Hysteresis-Free, Elastic, and Tough Hydrogel with Stretch-Rate Independence and High Stability in Physiological Conditions.

Many existing synthetic hydrogels are inappropriate for repetitive motions because of large hysteresis, and their mechanical properties in warm and saline physiological conditions remain understudied. In this study, a stretch-rate-independent, hysteresis-free, elastic, and tough nanocomposite hydrogel that can maintain its mechanical properties in phosphate-buffered saline of 37 °C similar to warm and saline conditions of the human body is developed. The strength, stiffness, and toughness of the hydrogel are simultaneously reinforced by biomimetic silica nanoparticles with a surface of embedded circular polyamine chains. Such distinctive surfaces form robust interfacial interactions by local topological folding/entanglement with the polymer chains of the matrix. Load transfer from the soft polymer matrix to stiff nanoparticles, along with the elastic sliding/unfolding/disentanglement of polymer chains, overcomes the traditional trade-off between strength/stiffness and toughness and allows for hysteresis-free, strain-rate-independent, and elastic behavior. This robust reinforcement is sustained in warm phosphate-buffered saline. These properties demonstrate the application potential of the developed hydrogel as a soft, elastic, and tough bio-strain sensor that can detect dynamic motions across various deformation speeds and ranges. The findings provide a simple yet effective approach to developing practical hydrogels with a desirable combination of strength/stiffness and toughness, in a fully swollen and equilibrated state.

Solvent-Free Fabrication of Anisotropic Microparticles with Precise 3D Shape Control Using Dipping-Based Micromolding.

We present an innovative solvent-free micromolding technique for rapidly fabricating complex polymer microparticles with three-dimensional (3D) shapes utilizing a surface tension-induced dipping process. Our fabrication process involves loading a photocurable solution into micromolds through mold dipping. The loaded solution, induced by surface tension, undergoes spatial deformation upon mold removal caused by surface forces, ultimately acquiring an anisotropic shape before photopolymerization. Results show that the amount of photocurable solution loaded depends on the degree of capillary penetration, which can be adjusted by varying the dipping time and mold height. It enables the production of polymer particles with precisely controlled 3D shapes without diluting them with volatile organic solvents. Sequential micromolding enables the spatial stacking of the polymer domain through a bottom-up approach, facilitating the creation of complex multicompartmental microparticles with independently controlled compartments. Finally, we demonstrated the successful simultaneous conjugation of multiple model-fluorescent proteins through the biofunctionalization of microparticles, indicating functional stability and effective conjugation of hydrophilic molecules such as proteins. We also extend our capacity to create bicompartmental microparticles with distinct functionalities in each compartment, revealing spatially controlled functional structures. In summary, these findings demonstrate a straightforward, rapid, and reliable method for producing highly uniform complex particles with precise control over the 3D shape and compartmentalization, all accomplished without the use of organic solvents.

Development of fluorescence-linked immunosorbent assay for rapid detection of Staphylococcus aureus.

Staphylococcus aureus is a major pathogen that causes infections and life-threatening diseases. Although antibiotics, such as methicillin, have been used, methicillin-resistant S. aureus (MRSA) causes high morbidity and mortality rates, and conventional detection methods are difficult to be used because of time-consuming process. To control the spread of S. aureus, a development of a rapid and simple detection method is required. In this study, we generated a fluorescent anti-S. aureus antibody, and established a novel fluorescence-linked immunosorbent assay (FLISA)-based S. aureus detection method. The method showed high sensitivity and low limit of detection toward MRSA detection. The assay time for FLISA was 5 h, which was faster than that of conventional enzyme-linked immunosorbent assay (ELISA) or rapid ELISA. Moreover, the FLISA-based detection method was applied to diagnose clinically isolated MRSA samples that required only 5.3 h of preincubation. The FLISA method developed in this study can be widely applied as a useful tool for convenient S. aureus detection. KEY POINTS: • A fluorescence-linked immunosorbent assay-based S. aureus detection method • Simultaneous quantification of a maximum of 96 samples within 5 h • Application of the novel system to diagnosis clinical isolates.

Fabrication and Morphological Control of Nonspherical Alginate Hydrogel Particles.

We report a simple platform for the fabrication of nonspherical alginate hydrogel particles using a dripping method. Hydrogel particles with novel morphologies, such as vortex ring, teardrop, disk, sphere, and mushroom, are fabricated by controlling various parameters. We monitored the deformation process of the hydrogel particles after they penetrated the crosslinking solution using a high-speed camera. Then, we proposed a mechanism showing a unique morphological transformation from a spherical to a disk shape. We demonstrated how controlling the collecting height that causes the drop impact force against the crosslinking solution surface was critical to producing hydrogel particles with these intriguing shapes. In particular, disk-shaped alginate particles show their ability as potential platforms for culturing mouse adrenocortical tumor cells (Y1) and a hippocampal neuronal cell (HT-22). To modify alginate particles, cell-adhesive gelatin is incorporated into the alginate matrix and then alginate particles are coated with poly(allylamine hydrochloride). Two modified alginate particles show good adhesion and proliferation rates on their surfaces. In particular, the hybrid hydrogel particles provide great potential to be developed into promising materials for cell culture, drug delivery, and tissue engineering.

Generation of a novel monoclonal antibody against inflammatory biomarker S100A8 using hybridoma technology.

OBJECTIVES: S100A8 is highly expressed in several inflammatory and oncological conditions. To address the current lack of a reliable and sensitive detection method for S100A8, we generated a monoclonal antibody with a high binding affinity to human S100A8 to enable early disease diagnosis. RESULTS: A soluble recombinant S100A8 protein with a high yield and purity was produced using Escherichia coli. Next, mice were immunized with recombinant S100A8 to obtain anti-human S100A8 monoclonal antibodies using hybridoma technology. Lastly, the high binding activity of the antibody was confirmed and its sequence was identified. CONCLUSIONS: This method, including the production of antigens and antibodies, will be useful for the generation of hybridoma cell lines that produce anti-S100A8 monoclonal antibodies. Moreover, the sequence information of the antibody can be used to develop a recombinant antibody for use in various research and clinical applications.

Hemicyanine-Based Near-Infrared Fluorescence Off-On Probes for Imaging Intracellular and In Vivo Nitroreductase Activity.

Nitroreductase (NTR) has the ability to activate nitro group-containing prodrugs and decompose explosives; thus, the evaluation of NTR activity is specifically important in pharmaceutical and environmental areas. Numerous studies have verified effective fluorescent methods to detect and image NTR activity; however, near-infrared (NIR) fluorescence probes for biological applications are lacking. Thus, in this study, we synthesized novel NIR probes (NIR-HCy-NO2 1-3) by introducing a nitro group to the hemicyanine skeleton to obtain fluorescence images of NTR activity. Additionally, this study was also designed to propose a different water solubility and investigate the catalytic efficiency of NTR. NIR-HCy-NO2 inherently exhibited a low fluorescence background due to the interference of intramolecular charge transfer (ICT) by the nitro group. The conversion from the nitro to amine group by NTR induced a change in the absorbance spectra and lead to the intense enhancement of the fluorescence spectra. When assessing the catalytic efficiency and the limit of detection (LOD), including NTR activity imaging, it was demonstrated that NIR-HCy-NO2 1 was superior to the other two probes. Moreover, we found that NIR-HCy-NO2 1 reacted with type I mitochondrial NTR in live cell imaging. Conclusively, NIR-HCy-NO2 demonstrated a great potential for application in various NTR-related fields, including NTR activity for cell imaging in vivo.

Generation of a recombinant antibody for sensitive detection of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes nosocomial infections and often exhibits antibiotic resistance. Therefore, the development of an accurate method for detecting P. aeruginosa is required to control P. aeruginosa-related outbreaks. In this study, we established an enzyme-linked immunosorbent assay method for the sensitive detection of three P. aeruginosa strains, UCBPP PA14, ATCC 27853, and multidrug-resistant ATCC BAA-2108. We produced a recombinant antibody (rAb) against P. aeruginosa V-antigen (PcrV), which is a needle tip protein of the type III secretion system of P. aeruginosa using mammalian cells with high yield and purity, and confirmed its P. aeruginosa binding efficiency. The rAb was paired with commercial anti-P. aeruginosa Ab for a sandwich ELISA, resulting in an antigen-concentration-dependent response with a limit of detection value of 230 CFU/mL. These results suggest that the rAb produced herein can be used for the sensitive detection of P. aeruginosa with a wide range of applications in clinical diagnosis and point-of-care testing.

Proteomic profiling of cryopreserved Trichormus variabilis using various cryoprotectants.

Algae, which may be unicellular or multicellular, can carry out photosynthesis just like plants as they effectively utilize light energy. They contain various physiologically active substances and are, therefore, widely used commercially to produce healthy food and feed additives, cosmetics, and energy supplements. For useful applications, the cryopreservation technique has been used in various fields. Recently, to develop suitable cryopreservation methods for algal applications, various studies have been performed. However, adequate investigations have not been conducted to understand the mechanism underlying algal cryopreservation at the molecular level. Therefore, this study examined the profile alteration of the proteome using cryopreservation with various cryoprotectants (CPAs). Trichormus variabilis was cultured and then cryopreserved with 10% dimethyl sulfoxide, methanol, and glycerol, after which, proteome profiling was done. Finally, signaling pathway search was performed, and a new signaling pathway was established based on differentially expressed proteins. As a result, the expression levels of 17 proteins were observed. Additionally, it was confirmed that the differentially expressed proteins were related to 16 signaling pathways and that they were capable of interacting with each other. The findings suggest that the differentially expressed proteins may be applied as biomarkers for algal cryopreservation and to understand the mechanism underlying T. variabilis cryopreservation. Moreover, it is anticipated that the results from this study would be useful in selecting suitable CPAs and in upgrading the cryopreservation techniques.

Expression of soluble recombinant human matrix metalloproteinase 9 and generation of its monoclonal antibody.

We have successfully produced a recombinant human matrix metalloproteinase 9 (hMMP9) antigen with high yield and purity and used it to generate a hybridoma cell-culture-based monoclonal anti-hMMP9 antibody. We selected the most effective antibody for binding antigens and successfully identified its nucleotide sequence. The entire antigen and antibody developmental procedures described herein can be a practical approach for producing large amounts of monoclonal antibodies against hMMP9 and other antigens of interest. Additionally, the nucleotide sequence information of the anti-hMMP9 monoclonal antibody revealed herein will be useful for the generation of recombinant antibodies or antibody fragments against hMMP9.

Effect of cryopreservation on the bacterial community structure of filamentous cyanobacteria, Trichormus variabilis (Nostocales, Cyanobacteria).

Cryopreservation is an efficient method used to preserve microorganisms for long periods of time, such as up to 30 years, without changes in genetic and physiological characteristics. As cyanobacteria and microalgae are usually maintained as both axenic and xenic cultures, knowledge of co-cultured bacteria and changes in their community structure is important for the successful maintenance of microbial culture collections. In this study, research on the changes in co-cultured bacterial community structure during cyanobacterial cryopreservation were investigated using three different experimental groups by next generation sequencing (NGS): 1) cultured Trichormus variabilis without cryopreservation (control group), 2) cultured T. variabilis after cryopreservation in 10% dimethyl sulfoxide (Me2SO) for 14 days (cryo-cell group), and 3) cultured T. variabilis after cryopreservation in 10% Me2SO for 14 days within alginate beads (cryo-bead group). The results showed that the abundance of Sphingomonas and Hydrogenophaga (belonging to phylum Proteobacteria) was significantly increased in the cryo-bead group (Sphingomonas, control: 0.25%, cryo-cell: 1.32%, cryo-bead: 41.70%; Hydrogenophaga, control: 5.47%, cryo-cell: 5.24%, cryo-bead: 12.32%). However, the abundance of the phylum Bacteroidetes was significantly decreased in the cryo-bead group compared to that in the other groups (control: 26.29%, cryo-cell: 38.84%, cryo-bead: 11.43%). Bacterial diversity was generally reduced after cryopreservation in the cryo-bead group, where the overgrowth of a few unique bacteria was observed in the co-cultured bacterial community. These results imply that changes in the co-cultured bacterial community during preservation should be considered as an important factor for the development of methods for cyanobacterial cryopreservation.

Genetically Modified Ferritin Nanoparticles with Bone-Targeting Peptides for Bone Imaging.

Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget's disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.

Diagnostic ability of salivary matrix metalloproteinase-9 lateral flow test point-of-care test for periodontitis.

AIM: This cross-sectional study aimed to examine the diagnostic ability of salivary matrix metalloproteinase (MMP)-9 lateral flow test (LFT) point-of-care (POC) kit and develop an algorithm for diagnosis of periodontitis. MATERIALS AND METHODS: Through Seoul National Dental Hospital, 137 participants (46 LFT negatives, 91 LFT positives) were recruited. For salivary diagnostics, 150 µl of the unstimulated saliva was applied to LFT-POC kit. To make a diagnosis of periodontitis, stage II-IV in modified new international classification system was used. Covariates encompassing age, sex, smoking and obesity were evaluated through face-to-face interview. Enzyme-linked immunosorbent assay was used for quantification of salivary MMP-9. To develop a diagnostic algorithm, multivariable logistic regression analysis was used. Receiver operating characteristic curve was applied for evaluating diagnostic ability. RESULTS: Diagnostic ability of salivary MMP-9 LFT-POC test was 0.82 (sensitivity of 0.92, specificity of 0.72) in total participants. Diagnostic algorithm using POC test resulted in a response equation, that is algorithm score = -3.675 + 2.877*LFT + 0.034*age + 0.121*sex + 0.372*smoking + 0.192*obesity. Diagnostic ability of the algorithm was 0.88 (sensitivity of 0.92, specificity of 0.85) with cut-off score of 0.589. CONCLUSIONS: Salivary MMP-9 LFT-POC kit showed appropriate diagnostic ability for periodontitis and would be an efficient tool for screening of periodontitis.

Epitaxial van der Waals Contacts between Transition-Metal Dichalcogenide Monolayer Polymorphs.

We have achieved heteroepitaxial stacking of a van der Waals ( vdW) monolayer metal, 1T'-WTe2, and a semiconductor, 2H-WSe2, in which a distinctively low contact barrier was established across a clean epitaxial vdW gap. Our epitaxial 1T'-WTe2 films were identified as a semimetal by low temperature transport and showed the robust breakdown current density of 5.0 × 107 A/cm2. In comparison with a series of planar metal contacts, our epitaxial vdW contact was identified to possess intrinsic Schottky barrier heights below 100 meV for both electron and hole injections, contributing to superior ambipolar field-effect transistor (FET) characteristics, i.e., higher FET mobilities and higher on-off current ratios at smaller threshold gate voltages. We discuss our observations around the critical roles of the epitaxial vdW heterointerfaces, such as incommensurate stacking sequences and absence of extrinsic interfacial defects that are inaccessible by other contact methods.

Ephemeroptericola cinctiostellae gen. nov., sp. nov., isolated from gut of an aquatic insect.

A Gram-stain-negative, catalase- and oxidase-positive, non-motile bacterium, designated F02T, was isolated from of gut of Cincticostellalevanidovae (Tshernova). Growth occurred at a temperature range of 4-30 °C, at pH 6-9 and in the presence of 0-0.5 % (w/v) NaCl. Phylogenetic analysis demonstrated that the 16S rRNA gene sequence of strain F02T shared the highest similarity to that of the type strain of Hydromonas duriensis A2P5T (96.82 %). The major isoprenoid quinone was Q-8. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major cellular fatty acids were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and iso-C13 : 0 3-OH. The polyamines were cadaverine and putrescine. Combined data from phylogenetic, phenotypic and chemotaxonomic analyses demonstrated that strain F02T represents a novel genus and species, for which the name Ephemeroptericolacinctiostellae gen. nov., sp. nov. is proposed. The type strain of Ephemeroptericola cinctiostellae gen. nov., sp. nov. is F02T (=FBCC 500047T=KCTC 62567T=JCM 32722T).

Improvement in the Reproducibility of a Paper-based Analytical Device (PAD) Using Stable Covalent Binding between Proteins and Cellulose Paper.

Paper-based analytical devices (PADs) have been widely used in many fields because they are affordable and portable. For reproducible quantitative analysis, it is crucial to strongly immobilize proteins on PADs. Conventional techniques for immobilizing proteins on PADs are based on physical adsorption, but proteins can be easily removed by weak physical forces. Therefore, it is difficult to ensure the reproducibility of the analytical results of PADs using physical adsorption. To overcome this limitation, in this study, we showed a method of covalent binding of proteins to cellulose paper. This method consists of three steps, which include periodate oxidation of paper, the formation of a Schiff base, and reductive amination. We identified aldehyde and imine groups formed on paper using FT-IR analysis. This covalent bonding approach enhanced the binding force and binding capacity of proteins. We confirmed the activity of an immobilized antibody through a sandwich immunoassay. We expect that this immobilization method will contribute to the commercialization of PADs with high reproducibility and sensitivity.

Improvement of the chemical recycling process of waste carbon fibre reinforced plastics using a mechanochemical process: Influence of process parameters.

We present the experimental results of an optimal recycling method for waste carbon fibre reinforced plastics (CFRPs) that is based on the application of a set of unit mechanochemical processes. The objectives of this study were to highlight the influence of process factors that are inherent in the chemical recycling process of waste CFRP. We investigated the influence of the soaking period, the application of a catalyst and impurities on the recycling process and recovery efficiency of the waste CFRP. Different combinations of the unit mechanochemical processes were investigated, and the effectiveness of the combination was analysed. The chemical recycling process was conducted using benzyl alcohol under ordinary pressure at initial solvent temperatures lower/equivalent to its flash point temperature. Experimental results showed that the solvent temperature increased up to boiling temperature levels when the mechanochemical process was initiated, thereby enhancing the mechanochemical process. The presence of impurities did not influence the recovery rate. Likewise, this experimental study highlighted the importance of accounting for the soaking period during the chemical recycling process: an extended soaking period resulted in a higher recovery rate, a lower portion of undissolved solids and recovered fibres of better quality. This research highlighted the significance of choosing the proper combination for the chemical recycling process as well as the benefits of recycling the waste CFRP with negligible application of the catalyst.

Programmable Static Droplet Array for the Analysis of Cell-Cell Communication in a Confined Microenvironment.

Direct cell-cell communication can occur through various chemical and mechanical signals. However, available cell culture systems lack single-cell resolution and are often limited by sensitivity and accuracy. In this study, we present an accurate, efficient and controllable microfluidic device that can be used for in situ monitoring of natural cell-cell contact and signaling processes in a confined microenvironment. This innovative static droplet array (SDA) enables highly efficient trapping, encapsulation, arraying, storage, and incubation of defined cell populations. For proof-of-principle experiments, we monitored the response of budding yeast to peptide mating pheromones, as it is one of the best understood examples of eukaryotic cell-cell communication. Specifically, we measured the yeast response to varying concentration of synthetic MATα-type mating factor, as well as varying the cell number ratio of MATα and MATa in a confined space. We found clear morphological and doubling-time changes during the mating reaction with a significantly higher accuracy than conventional methods. Further, phenotypic analysis of data generated with the microfluidic static droplet array allowed distinguishing the function of genes in yeast mutants defective for different aspects of pheromone signaling. Taken together, the microfluidic platform provides a valuable research tool to study cell-cell communication and signaling in a controlled microenvironment with the sensitivity and accuracy required for screening and long-term phenotypic analysis.

Directed Assembly of Janus Cylinders by Controlling the Solvent Polarity.

This study demonstrates the possibility of controlling the directed self-assembly of microsized Janus cylinders by changing the solvent polarity of the assembly media. Experimental results are analyzed and theoretical calculations of the free energy of adhesion (ΔGad) are performed to elucidate the underlying basic principles and investigate the effects of the solvent on the self-assembled structures. This approach will pave a predictive route for controlling the structures of assembly depending on the solvent polarity. In particular, we find that a binary solvent system with precisely controlled polarity induces directional assembly of the microsized Janus cylinders. Thus, the formation of two-dimensional (2D) and three-dimensional (3D) assembled clusters can be reliably tuned by controlling the numbers of constituent Janus cylinders in a binary solvent system. Finally, this approach is expanded to stepwise assembly, which forms unique microstructures via secondary growth of primary seed clusters formed by the Janus cylinders. We envision that this investigation is highly promising for the construction of desired superstructures using a wide variety of polymeric Janus microparticles with chemical and physical multicompartments.

Asprobacter aquaticus gen. nov., sp. nov., a prosthecate alphaproteobacterium isolated from fresh water.

A novel Gram-negative bacterium strain, DRW22-8T, was isolated from fresh water taken at a depth of 22 m at Daechung Reservoir, Republic of Korea. The cells of strain DRW22-8T were aerobic and motile with a single polar flagellum or non-motile (stalked), and formed creamy-white colonies on R2A agar. The phylogenetic analysis based on 16S rRNA gene sequencing indicated that the strain formed a separate lineage within the order Rhodobacterales, showing similarity values under 91.8 % with its closest phylogenetic neighbours, Hirschia litorea, Hirschia baltica and Hirschia maritima. The chemotaxonomic results showed Q-10 as the predominant respiratory ubiquinone, three unidentified glycolipids, an unidentified lipid and phosphatidylglycerol as the major polar lipids, and C16 : 0, 11-methyl C18 : 1, C18 : 1 ω7c and/or C18 : 1 ω6c as the major fatty acids. The DNA G+C content was 64.4 mol%. The combined genotypic and phenotypic data showed that strain DRW22-8T could be distinguished from all genera within the family Hyphomonadaceae and represented a novel genus, Asprobacter gen. nov., with the name Asprobacter aquaticus sp. nov., in the family Hyphomonadaceae. The type strain is DRW22-8T (=KCTC 42356T=JCM 30469T).

Blastomonas fulva sp. nov., aerobic photosynthetic bacteria isolated from a Microcystis culture.

Two Gram-stain-negative, aerobic, non-motile, non-spore-forming and rod-shaped bacteria, designated strains T2T and T5, were isolated from a culture of Microcystis from Daejeon, Republic of Korea. Comparative 16S rRNA gene sequence studies placed the new isolates in the class Alphaproteobacteria and, notably, most closely related to Blastomonasaquatica PE 4-5T, Blastomonas natatoria DSM 3183T and Blastomonas ursincola KR-99T showing 99.4 %, 98.2 % and 97.9 % 16S rRNA gene sequence similarities, respectively. The two novel strains shared 100 % similarity with each other. The cells of strains T2T and T5 formed yellow colonies on R2A agar and contained Q-10 as the only ubiquinone, sphingoglycolipid, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol as major polar lipids, and C17 : 1ω6c, summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), C17 : 1ω8c and C17 : 0 as the major fatty acids (>5 %). The DNA G+C content of the genomes was determined to be 64.2 mol% for strain T2T and 64.4 mol% for strain T5. The DNA-DNA hybridization values of strains T2T and T5 with B.aquatica PE 4-5T, B. natatoria DSM 3183T, and B. ursincola KR-99T were 19.7-42.4 %. Based on the combined genotypic and phenotypic data, we propose that strains T2T and T5 represent a novel species of the genus Blastomonas, for which the name Blastomonas fulvasp. nov. is proposed. The type strain is T2T (=KCTC 42354T=JCM 30467T).