

Three new polyketides, cyathuscavins A (1), B (2), and C (3) were isolated from the mycelium culture of Cyathus stercoreus. The structures of the compounds were elucidated on the basis of NMR and mass spectroscopic data. Antioxidant activities of the compounds were evaluated by the scavenging ability against ABTS+, DPPH, and superoxide anion radicals. Cyathuscavins A–C showed significant antioxidant activity comparable to those of reference antioxidants, BHA and Trolox. Cyathuscavins A–C protected supercoiled plasmid DNA from Fe2+/H2O2-induced breakage.

Design and evaluation of new illumination system utilizing white light emitting diode (LED) and plastic light-guide for letter-size (216mm width) document scanner and copier have been performed. We investigated the best condition for illumination on document plane through the optical analysis of scanner imaging system. To compensate the relative illumination fall-off caused by imaging lens, illumination on main-direction was designed to edge enhanced distribution. The illumination on sub-direction was designed to flat-top shape for stable performance against mechanical displacement of optics. The light-guide was designed dual-angled sidewalls for the decrease of light leakage and the increase of the degree of freedom for illumination shaping. The rear area of a light-guide is formed with crossed micro-prism patterns to axial direction to induce scattering of guided light, and the front area is formed with lens-shape for efficient lighting. Total 12 parameters were used for the optimization of design, and the various relationships between parameters and performances were also investigated. The optical characteristics of the proposed illumination system utilizing a white LED and light-guide, fabricated by mold process, were also experimentally evaluated in terms of illumination distribution against main- and sub-direction on image plane. They showed excellent agreement with simulated result and showed good performance of uniformity above 91%, and 89% in sub-direction and main-direction, respectively.

Three new polyketides, cyathuscavins A (1), B (2), and C (3) were isolated from the mycelium culture of Cyathus stercoreus. The structures of the compounds were elucidated on the basis of NMR and mass spectroscopic data. Antioxidant activities of the compounds were evaluated by the scavenging ability against ABTS+, DPPH, and superoxide anion radicals. Cyathuscavins A–C showed significant antioxidant activity comparable to those of reference antioxidants, BHA and Trolox. Cyathuscavins A–C protected supercoiled plasmid DNA from Fe2+/H2O2-induced breakage.

Statement of problem Different assessment methods have been used to measure the accuracy of implant impression techniques; therefore, the readers should understand the benefits and limitations of each assessment method used. Purpose The purpose of this systematic review was to classify the implant impression studies by the assessment methods and techniques used and to understand the characteristics of each assessment method. The results of published studies were also analyzed to draw meaningful conclusions about the accuracy of the implant impressions. Material and methods An electronic search of the MEDLINE/PubMed database was performed in February 2013 using specific search terms and predetermined criteria to identify and assess laboratory studies of the accuracy of implant impression techniques. A final list of articles deemed to be of interest was comprehensively reviewed by 2 reviewers to ensure that these were suitable for the purpose of this review. The results of the current review were also compared with results from a previous systematic review. Results A total of 56 studies met the inclusion criteria for this review. Thirty-seven studies measured the amount of linear distortion, and 17 studies compared the angular change to assess the accuracy. Most linear or angular distortions were only measured in 2 dimensions, and 3-dimensional analysis was rare. More than 80% of the studies compared nonsplinting versus splinting, direct versus indirect techniques, and different impression materials. Conclusions In recent publications, the direct or splint technique showed more accurate results than the indirect or nonsplinted technique. In contrast to external connection implants, inconsistent results were reported for internal connection implants.