Design, synthesis, and biological evaluation of 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides as a potential EGR-1 inhibitor for targeted therapy of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.

Effect of Coating Thickness on Abrasion and Cutting Performance of NCD-Coated Ball Endmills on Graphite Machining.

Nano-crystalline diamond (NCD) coating to improve the performance of cutting tools, as the coating thickness varies, the cutting performance and lifespan of the tool varies because the radius of its cutting edge and coating surface roughness are altered. Therefore, an in-depth analysis on the impact of the variations in coating thickness on the cutting tool abrasion and quality of machined surface is necessary. In this study, two NCD ball endmills were coated with 8 and 12 µm thicknesses, and the tool abrasion and roughness of the machined plane were observed after milling. Furthermore, the morphology of the coated surface and abrased cutting edge were observed using a 3D confocal microscope. Consequently, we observed that individual nodules were formed on the continuous aggregates as the coating thickness increased, which increased the coated surface roughness. The two damage modes of the aggregation determined the dominant abrasion that occurred on the cutting edges of both types of coating thicknesses. Delamination and crater wear caused a sharp increase in the roughness of the machined surface. In summary, the increase in coating thickness delayed the delamination of the coating but increased the roughness of the cutting edge, which reduced the machined surface roughness.

Two-Step Glass Molding Process for Forming Glass Edges with Obtuse Angles for Mobile Displays.

The domain of edge displays with 2.5D or 3D curved designs has been expanded to improve user convenience. The currently available 3D cover glass offers a limited curvature radius of at least 5 mm and a curvature less than 88°, due to limitations in the undercuts and formability of parts. The development of a full 3D cover, applicable to next-generation displays, requires cover glass molding technology with a curvature exceeding 90°. Here, a mold design and molding process, which addresses the current limitations by dividing the existing glass molding press (GMP) process into two stages, is proposed. The bending geometry of the glass prepared on the basis of the proposed mold design plan during single-step compression forming and two-step compression forming was predicted using commercial analysis software. A molding product with a curvature radius of 2.5 mm and an angle of curvature of 138.9° was produced when process conditions with bending by up to 180° with no damage were applied during actual forming experiments. Further research on annealing and cooling processes of GMP is expected to enable the design and process implementation to manufacture curved glass with a single curvature of at least 90° and multiple curvatures.

Analysis of Tool Wear and Roughness of Graphite Surfaces Machined Using MCD and NCD-Coated Ball Endmills.

The high-purity G5 graphite material is widely used for glass moulding and provides high hardness and brittleness because it is sintered to fine particles unlike other graphite materials. Hence, tool cutting of a G5 workpiece is performed by local fracture instead of plastic deformation of the machined surface. Although a diamond-coated tool with outstanding hardness is used to machine very hard graphite, the tool shows variability regarding the service life and machining performance depending on the grain size, even in the same machining environment. We investigated the wear and change trend of machined surface roughness considering microcrystalline diamond (MCD) and nanocrystalline diamond (NCD)-coated tools, which are generally used to machine graphite materials, and analysed their relation with coating. For rough machining, the MCD-coated tool, for which the delamination of coating occurred later, showed less wear and improved machined surface roughness. For precision machining, the NCD tool showed less tool wear rate relative to the cutting length, leading to a small difference in the machined surface roughness between the two tools. We conclude that if rough and precision machining processes are performed using the same cutting tool, the MCD-coated tool is advantageous in terms of service life, while the difference in roughness of the final machined surface between the tools is negligible.

Analytical method validation and monitoring of diacetyl in liquors from Korean market.

Diacetyl is a natural fermentation by-product and is an important flavor component of certain liquors. This paper aims to validate the high performance liquid chromatography (HPLC) method based on derivatization with 1,2-diaminobenzene for diacetyl quantification in liquor samples. A limit of quantitation of 0.039 mg/L was obtained. Coefficient regression (R2) of calibration curve for the HPLC-UV method exceeded 0.999, showing adequate linearity on the standard curve. Relative standard deviation values obtained from intraday and interday analysis for precision were 2.5 and 4.1%, respectively. Using the validated method, the contents of diacetyl in liquors (12 types, 389 samples) distributed throughout Korea were monitored. The average diacetyl content of all analyzed liquor samples ranged from trace amounts to 3.655 mg/L (microbrewery beer). The highest average diacetyl content was found in fruit wines (0.432 mg/L), followed by red wine (0.320 mg/L) and general distilled spirits (0.249 mg/L). In takju and yakju, no distinctive effect of sterilization on diacetyl content was found.

Synthesis of organic-inorganic hybrid sols using trifunctional organoalkoxysilanes for dispersion agents.

We investigated the properties of synthetically produced organic-inorganic hybrid coatings by a sol-gel process. The properties of organic-inorganic hybrid materials arise from the synergism between the properties of the individual components. One of the typical way to synthesize the organic-inorganic hybrid materials is to use silica and silanes. A colloidal silica sol was used as an inorganic material. Methyltrimethoxysilane and phenyltrimethoxysilane were used as the trifunctional organoalkoxysilanes. Hybrid sols of colloidal silica and silanes were synthesized as a function of reaction time and methyltrimethoxysilane/phenyltrimethoxysilane ratio by a sol-gel process. Physical properties of sol solutions such as stability, viscosity, and transmittance were investigated. The surface roughness and surface free energy of the coatings were also measured.

Synthesis of organic-inorganic hybrid sols with nano silica particles and organoalkoxysilanes for transparent and high-thermal-resistance coating films using sol-gel reaction.

Organic-inorganic hybrid sols were synthesized from nano silica particles dispersed in water and from organoalkoxysilanes, using the sol-gel reaction. This work focuses on the effects of the three multifunctional organoalkoxysilanes dimethyldimethoxysilane (DMDMS), methyltrimethoxysilane (MTMS), and tetramethoxysilane (TMOS) to form a transparent and high-thermal-resistance coating film. The stability of the hybrid sol was evaluated as a function of the reaction time for 10 d through the variation of the viscosity. The viscosity of the silica/DMDMS and silica/MTMS sol was slightly increased for 10 d. The multifunctional organoalkoxysilanes formed dense silica networks through hydrolysis and condensation reaction, which enhanced the thermal resistance of the coating films. No thermal degradation of the silica/DMDMS sample occurred up to 600 degrees C, and none of the silica/MTMS and silica/TMOS samples occurred either up to 700 degrees C. The organic-inorganic hybrid sols were coated on the glass substrate using a spin-coating procedure. The organic-inorganic hybrid sols formed flat coating films without cracks. The transmittance of the hybrid sol coating films using MTMS and DMDMS was shown to be over 90%. The transmittance of the silica/TMOS sol coating film reacted for 10 d abruptly decreased due to faster gelation. The silica/DMDMS and silica/MTMS hybrid sols formed smooth coating films while the surface roughness of the silica/TMOS coating film markedly increased when the hybrid sol reacted for 10 d. The increase of the surface roughness of the silica/TMOS coating film can be attributed to the degradation of the stability of the hybrid sol and to the loss of transmittance of the coating film. It was confirmed in this study that the use of organic-inorganic hybrid sol can yield transparent and high-thermal-resistance coating films.

A three-step proteolytic cascade mediates the activation of the peptidoglycan-induced toll pathway in an insect.

The recognition of lysine-type peptidoglycans (PG) by the PG recognition complex has been suggested to cause activation of the serine protease cascade leading to the processing of Spätzle and subsequent activation of the Toll signaling pathway. So far, two serine proteases involved in the lysine-type PG Toll signaling pathway have been identified. One is a modular serine protease functioning as an initial enzyme to be recruited into the lysine-type PG recognition complex. The other is the Drosophila Spätzle processing enzyme (SPE), a terminal enzyme that converts Spätzle pro-protein to its processed form capable of binding to the Toll receptor. However, it remains unclear how the initial PG recognition signal is transferred to Spätzle resulting in Toll pathway activation. Also, the biochemical characteristics and mechanism of action of a serine protease linking the modular serine protease and SPE have not been investigated. Here, we purified and cloned a novel upstream serine protease of SPE that we named SAE, SPE-activating enzyme, from the hemolymph of a large beetle, Tenebrio molitor larvae. This enzyme was activated by Tenebrio modular serine protease and in turn activated the Tenebrio SPE. The biochemical ordered functions of these three serine proteases were determined in vitro, suggesting that the activation of a three-step proteolytic cascade is necessary and sufficient for lysine-type PG recognition signaling. The processed Spätzle by this cascade induced antibacterial activity in vivo. These results demonstrate that the three-step proteolytic cascade linking the PG recognition complex and Spätzle processing is essential for the PG-dependent Toll signaling pathway.

Rat liver 10-formyltetrahydrofolate dehydrogenase, carbamoyl phosphate synthetase 1 and betaine homocysteine S-methytransferase were co-purified on Kunitz-type soybean trypsin inhibitor-coupled sepharose CL-4B.

An Asp/His catalytic site of 10-formyltetrahydrofolate dehydrogenase (FDH) was suggested to have a similar catalytic topology with the Asp/His catalytic site of serine proteases. Many studies supported the hypothesis that serine protease inhibitors can bind and modulate the activity of serine proteases by binding to the catalytic site of serine proteases. To explore the possibility that soybean trypsin inhibitor (SBTI) can recognize catalytic sites of FDH and can make a stable complex, we carried out an SBTI-affinity column by using rat liver homogenate. Surprisingly, the Rat FDH molecule with two typical liver proteins, carbamoyl-phosphate synthetase 1 (CPS1) and betaine homocysteine S-methyltransferase (BHMT) were co-purified to homogeneity on SBTI-coupled Sepharose and Sephacryl S-200 followed by Superdex 200 FPLC columns. These three liver-specific proteins make a protein complex with 300 kDa molecular mass on the gel-filtration column chromatography in vitro. Immuno-precipitation experiments by using anti-FDH and anti-SBTI antibodies also supported the fact that FDH binds to SBTI in vitro and in vivo. These results demonstrate that the catalytic site of rat FDH has a similar structure with those of serine proteases. Also, the SBTI-affinity column will be useful for the purification of rat liver proteins such as FDH, CPS1 and BHMT.

Mannose-binding lectin without the aid of its associated serine proteases alters lipopolysaccharide-mediated cytokine/chemokine secretion from human endothelial cells.

Coupling between certain pathogen-associated molecular patterns and corresponding pattern recognition receptors of endothelial cells is important for the mediation of vascular inflammatory responses. Mannose-binding lectin (MBL) recognizes certain carbohydrate structures of microbes and subsequently activates the complement system as well as facilitates the phagocytosis of targets. We investigated whether MBL can intervene in the interaction between bacterial lipopolysaccharide (LPS) and endothelial cells to modulate subsequent inflammatory responses. In response to LPS, human umbilical vein endothelial cells (HUVEC) produced various cytokines/chemokines. Addition of the purified human MBL/MBL-associated serine proteases (MASP) complex or recombinant human MBL enhanced LPS-mediated cytokine/chemokine secretion by HUVEC, including interleukin-8 (IL-8), IL-6 and monocyte chemoattractant protein-1 in a dose-dependent manner. This enhancing effect was ameliorated by the addition of anti-MBL antibody or mannan. Among the cytokines/chemokines we analysed, IL-6 showed the greatest increase of secretion in the presence of native MBL/MASP complex or recombinant MBL. MBL, regardless of its association with MASP, alters LPS-mediated cytokine/chemokine secretion of HUVEC. Besides the well-known functions of MBL, to activate the lectin-complement pathway and to facilitate clearance of targets, alteration of cytokine/chemokine secretion may provide an additional role for MBL in modulating vascular inflammation.

Clustering of peptidoglycan recognition protein-SA is required for sensing lysine-type peptidoglycan in insects.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor. Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.