• Title/Summary/Keyword: substrate binding

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Molecular Cloning and Sequence Analysis of Coelomic Cytolytic Factor-like Gene from the Midgut of the Earthworm, Eisenia Andrei (줄지렁이 중장에서 분리한 Coelomic cytolytic factor-유사 유전자의 클로닝 및 염기서열 분석에 관한 연구)

  • Baek, Nam Sook;Lee, Myung-Sik;Park, Sang-Kil;Kim, Dae-hwan;Tak, Eun-Sik;Ahn, Chi-Hyun;Sun, Zhenjun;Park, Soon Cheol
    • Journal of the Korea Organic Resources Recycling Association
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    • v.16 no.4
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    • pp.64-73
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    • 2008
  • The cDNA of CCF (coelomic cytolytic factor)-like gene (EC 3.2.1.16), a kind of glycosyl hydorlase, was isolated and cloned from the midgut of the earthworm Eisenia anderi. The size of nucleotide sequence appeared to be 1,152 bp and its predicted coding region was composed of 384 amino acid residues including the initiation methionine. The 17 residues at N-terminal end in the deduced amino acid sequence were regarded to be a signal peptide. Based on the amino acid sequence analysis, it appeared that this CCF-like protein could belong to glycosyl hydrolase family 16 (GHF16) and showed a high sequence homology of about 79~99% with CCF and CCF-like proteins from other earthworm species. The CCFs and CCF-like proteins from various earthworm species exhibited a 100% homology in the polysacchride-binding motif and glucanase motif. It has been reported that the CCFs isolated from E. fedita appeared to show a broader pattern recognition specificity than those from other earthworm species because this species resides in decaying organic matter showing very high microbial activity, implying that CCF-like protein isolated in this study from E. andrei might exhibit a broad substrate specificity that is a useful characteristic for industrial application. A phylogenetic analysis using the deduced amino acid sequences of CCF-related proteins through the BLASTX revealed that GHF16 families could be divided into three groups of metazoa, viriplantae and eubacteria subfamily. Subsequently the CCF-related proteins of metazoa subfamily could clearly be subgroup into lophotrochozoan and edysozoan type including a deuterostome origin. Further understanding of the biological properties of E. andrei CCF-like protein should be addressed to regulate the ${\beta}$-D-glucan hydrolysis and production for the industrial uses.

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Growth and Characterization of AgGa$Se_2$ Single Crystal Thin Films by Hot Wall Epitaxy (Hot Wall Epitaxy (HWE)법에 의한 AgGa$Se_2$ 단결정 박막 성장과 특성)

  • Hong, Gwang-Jun;Lee, Gwan-Gyo;Park, Jin-Seong
    • Korean Journal of Materials Research
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    • v.11 no.5
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    • pp.419-426
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    • 2001
  • The stochiometric $AgGaSe_2$ polycrystalline mixture of evaporating materials for the $AgGaSe_2$ single crystal thin film was prepared from horizontal furnace. To obtain the single crystal thin films, $AgGaSe_2$ mixed crystal and semi-insulating GaAs(100) wafer were used as source material and substrate for the Hot Wall Epitaxy (HWE) system, respectively. The source and substrate temperature were fixed at$ 630^{\circ}C$ and $420^{\circ}C$, respectively. The thickness of grown single crystal thin films is 2.1$\mu\textrm{m}$. The single crystal thin films were investigated by photoluminescence and double crystal X-ray diffraction(DCXD) measurement. The carrier density and mobility of AgGaSe$_2$ single crystal thin films measured from Hall effect by van der Pauw method are $4.89\Times10^{17}$ cm$^{-3}$ , 129cm2/V.s at 293K, respectively. From the Photocurrent spectrum by illumination of perpendicular light on the c-axis of the AgGaSe$_2$ single crystal thin film, we have found that the values of spin orbit splitting $$\Delta$S_{o}$ and the crystal field splitting $\Delta$C$_{r}$, were 0.1762eV and 0.2474eV at 10K, respectively. From the photoluminescence measurement of AgGaSe$_2$ single crystal thin film, we observed free excision (EX) observable only in high quality crystal and neutral bound exciton ($D^{o}$ , X) having very strong peak intensity. And, the full width at half maximum and binding energy of neutral donor bound excition were 8mev and 14.1meV, respectively. By Haynes rule, an activation energy of impurity was 141 meV.ion energy of impurity was 141 meV.

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The study of growth and characterization of CuGaTe$_2$single crystal thin films by hot wall epitaxy (Hot wall epitaxy(HWE) 방법에 의한 CuGaTe$_2$단결정 박막 성장과 특성에 관한 연구)

  • 홍광준;이관교;이상열;유상하;정준우;정경아;백형원;방진주;신영진
    • Journal of the Korean Crystal Growth and Crystal Technology
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    • v.10 no.6
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    • pp.425-433
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    • 2000
  • The stochiometric mix of evaporating materials for the $CuGaTe_2$single crystal thin films was prepared from horizontal furnance. Using extrapolation method of X-ray diffraction patterns for the $CuGaTe_2$polycrystal, it was found tetragonal structure whose lattice constant $a_0 and c_0$ were 6.025 $\AA$ and 11.931 $\AA$, respectively. To obtain the single crystal thin films, $CuGaTe_2$mixed crystal was deposited on throughly etched semi-insulator GaAs(100) substrate by the Hot Wall Epitaxy (HWE) system. The source and substrate temperature were $670^{\circ}C$ and $410^{\circ}C$ respectively, and the thickness of the single crystal thin films is 2.1$\mu\textrm{m}$. The crystalline structure of single crystal thin films was investigated by the photoluminescence and double crystal X-ray diffraction (DCXD). Hall effect on this sample was measured by the method of van der Pauw and studied on carrier density and mobility dependence on temperature. The carrier density and mobility of $CuGaTe_2$single crystal thin films deduced from Hall data are $8.72{\times}10{23}$$\textrm m^3$, $3.42{\times}10^{-2}$ $\textrm m^2$/V.s at 293K, respectively. From the photocurrent spectrum by illumination of perpendicular light on the c-axis of the $CuGaTe_2$single crystal thin film, we have found that the values of spin orbit coupling $\Delta$s.o and the crystal field splitting $\Delta$cr were 0.0791 eV and 0.2463 eV at 10 K, respectively. From the PL spectra at 10 K, the peaks corresponding to free bound excitons and D-A pair and a broad emission band due to SA is identified. The binding energy of the free excitons are determined to be 0.0470 eV and the dissipation energy of the donor-bound exciton and acceptor-bound exciton to be 0.0490 eV, 0.0558 eV, respectively.

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Protein Engineering of Flavin-containing Monooxygenase from Corynebacterium glutamicum for Improved Production of Indigo and Indirubin (인디고와 인디루빈의 생산을 증대하기 위한 플라빈-함유 모노옥시게나제의 단백질공학)

  • Jung, Hye Sook;Jung, Hae Bin;Kim, Hee Sook;Kim, Chang Gyeom;Lee, Jin Ho
    • Journal of Life Science
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    • v.28 no.6
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    • pp.656-662
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    • 2018
  • Flavin-containing monooxygenases from Corynebacterium (cFMOs) were mutagenized based on homology modeling to develop variants with an enhanced indigoid production capability. The four mutants, F170Y, A210G, A210S, and T326S, which fused to a maltose-binding protein (MBP), were constructed, and their biochemical properties were characterized. Of these, purified MBP-T326S required a higher concentration of exogenous FAD (100 mM) than the wild-type MBP-cFMO for optimal activity and showed a 3.8-fold increase in the $k_{cat}/K_m$ value at $100{\mu}M$ FAD compared to that of MBP-cFMO at $2{\mu}M$ FAD. The indole oxygenase activities of MBP-T326S decreased to 63-77% compared to that of the MBP-cFMO In addition, MBP-T326S displayed a very low level of futile NADPH oxidase activities (21-24%) in the absence of a substrate. Mutant proteins except for T326S displayed similar $K_m$ and increased $k_{cat}/K_m$ values compared to the wild-type. MBP-F170Y and -A210S mutants showed elevated indole oxygenase activity higher than 3.1- and 2.9-fold, respectively, in comparison with MBP-cFMO. When indigoid production was carried out in LB broth with 2.5 g/l of tryptophan, Escherichia coli expressing cFMO produced 684 mg/l of indigo and 104 mg/l of indirubin, while cells harboring T326S produced 1,040 mg/l of indigo and 112 mg/l of indirubin. The results indicate that the production of indigo was 13% higher when compared to a previous report in which an E. coli expressing FMO from Methylophaga produced 920 mg/l of indigo. The protein engineering of cFMO based on homology modeling provided a more rational strategy for developing indigoid-producing strains.

Growth and characterization ofZnIn$_2S_4$ single crystal thin film using hot wall epitaxy method (Hot Wall Epitaxy(HWE)에 의한 ZnIn$_2S_4$ 단결정 박막 성장과 특성)

  • 최승평;홍광준
    • Journal of the Korean Crystal Growth and Crystal Technology
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    • v.11 no.4
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    • pp.138-147
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    • 2001
  • The stochiometric mixtures mixture of evaporating materials for the $ZnIn_{2}S_{4}$ single crystal thin film was prepared from horizontal furnace. To obtain the $ZnIn_{2}S_{4}$ single crystal thin film, $ZnIn_{2}S_{4}$ mixed crystal was deposited on throughly etched semi-insulting GaAs(100) in the Hot Wall Epitaxy(HWE) system. The sourceand substrate temperature were $610^{\circ}C$ and $450^{\circ}C$, respectively and the growth rate of the $ZnIn_{2}S_{4}$ single crystal thin film was about 0.5$\mu\textrm{m}$/hr. The crystalline structure of $ZnIn_{2}S_{4}$ single crystal thin film was investigated by photoluminescence and double crystal X-ray diffraction (DCXD) measurement. The carrier density and mobility of $ZnIn_{2}S_{4}$ single crystal thin film measured from Hal effect by van der Pauw method are $8.51{\times}10^{17}{\textrm}{cm}^{-3}$, 291$\textrm{cm}^2$/V.s at $293^{\circ}$K, respectively. From the photocurrent spectrum by illumination of perpendicular light on the c-axis of the $ZnIn_{2}S_{4}$ single crystal thin film, we have found that the values of spin orbit splitting $\Delta$So and the crystal filed splitting DCr were 0.0148eV and 0.1678 eV at $10^{\circ}$K, respectively. From the photoluminescence measurement of $ZnIn_{2}S_{4}$ single crystal thin film, we observed free excition($E_{X}$) typically observed only in high quality crystal and neutral donor bound exicton ($D^{\circ}$, X) having very strong peak intensity. The full width at half maximum and binding energy of neutral donor bound excition were 9meV and 26meV, respectively. The activation energy of impurity measured by Haynes rule was 130meV.

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The study of growth and characterization of CuGaSe$_2$ single crystal thin films by hot wall epitaxy (HWE(Hot wall epitaxy)에 의한 CuGaSe$_2$단결정 박막 성장과 특성에 관한 연구)

  • 홍광준;백형원
    • Journal of the Korean Crystal Growth and Crystal Technology
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    • v.10 no.3
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    • pp.189-198
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    • 2000
  • The stochiometric mixture of evaporating materials for the $CuGaSe_2$single crystal thin films were prepared from horizontal furnace. Using extrapolation method of X-ray diffraction patterns for the polycrystal $CuGaSe_2$, it was found tetragonal structure whose lattice constant $a_0}$ and $c_0$ were 5.615 $\AA$ and 11.025 $\AA$, respectively. To obtains the single crystal thin films, $CuGaSe_2$mixed crystal was deposited on throughly etched GaAs(100) by the Hot Wall Epitaxy (HWE) system. The source and substrate temperature were $610^{\circ}C$ and $450^{\circ}C$ respectively, and the growth rate of the single crystal thin films was about 0.5$\mu\textrm{m}$/h. The crystalline structure of single crystal thin films was investigated by the double crystal X-ray diffraction (DCXD). Hall effect on this sample was measured by the method of van der Pauw and studied on carrier density and mobility depending on temperature. From Hall data, the mobility was likely to be decreased by pizoelectric scattering in the temperature range 30 K to 150 K and by polar optical scattering in the temperature range 150 K to 293 K. The optical energy gaps were found to be 1.68 eV for CuGaSe$_2$sing1e crystal thin films at room temperature. The temperature dependence of the photocurrent peak energy is well explained by the Varshni equation then the constants in the Varshni equation are given by $\alpha$ = $9.615{\times}10^{-4}$eV/K, and $\beta$ = 335 K. From the photocurrent spectra by illumination of polarized light of the $CuGaSe_2$single crystal thin films. We have found that values of spin orbit coupling $\Delta$So and crystal field splitting $\Delta$Cr was 0.0900 eV and 0.2498 eV, respectively. From the PL spectra at 20 K, the peaks corresponding to free bound excitons and D-A pair and a broad emission band due to SA is identified. The binding energy of the free excitons are determined to be 0.0626 eV and the dissipation energy of the acceptor-bound exciton and donor-bound exciton to be 0.0352 eV, 0.0932 eV, respectively.

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Overexpression and Activity Analysis of Cystathionine γ-Lyase Responsible for the Biogenesis of H2S Neurotransmitter (새로운 신경전달물질 H2S 발생 효소, cystathionine γ-lyase의 대량발현 조건과 활성측정)

  • Kim, Kyoung-Ran;Byun, Hae-Jung;Cho, Hyun-Nam;Kim, Jung-Hyun;Yang, Seun-Ah;Jhee, Kwang-Hwan
    • Journal of Life Science
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    • v.21 no.1
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    • pp.119-126
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    • 2011
  • There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

Sesquiterpenoids Bioconversion Analysis by Wood Rot Fungi

  • Lee, Su-Yeon;Ryu, Sun-Hwa;Choi, In-Gyu;Kim, Myungkil
    • 한국균학회소식:학술대회논문집
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    • 2016.05a
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    • pp.19-20
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    • 2016
  • Sesquiterpenoids are defined as $C_{15}$ compounds derived from farnesyl pyrophosphate (FPP), and their complex structures are found in the tissue of many diverse plants (Degenhardt et al. 2009). FPP's long chain length and additional double bond enables its conversion to a huge range of mono-, di-, and tri-cyclic structures. A number of cyclic sesquiterpenes with alcohol, aldehyde, and ketone derivatives have key biological and medicinal properties (Fraga 1999). Fungi, such as the wood-rotting Polyporus brumalis, are excellent sources of pharmaceutically interesting natural products such as sesquiterpenoids. In this study, we investigated the biosynthesis of P. brumalis sesquiterpenoids on modified medium. Fungal suspensions of 11 white rot species were inoculated in modified medium containing $C_6H_{12}O_6$, $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ for 20 days. Cultivation was stopped by solvent extraction via separation of the mycelium. The metabolites were identified as follows: propionic acid (1), mevalonic acid lactone (2), ${\beta}$-eudesmane (3), and ${\beta}$-eudesmol (4), respectively (Figure 1). The main peaks of ${\beta}$-eudesmane and ${\beta}$-eudesmol, which were indicative of sesquiterpene structures, were consistently detected for 5, 7, 12, and 15 days These results demonstrated the existence of terpene metabolism in the mycelium of P. brumalis. Polyporus spp. are known to generate flavor components such as methyl 2,4-dihydroxy-3,6-dimethyl benzoate; 2-hydroxy-4-methoxy-6-methyl benzoic acid; 3-hydroxy-5-methyl phenol; and 3-methoxy-2,5-dimethyl phenol in submerged cultures (Hoffmann and Esser 1978). Drimanes of sesquiterpenes were reported as metabolites from P. arcularius and shown to exhibit antimicrobial activity against Gram-positive bacteria such as Staphylococcus aureus (Fleck et al. 1996). The main metabolites of P. brumalis, ${\beta}$-Eudesmol and ${\beta}$-eudesmane, were categorized as eudesmane-type sesquiterpene structures. The eudesmane skeleton could be biosynthesized from FPP-derived IPP, and approximately 1,000 structures have been identified in plants as essential oils. The biosynthesis of eudesmol from P. brumalis may thus be an important tool for the production of useful natural compounds as presumed from its identified potent bioactivity in plants. Essential oils comprising eudesmane-type sesquiterpenoids have been previously and extensively researched (Wu et al. 2006). ${\beta}$-Eudesmol is a well-known and important eudesmane alcohol with an anticholinergic effect in the vascular endothelium (Tsuneki et al. 2005). Additionally, recent studies demonstrated that ${\beta}$-eudesmol acts as a channel blocker for nicotinic acetylcholine receptors at the neuromuscular junction, and it can inhibit angiogenesis in vitro and in vivo by blocking the mitogen-activated protein kinase (MAPK) signaling pathway (Seo et al. 2011). Variation of nutrients was conducted to determine an optimum condition for the biosynthesis of sesquiterpenes by P. brumalis. Genes encoding terpene synthases, which are crucial to the terpene synthesis pathway, generally respond to environmental factors such as pH, temperature, and available nutrients (Hoffmeister and Keller 2007, Yu and Keller 2005). Calvo et al. described the effect of major nutrients, carbon and nitrogen, on the synthesis of secondary metabolites (Calvo et al. 2002). P. brumalis did not prefer to synthesize sesquiterpenes under all growth conditions. Results of differences in metabolites observed in P. brumalis grown in PDB and modified medium highlighted the potential effect inorganic sources such as $C_4H_{12}N_2O_6$, $KH_2PO_4$, $MgSO_4$, and $CaCl_2$ on sesquiterpene synthesis. ${\beta}$-eudesmol was apparent during cultivation except for when P. brumalis was grown on $MgSO_4$-free medium. These results demonstrated that $MgSO_4$ can specifically control the biosynthesis of ${\beta}$-eudesmol. Magnesium has been reported as a cofactor that binds to sesquiterpene synthase (Agger et al. 2008). Specifically, the $Mg^{2+}$ ions bind to two conserved metal-binding motifs. These metal ions complex to the substrate pyrophosphate, thereby promoting the ionization of the leaving groups of FPP and resulting in the generation of a highly reactive allylic cation. Effect of magnesium source on the sesquiterpene biosynthesis was also identified via analysis of the concentration of total carbohydrates. Our current study offered further insight that fungal sesquiterpene biosynthesis can be controlled by nutrients. To profile the metabolites of P. brumalis, the cultures were extracted based on the growth curve. Despite metabolites produced during mycelia growth, there was difficulty in detecting significant changes in metabolite production, especially those at low concentrations. These compounds may be of interest in understanding their synthetic mechanisms in P. brumalis. The synthesis of terpene compounds began during the growth phase at day 9. Sesquiterpene synthesis occurred after growth was complete. At day 9, drimenol, farnesol, and mevalonic lactone (or mevalonic acid lactone) were identified. Mevalonic acid lactone is the precursor of the mevalonic pathway, and particularly, it is a precursor for a number of biologically important lipids, including cholesterol hormones (Buckley et al. 2002). Farnesol is the precursor of sesquiterpenoids. Drimenol compounds, bi-cyclic-sesquiterpene alcohols, can be synthesized from trans-trans farnesol via cyclization and rearrangement (Polovinka et al. 1994). They have also been identified in the basidiomycota Lentinus lepideus as secondary metabolites. After 12 days in the growth phase, ${\beta}$-elemene caryophyllene, ${\delta}$-cadiene, and eudesmane were detected with ${\beta}$-eudesmol. The data showed the synthesis of sesquiterpene hydrocarbons with bi-cyclic structures. These compounds can be synthesized from FPP by cyclization. Cyclic terpenoids are synthesized through the formation of a carbon skeleton from linear precursors by terpene cyclase, which is followed by chemical modification by oxidation, reduction, methylation, etc. Sesquiterpene cyclase is a key branch-point enzyme that catalyzes the complex intermolecular cyclization of the linear prenyl diphosphate into cyclic hydrocarbons (Toyomasu et al. 2007). After 20 days in stationary phase, the oxygenated structures eudesmol, elemol, and caryophyllene oxide were detected. Thus, after growth, sesquiterpenes were identified. Per these results, we showed that terpene metabolism in wood-rotting fungi occurs in the stationary phase. We also showed that such metabolism can be controlled by magnesium supplementation in the growth medium. In conclusion, we identified P. brumalis as a wood-rotting fungus that can produce sesquiterpenes. To mechanistically understand eudesmane-type sesquiterpene biosynthesis in P. brumalis, further research into the genes regulating the dynamics of such biosynthesis is warranted.

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