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Garlic sprouts can provide data on functional and food processing materials. This study compared the leaves, bulbs, and roots of garlic sprouts grown on smart farms during two growth periods (20 and 25 days). In addition, data for garlic bulbs grown in open fields were presented as reference materials. All garlic sprouts’ total free sugar content decreased as the growth period increased. All plant parts’ total organic acid content decreased as the growth period progressed, except for the root section. Potassium, phosphorus, and sulfur content increased during growth in all parts of the garlic sprouts. Alliin content decreased in all parts of the plant over time, whereas thiosulfinate content increased in the roots but decreased in the leaves and bulbs. Total polyphenol content increased in all parts of the plant during the growth period, except for the bulb, whereas the flavonoid content did not change significantly over time. The 2,2-diphenyl-1-picrylhydrazy (DPPH) and 2,2′-azinobis (3-ethylben-zothiazoline 6-sulfonate) (ABTS) free radical scavenging activities, as well as the superoxide dismutase (SOD)-like activity of garlic sprouts were 37.45-65.47%, 59.12-89.81%, and 89.52-98.59%, respectively. These activities tend to decrease during the growth period. Here, we showed that garlic sprouts have higher levels of functional substances and physiological activities than general garlic sprouts. It was also determined that a growth period of 20 days was suitable for garlic sprouts. Data for research on functional and food-processing materials can be obtained by analyzing garlic sprouts produced by smart farms.

The aim of this study was to improve the quality and functionality of Korean soy sauce made with onion juice. At the end of fermentation, the titratable acidity of the soy sauce containing 100% onion juice was the highest at 2.90%. The salt (NaCl) content was highest and lowest in the samples without and with onion juice supplementation, respectively. The protein content ranged from 11.09-12.50%. The sensory evaluation revealed that the soy sauce sample containing 70% onion juice had the highest scove at 5.0-5.8 levels in terms of sweetness, salty taste, delicate flavor, and overall preference. Quercetin content was 1.73 mg/100 g and 1.68 mg/100 g in soy sauce containing 70% and 100% onion juice, respectively. However, quercetin was not detected in the soy sauce without onion juice supplementation. Total polyphenol and flavonoid contents, as well as antioxidant activity, increased proportionally to the amount of onion juice added. Overall, it was confirmed that the addition of onion juice improved the quality and functionality of Korean soy sauce.

This study aimed to select natural materials with antibacterial activity against Streptococcus mutans (S. mutans) KCCM 40105 and determine their most efficient extraction conditions. The antibacterial activity was stable under various treatment conditions. Of the 17 material groups, extracts of Sophora flavescens (SF) and Schisandra chinensis (SC) showed antibacterial activity against the S. mutans strain. Measuring the antibacterial activity based on ethanol concentration revealed that the 30% ethanol extracts of SF and SC showed the highest antibacterial activity as observed by the inhibition zones of 10.69 and 14.49 mm, respectively. The minimum inhibitory concentrations of the 30% ethanol extracts of SF and SC against the S. mutans strain were 2.0 and 5.0 mg/mL, respectively. Heat stability was confirmed; however, the 30% ethanol extract of SC was inactive at pH values ≥6.0. The antibacterial activity of the 30% ethanol extract of SF and SC treated with nine enzymes was the same at 10 ppm and 100 ppm. However, the activity tended to decrease marginally in the 1,000 ppm concentration treatment group.

This study aimed to improve the physiological activity and processing properties of ginseng sprouts. We first determined the effect of extraction and fermentation conditions on the antioxidant activity of ginseng sprouts. The optimal extraction conditions were 2 h at 80°C, with water as a solvent. The fermentation strains used were Lactobacillus delbrueckii subsp. bulgaricus KCTC 3635 and Enterococcus faecalis KCTC 3206. We then analyzed the changes in the reducing sugar and alcohol content. In fermented samples, the reducing sugar content was lower and the alcohol content was higher than those in control sample. The cell counts of lactic acid bacteria were higher in the fermented samples than those in the control sample. Fermented samples contained more ginsenoside than the extract samples, and ginsenoside was not detected in the control sample. The total polyphenol and total flavonoid contents were relatively high in the fermented samples, particularly in the sample fermented by E. faecalis. Finally, sensory evaluation revealed that the Yakju prepared using ginseng sprouts fermented by lactic acid bacteria was preferable to other drinks control and ginseng sprouts extracted sample. Overall, it was confirme that the addition of fermented ginseng improved the physiological activity and palatability of yakju, there by improving the quality of yakju.

Soybean mosaic virus (SMV) is a member of the genus Potyvirus in the family Potyviridae. Legume crops are often infected by SMV. SMV has not been naturally isolated from sword bean (Canavalia gladiata) in South Korea. In July 2021, 30 samples of sword bean were collected at the field located in Hwasun and Muan, Jeonnam, Korea to investigate viruses infecting sword bean. The samples exhibited symptoms typical of viral infection such as mosaic pattern and, mottling of leaves. Reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) techniques were employed to identify the agent of viral infection in sword bean samples. Total RNA was extracted from the samples using the Easy-SpinTM Total RNA Extraction Kit (Intron, Seongnam, Korea). Out of the 30 samples, seven were found to be infected by the SMV. RT-PCR was performed using RT-PCR Premix (GeNet Bio, Daejeon, Korea) with SMV-specific primer set, forward primer (SM-N40, 5′-CATATCAGTTTGTTGGGCA-3′) and the reverse primer (SM-C20, 5′-TGCCTATACCCTCAACAT-3′), yielding a product of 492 bp (Lim et al., 2014). RT-LAMP was performed using RT-LAMP Premix (EIKEN Chemical, Tokyo, Japan) with SMV-specific primer set, the forward primer (SML-F3, 5′-GACGATGAACAGATGGGC-3′, SML-FIP, 5′-GCATCTGGAGATGTGCTTTTGTGGTTATGAATGGTTTCATGG-3′) and reverse primer (SML-B3, 5′-TCTCAGAGTTGGTTTTGCA-3′, SML-BIP, 5′-GCGTGTGGGTGATGATGGATTTTTTCGACAATGGGTTTCAGC-3′) for diagnosis of viral infection (Lee et al., 2015). The full coat protein genes of seven isolates were amplified using RT-PCR to determine their nucleotide sequence. The standard nucleotide BLAST (blastn suite) showed that the seven isolates had approximately 98.2–100% homology with SMV isolates (FJ640966, MT603833, MW079200, and MK561002) in NCBI GenBank. The sequences of seven isolates were deposited in the GenBank database under the accession numbers: OP046403–9. For the pathogenicity assay of the isolate, the crude saps from SMV-infected samples were mechanically inoculated into sword bean. Fourteen days after inoculation, the mosaic symptoms were observed on the upper leaves of sword bean. As a result of the RT-PCR diagnosis in the upper leaves, SMV was reconfirmed in sword bean. This is the first report of natural SMV infection in sword bean. As sword beans are increasingly consumed for teas, transmitted seeds are resulting in a decrease in pod production and quality. It is necessary to develop efficient methods of seed processing and management strategies to control SMV infection in sword bean.

In this study, the quality characteristics of kombucha according to the mixing ratio of green tea extract and yuzu juice during fermentation were compared. The pH of the kombucha decreased as the amount of yuzu juice increased. The titratable acidity increased in a juice-concentration dependent manner during fermentation. The reducing sugar content was 1.68% in kombucha made with a mixture of 70% green tea extract and 30% yuzu juice on the 20th day of fermentation, showing the lowest reducing sugar content. The alcohol content did not change until the 10th day of fermentation and increased slightly thereafter. The number of lactic acid bacteria and the quantity of yeast were the highest in the samples of kombucha made with a mixture of 70% green tea extract and 30% yuzu juice. The total polyphenol content was 40.79 mg TAE/mL in the kombucha made with this combination and this also showed the highest total flavonoid content of 37.15 mg QE/mL. In addition, the sensory evaluation results showed the highest preference in all the measured parameters for kombucha made with a mixture of 70% green tea extract and 30% yuzu juice.

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea that provide resistance against phages and other mobile genetic elements. To fight against CRISPR-Cas systems, phages and archaeal viruses encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas systems. The expression of acr genes is controlled by anti-CRISPR-associated (Aca) proteins encoded within acr-aca operons. AcrIF24 is a recently identified Acr that inhibits the type I-F CRISPR-Cas system. Interestingly, AcrIF24 was predicted to be a dual-function Acr and Aca. Here, we elucidated the crystal structure of AcrIF24 from Pseudomonas aeruginosa and identified its operator sequence within the regulated acr-aca operon promoter. The structure of AcrIF24 has a novel domain composition, with wing, head and body domains. The body domain is responsible for recognition of promoter DNA for Aca regulatory activity. We also revealed that AcrIF24 directly bound to type I-F Cascade, specifically to Cas7 via its head domain as part of its Acr mechanism. Our results provide new molecular insights into the mechanism of a dual functional Acr-Aca protein.

In this study, natural materials with antibacterial activity against the Streptococcus mutans KCCM 40105 strain were selected, and the most efficient extraction conditions were observed. The active substance was stable under various treatment conditions. Of the 10 food materials groups, only Prunus mume fruit showed antibacterial activity against S. mutans strains. The antimicrobial activity by the various extraction solvents was 18.66 mm (clear zone diameter) in the ethanol extract and 20.18 mm in the water extract. Measurements of the antibacterial activity according to the mixing ratio of the two solvents revealed the 30% ethanol extract to have the highest activity (20.49 mm). Measurements of the minimum inhibitory concentration (MIC) showed activity at a concentration of 3.0 mg/mL. The stability to heat was observed, but the extract was not active at pH 6.0 or higher. The antimicrobial activity of the 30% ethanol extract of P. mume treated with enzymes was the same with both 10 ppm and 100 ppm of the nine enzymes. On the other hand, the activity tended to decrease slightly at 1,000 ppm. The activity of the catazyme enzyme and hydrogen peroxide treatment decreased at the treatment concentration of 100 ppm.

A simple technology to manufacture ‘Nuruk’ with pigmented rice and ‘Yakju’ with improved quality and functionality was developed. Yakju (designated WY and PY, respectively) was prepared with white rice flour Nuruk (WN) and pigmented rice flour Nuruk (PN), and their qualities were compared. The enzyme activities of Nuruk revealed that PN had higher β-amylase activity (1,186.67 Saccharogenic acitivty, SP/g) and higher protease activity (378.90 tyrosine mg/min) than WN. During 11 days of fermentation, the pH of PY was steady in the range of 4.77-4.58, whereas the pH (4.82) of WY decreased to 3.90 on day 3 of fermentation and then increased to similar pH of PY until the fermentation was completed. The titratable acidities of PY and WY increased to 0.40-0.48%. The ethanol content of PY and WY increased to 13.43% ad 13.23%, respectively, on day 5, after then PY showed higher ethanol content than WY until day 11 of fermentation. The organic acid content of PY (1,225.87 mg%) was higher than that of WY (1,021.69 mg%). The antioxidant activities of PY were twice as high as those of WY, and the total polyphenolics and total flavonoid contents of PY were also higher (35.96 mg% and 19.01 mg%, respectively) than those of WY (16.29 mg% and 6.36 mg%, respectively). Sensory scores on color, flavor and taste, and overall preference were higher in PY than in WY.

Immunity-related GTPase B10 (IRGB10) belongs to the interferon (IFN)-inducible GTPases, a family of proteins critical to host defense. It is induced by IFNs after pathogen infection, and plays a role in liberating pathogenic ligands for the activation of the inflammasome by directly disrupting the pathogen membrane. Although IRGB10 has been intensively studied owing to its functional importance in the cell-autonomous immune response, the molecular mechanism of IRGB10-mediated microbial membrane disruption is still unclear. In this study, we report the structure of mouse IRGB10. Our structural study showed that IRGB10 bound to GDP forms an inactive head-to-head dimer. Further structural analysis and comparisons indicated that IRGB10 might change its conformation to activate its membrane-binding and disruptive functions. Based on this observation, we propose a model of the working mechanism of IRGB10 during pathogen membrane disruption., Ha et al. present a crystal structure of mouse IRGB10, a mouse interferon-inducible GTPase that mediates bacteriolysis in cell autonomous immunity. With further mutagenesis studies, they show that IRGB10 bound to GDP forms an inactive head-to-head dimer, which changes its conformation to activate its membrane-binding and disruptive functions.

In this study, pigmented rice flour ‘Nuruk’ was prepared using various fungal strains to improve the quality of Korean traditional ‘Kochujang’, and the quality characteristics during the aging of ‘Kochujang’ made with the ‘Nuruk’ were confirmed. The pH of ‘Nuruk’ was highest using Aspergillus oryzae pigmented rice flour ‘Nuruk’ (N2) at 5.83, and the highest acidity was Aspergillus niger pigmented rice flour ‘Nuruk’ (N3) at 2.69. The crude ash content was 1.10-1.14% from the ‘Nuruk’ made with pigmented rice flour, which was higher than the 0.42% from ‘Nuruk’ made with white rice flour. The pH of ‘Kochujang’ decreased gradually from 0 to 90 days as aging progressed. The titratable acidity was 2.76-3.71%, which increased with increasing aging period. During the aging of ‘Kochujang’, the water content increased by 2-3%. The reducing sugar content decreased from 0.33 to 3.25% after 60 days of aging and increased to 3.85 to 4.94% at the end of aging. Regarding the color values, the ‘Kochujang’ made with white rice flour ‘Nuruk’ showed higher overall L, a, and b values than the ‘Kochujang’ made with pigmented rice flour ‘Nuruk’. A sensory evaluation of six kinds of ‘Kochujang’ revealed the ‘Kochujang’ made with N2 ‘Nuruk’ to have the highest preference in all evaluation items.

This study aimed to isolate and identify antibacterial compounds from Schisandra chinensis (S. chinensis) that are effective against the Streptococcus mutans KCCM 40105 strain. First, S. chinensis was extracted using varying concentrations of ethanol, and the resulting antibacterial activity was evaluated. The 30% ethanol extract of S. chinensis showed high activity. The fractionation and antibacterial activity of a 30% ethanol extract from S. chinensis were examined using five different solvents. Upon investigation of the antibacterial activity of the solvent fraction, the water and butanol fractions showed high activity, and no significant difference was found. Therefore, the butanol fraction was chosen for material exploration using silica gel column chromatography. A total of 24 fractions were obtained from the butanol portion using silica gel chromatography. The fraction with the highest antibacterial activity was Fr 7. From Fr 7, thirty-three sub-fractions were isolated, and sub-fraction 17 showed the highest level of antibacterial activity. A total of five peaks were obtained through the pure separation of sub-fraction 17 using HPLC. Peak 2 was identified as a substance exhibiting a high level of antibacterial activity. Based on the results of UV spectrometry, 13C-NMR, 1H-NMR, LC-MS, and HPLC analyses, the compound corresponding to peak number 2 was identified as tartaric acid.

In this study, we examined the quality of Tenebrio molitor L. that ingested yuzu supplemented feed at four feed compositions: C120 (120 g Chinese cabbage), C80+Y40 (80 g Chinese cabbage+40 g yuzu peel), C40+Y80 (40 g Chinese cabbage+80 g yuzu peel) and Y120 (120 g yuzu peel). Tenebrio molitor L. was fed for 90 d at a rate according to age, after which the moisture, crude protein, crude fat, and crude ash contents of the samples were determined to be 0.49-0.50%, 52.58-53.90%, 30.76-32.34%, and 3.32-3.45%, respectively. Color values, namely yellowness (b) and redness (a), were observed to decrease with increasing yuzu ingestion, and the C80+Y40 sample exhibited the highest lightness (L=36.28). The K and Mg contents were observed to increase with increasing yuzu content, and Pb and As were not detected in any Tenebrio molitor L. sample that ingested the yuzu supplemented feed. The free amino acid (serine and alanine) contents also increased with increasing yuzu content, while the histidine and arginine contents were observed to decrease with increasing yuzu ingestion. Naringin and hesperidin were detected at 1.77 mg% and 1.63 mg% in sample Y120, respectively, while trace amounts were detected in samples C80+Y40 and C40+Y80, but none were detected in the control. The total polyphenol and flavonoid contents increased with increasing level of yuzu ingestion, and were highest in sample Y120 (123.11 mg% and 5.69 mg%, respectively).

An easy and simple method of producing Nuruk using rice was developed. In addition, the quality characteristics of the prepared Nuruk were evaluated for Makgeolli production. A comparison of the enzyme activities between A. kawachii rice Koji (RK) and non-steamed rice flour Nuruk according to the fungal strain revealed the β-amylase activity of A. oryzae rice flour Nuruk (AON) to be the highest at 3697.85 SP/g. The α-amylase activity and protease activity were highest at 111.67 A.U and 33.23 tyrosine mg/min of P. candidum rice flour Nuruk (PCN), respectively. The ethanol contents of the four samples were similar at 16.1-16.4%. The sensory evaluation result showed that Makgeolli made with PCN had the highest preference score of 5.78 and showed a significant difference in overall preference. Moreover, Makgeolli made with PCN showed a higher preference than of Makgeolli made with RK. The titratable acidity and reducing sugar content of Makgeolli made with PCN were 0.48% and 4.14%, respectively, which were higher than those of Makgeolli made with RK. The ethanol content of Makgeolli made with PCN was 15.10%, which showed a 12.80% higher ethanol production yield than that of Makgeolli made with RK. Therefore the PCN produced in this study will be effective for Makgeolli production.

In this study, milk samples were fermented following the addition of Ligularia fischeri extracts. The aqueous extracts were obtained from L. fischer grown in the open field (OL) or in a greenhouse (GL). The quality related properties, and antioxidant activity of the fermented milk were evaluated, and the effects of storage on the fermented samples were assessed. The time required for the fermented milk to reach pH 4.6 was approximately four hours in all samples. The titratable acidity of the samples was in the range of 0.5-1%. The number of lactic acid bacteria in the fermented milks made with L. fischeri GL extract was slightly higher than it was in milks samples fermented with L. fischeri OL extract. The Hunter Lab color (L) and redness (a) values of the samples were lower after fermentation, but their yellowness (b) increased with increasing amounts of L. fischeri extracts. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity in milk fermented with 0.5%, 1%, 3%, and 8% L. fischeri OL extracts was higher than that in milk fermented with corresponding amounts of L. fischeri GL extract. However, the difference was not significant. DPPH activity in milk fermented with 5% L. fischeri GL extract was higher than it was in milk fermented with 5% L. fischeri OL extract. The pattern of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) radical cation (ABTS˙+) scavenging activity in the samples was similar to that observed during the DPPH assay. The pH and, titratable acidity, of the fermented samples were not significantly affected by storage, and the number of lactic acid bacteria in the stored and freshly fermented samples was similar.

This study was conducted to enhance the utilization of Ligularia fischeri as a food ingredient. The effects of the location of cultivation and the harvest time on the components were examined. The crude fats and proteins in greenhouse samples (GL) were 2% higher than those in open field samples (OL). The fat content was found to increase from 4.31% to 5.27% as the harvest time was delayed. The Na contents were more than two times higher in GL than in OL. Moreover, the amount of free sugars in leaves differed slightly according to the harvest time but decreased overall as the harvest time was delayed. The total polyphenols increased from 202.53 to 389.16 mg% in OL and 293.57 to 439.40 mg% in GL as the harvest time was delayed. The levels of total flavonoids in OL and GL ranged from 105.58 to 202.79 mg% and 135.84 to 216.96 mg%, respectively. These increased from 0.3 to 2 times upon delaying the harvest time. The samples were also analyzed for the presence of five catechin compounds, namely, (-)-epigallocatechin (EGC), (+)-catechin (CE), (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate (ECG), and (-)-gallocatechin gallate (GCG). EGC and CE were detected in both OL and GL, while EGCG, ECG, and GCG were detected only in OL at low concentrations. The 4-Ο-caffeoylquinic acid content in GL was higher than that in OL, except in the early stages of harvesting.

Vaccines and therapeutics are urgently needed for the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we screen human monoclonal antibodies (mAb) targeting the receptor binding domain (RBD) of the viral spike protein via antibody library constructed from peripheral blood mononuclear cells of a convalescent patient. The CT-P59 mAb potently neutralizes SARS-CoV-2 isolates including the D614G variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/RBD shows that CT-P59 blocks interaction regions of RBD for angiotensin converting enzyme 2 (ACE2) receptor with an orientation that is notably different from previously reported RBD-targeting mAbs. Furthermore, therapeutic effects of CT-P59 are evaluated in three animal models (ferret, hamster, and rhesus monkey), demonstrating a substantial reduction in viral titer along with alleviation of clinical symptoms. Therefore, CT-P59 may be a promising therapeutic candidate for COVID-19., Therapies and vaccines for COVID-19, caused by the SARS-CoV-2 viral pandemic, are urgently needed. Here the authors establish and screen an antibody library from a convalescent COVID-19 patient to isolate a neutralizing antibody with the ability to reduce viral titer and alleviate symptoms in ferret, hamster, and rhesus monkey infection models.

Significance Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a highly conserved family with diverse functions ranging from cell death to lipid homeostasis and synaptic regulation. Through structural determination of two CIDE family proteins, Drep2 and Drep4, we found that CIDE domains can form helical oligomers. Our results reveal that such higher-order structures not only are conserved in the CIDE family, but also are critically important for both DNA fragmentation and lipid droplet fusion. Therefore, our findings identify the CIDE domain as a scaffolding component for higher-order structure assembly. Our results expand the importance of higher-order structures from the established field of immune signaling to broader biological functions.

This study was carried out to investigate the quality characteristics of fermented milk containing Rhus verniciflua seed (RVS) extracts (0.0%, 0.1%, 0.3% and 0.5%) and antioxidant activity of RVS extracts. The proximate composition of RVS was moisture (4.76%), crude ash (1.40%), crude fat (5.33%) and crude protein (11.10%). A total polyphenol content of 70% ethanol extract of RVS (554±2.64 mg/g) was higher than that of RVS water extract (145±3.47 mg/g). 70% ethanol extract (1103±6.42 mg/g) of RVS showed higher content in the total flavonoids (37±2.30 mg/g) and activities on DPPH free radical scavenging (73.23%) and ABTS free radical scavenging (83.47%) when compared with water extracted of RVS. The quality characteristics, such as pH, titratable acidity, and the number of lactic acid producing bacteria were not remarkably different between the fermented milk samples subjected to treatments with and without the addition of RVS extracts.

Caspase recruitment domain (CARD)-only proteins (COPs), regulate apoptosis, inflammation, and innate immunity. They inhibit the assembly of NOD-like receptor complexes such as the inflammasome and NODosome, which are molecular complexes critical for caspase-1 activation. COPs are known to interact with either caspase-1 CARD or RIP2 CARD via a CARD-CARD interaction, and inhibit caspase-1 activation or further downstream signaling. In addition to the human COPs, Pseudo-ICE, INCA, and ICEBERG, several viruses also contain viral COPs that help them escape the host immune system. To elucidate the molecular mechanism of host immunity inhibition by viral COPs, we solved the structure of a viral COP for the first time. Our structure showed that viral COP forms a structural transformation-mediated dimer, which is unique and has not been reported in any structural study of a CARD domain. Based on the current structure, and the previously solved structures of other death domain superfamily members, we propose that structural transformation-mediated dimerization might be a new strategy for dimer assembly in the death domain superfamily.

β-Lactam antibiotics that inhibit penicillin-binding proteins (PBPs) have been widely used in the treatment of bacterial infections. However, the molecular basis underlying the different inhibitory potencies of β-lactams against specific PBPs is not fully understood. Here, we present the crystal structures of penicillin-binding protein D2 (PBPD2) from Listeria monocytogenes, a Gram-positive foodborne bacterial pathogen that causes listeriosis in humans. The acylated structures in complex with four antibiotics (penicillin G, ampicillin, cefotaxime, and cefuroxime) revealed that the β-lactam core structures were recognized by a common set of residues; however, the R1 side chains of each antibiotic participate in different interactions with PBPD2. In addition, the structural complementarities between the side chains of β-lactams and the enzyme were found to be highly correlated with the relative reactivities of penam or cephem antibiotics against PBPD2. Our study provides the structural basis for the inhibition of PBPD2 by clinically important β-lactam antibiotics that are commonly used in listeriosis treatment. Our findings imply that the modification of β-lactam side chains based on structural complementarity could be useful for the development of potent inhibitors against β-lactam-resistant PBPs.

This study was performed to improve the availability of reed and quality of traditional Yakju by using roasted reed. The pH of sprout Yakju was 3.43, whereas the pHs of other Yakjus were 3.57. No remarkable differences were observed in contents of total titratable acids and reducing sugars. Color changes were measured by Hunter’s color value; L value decreased, whereas redness (+a) and yellowness (+b) increased during fermentation. Ethanol content of Yakjus prepared with sprout and root were 15.30% and 15.28%, respectively, which were lower than that of control Yakju (15.31%). However, there were obvious differences of ethanol contents in three types of Yakjus. The major free sugar and organic acid were glucose and lactic acid, respectively. The total polyphenol contents of sprout Yakju (4.26 mg%) and root Yakju (4.21 mg%) were much higher than that of control Yakju (2.56 mg%). The DPPH (1,1-diphenyl-2-picryl- hydrazyl) free radical scavenging activities of sprout Yakju and root Yakju were higher than those of control Yakju. Yakju prepared from 0.3% addition of sprout showed the highest score in overall preference from the sensory test.

The type VI secretion system (T6SS) is a macromolecular complex that is conserved in Gram-negative bacteria. The T6SS secretes effector proteins into recipient cells in a contact-dependent manner in order to accomplish cooperative and competitive interactions with the cells. Although the composition and mechanism of the T6SS have been intensively investigated across many Gram-negative bacteria, to date structural information on T6SS components from the important pathogenVibrio choleraehas been rare. Here, the cloning, purification, crystallization and preliminary X-ray crystallographic analysis of the cytoplasmic domain of TssL, an inner membrane protein of the T6SS, fromV. choleraeare reported. Diffraction data were collected to 1.5 Å resolution using synchrotron radiation. The crystal belonged to the hexagonal space groupP61, with unit-cell parametersa= 78.4,b= 78.4, c= 49.5 Å. The successful structural characterization of TssL fromV. choleraewill contribute to understanding the role of the membrane-associated subunits of the T6SS in more detail.

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 A resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa. Proteins 2014; 82:1072–1078. © 2013 Wiley Periodicals, Inc.

Penicillin-binding proteins (PBPs), which catalyze the biosynthesis of the peptidoglycan chain of the bacterial cell wall, are the major molecular target of bacterial antibiotics. Here, we present the crystal structures of the bifunctional peptidoglycan glycosyltransferase (GT)/transpeptidase (TP) PBP4 from Listeria monocytogenes in the apo-form and covalently linked to two β-lactam antibiotics, ampicillin and carbenicillin. The orientation of the TP domain with respect to the GT domain is distinct from that observed in the previously reported structures of bifunctional PBPs, suggesting interdomain flexibility. In this structure, the active site of the GT domain is occluded by the close apposition of the linker domain, which supports the hypothesis that interdomain flexibility is related to the regulation of GT activity. The acylated structures reveal the mode of action of β-lactam antibiotics toward the class A PBP4 from the human pathogen L. monocytogenes . Ampicillin and carbenicillin can access the active site and be acylated without requiring a structural rearrangement. In addition, the active site of the TP domain in the apo-form is occupied by the tartrate molecule via extensive hydrogen bond interactions with the catalytically important residues; thus, derivatives of the tartrate molecule may be useful in the search for new antibiotics to inhibit PBPs.

Tumor necrosis factor receptor-associated factor 1 (TRAF1) is a multifunctional adaptor protein involved in important processes of cellular signaling, including innate immunity and apoptosis. TRAF family member-associated NF-kappaB activator (TANK) has been identified as a competitive intracellular inhibitor of TRAF2 function. Although TRAF recognition by various receptors has been studied extensively in the field of TRAF-mediated biology, molecular and functional details of TANK recognition and interaction with TRAF1 have not been studied. In this study, we report the crystal structure of the TRAF1/TANK peptide complex. Quantitative interaction experiments showed that TANK peptide interacts with both TRAF1 and TRAF2 with similar affinity in a micromolar range. Our structural study also reveals that TANK binds TRAF1 using a minor minimal consensus motif for TRAF binding, Px(Q/E)xT. Database Coordinate and structural factor were deposited in the Protein Data Bank under PDB ID code 5H10.

The synaptonemal complex protein 1 (SYCP1) is the main structural element of transverse filaments (TFs) of the synaptonemal complex (SC), which is a meiosis-specific complex structure formed at the synapse of homologue chromosomes to hold them together. The N-terminal domain of SYCP1 is known to be located within the central elements (CEs), whereas the C-terminal domain is located toward lateral elements (LEs). SYCP1 is a well-known meiosis marker that is also known to be a prognostic marker in the early stage of several cancers including breast, gliomas, and ovarian cancers. The structure of SC, especially the TF structure formed mainly by SYCP1, remains unclear without any structural information. To elucidate a molecular basis of SC formation and function, we first solved the crystal structure of C-terminal coiled-coil domain of SYCP1. The coiled-coil domain of SYCP1 forms asymmetric, anti-parallel dimers in solution.

Ketosteroid isomerase (3-oxosteroid Δ(5)-Δ(4)-isomerase, KSI) from Pseudomonas putida catalyzes allylic rearrangement of the 5,6-double bond of Δ(5)-3-ketosteroid to 4,5-position by stereospecific intramolecular transfer of a proton. The active site of KSI is formed by several hydrophobic residues and three catalytic residues (Tyr14, Asp38, and Asp99). In this study, we investigated the role of a hydrophobic Met112 residue near the active site in the catalysis, steroid binding, and stability of KSI. Replacing Met112 with alanine (yields M112A) or leucine (M112L) decreased the kcat by 20- and 4-fold, respectively. Compared with the wild type (WT), M112A and M112L KSIs showed increased KD values for equilenin, an intermediate analogue; these changes suggest that loss of packing at position 112 might lead to unfavorable steroid binding, thereby resulting in decreased catalytic activity. Furthermore, M112A and M112L mutations reduced melting temperature (Tm) by 6.4°C and 2.5°C, respectively. These changes suggest that favorable packing in the core is important for the maintenance of stability in KSI. The M112K mutation decreased kcat by 2000-fold, compared with the WT. In M112K KSI structure, a new salt bridge was formed between Asp38 and Lys112. This bridge could change the electrostatic potential of Asp38, and thereby contribute to the decreased catalytic activity. The M112K mutation also decreased the stability by reducing Tm by 4.1°C. Our data suggest that the Met112 residue may contribute to the catalytic activity and stability of KSI by providing favorable hydrophobic environments and compact packing in the catalytic core.

In situ neutron diffraction experiments were performed to measure the tensile deformation behavior of high manganese austenitic steels with different Al contents (0, 1.5, 2.0, 3.0 wt.%). Significant variations of peak shift, broadening and asymmetry of the diffraction peaks were observed in the plastic region with the measurement. Diffraction peak profile analysis was applied to determine microstructural parameters such as stacking/twinning fault probabilities, dislocation density and stacking fault energy (SFE). These parameters are quantitatively correlated to the yield strength, serrated flow and strain hardening rate during tensile deformation. The main results showed that the twin/stacking fault probability considerably decreases from 0.05 to 0.01 and dislocation density from 1016 to 4 × 1015 m−2 as a function of Al addition, while SFE (γ) increases from 20 to 45 mJ m−2 with the relationship of γ = 8.84 wt.% Al + 19.0 mJ m−2. Such microstructural parameters are also in good agreement with the results of the misorientation and pattern quality map obtained by the electron backscatter method.

We have investigated micro-structural parameters, twin and stacking fault probabilities and dislocation density, of twinning induced plasticity (TWIP) steels by using neutron diffraction profile analysis. It is observed that the addition of 1.4 wt.% Al in Fe–18Mn–0.6C steel leads to lower tensile strength and higher elongation, reducing twin formation rate. In other words, Al addition weakens the tendency of twin and stacking faults evolution under applied strain in the range of ɛ = 0.0–0.4, while the screw component of dislocations is increased. It is also confirmed that enlarged austenite grain size in TWIP steels disfavors twin and stacking fault formation.

Listeria monocytogenes is a facultative intracellular pathogen invading humans and animals with the highest fatality rate among the food-borne pathogens. The Listeria pathogenic processes, such as cell entry and escape from phagosomes, depend on the actions of diverse bacterial factors, including lipoproteins. Here, we report the crystal structure of Lmo2642, a conserved putative lipoprotein containing a Ser/Thr phosphatase domain. The protein consists of two distinct domains: a catalytic domain that belongs to the metallophosphoesterase superfamily and an auxiliary α-helical bundle domain. The active site in the catalytic domain of Lmo2642 contains a dinuclear metal center in which Mn²(+) and Fe³(+) are preferentially positioned at the site1 and site2, respectively. On the basis of the structural analysis and enzymatic assays, we identified the biochemical activity of the protein as a cyclic nucleotide phosphodiesterase toward 2',3'- and 3',5'-cyclic nucleotides. Considering the cNMP phosphodiesterase activity and the putative surface localization of Lmo2642, we speculate that Lmo2642 has some potential roles in the host-pathogen interactions by changing the cAMP concentration of host cells during L. monocytogenes infection.

TNF-receptor associated factor (TRAF) proteins are key adaptor molecules containing E3 ubiquitin ligase activity that play a critical role in immune cell signaling. TRAF1 is a unique family of TRAF lacking the N-terminal RING finger domain. TRAF1 is an important scaffold protein that participates in TNFR2 signaling in T cells as a negative or positive regulator via direct interaction with TRAF2, which has recently been identified as a pro-apoptotic regulator in neuronal cell death. Here, we report the first crystal structure of the TRAF1 TRAF domain containing both the TRAF-N coiled-coil domain and the TRAF-C domain. Our structure reveals both similarities and differences with other TRAF family members, which may be functionally relevant to TRAFs. We also found that the TRAF-N coiled-coil domain of TRAF1 is critical for the trimer formation and stability of the protein. Finally, we found that conserved surface residues on the TRAF1 TRAF domain that might be binding hot spots that are critical for interaction with signaling molecules.

Apoptosis repressor with caspase recruiting domain (ARC) is a multifunctional inhibitor of apoptosis that is unusually over-expressed or activated in various cancers and in the state of the pulmonary hypertension. Therefore, ARC might be an optimal target for therapeutic intervention. Human ARC is composed of two distinct domains, N-terminal caspase recruiting domain (CARD) and C-terminal P/E (proline and glutamic acid) rich domain. ARC inhibits the extrinsic apoptosis pathway by interfering with DISC formation. ARC CARD directly interacts with the death domains (DDs) of Fas and FADD, as well as with the death effector domains (DEDs) of procaspase-8. Here, we report the first crystal structure of the CARD domain of ARC at a resolution of 2.4 Å. Our structure was a dimer with novel homo-dimerization interfaces that might be critical to its inhibitory function. Interestingly, ARC did not exhibit a typical death domain fold. The sixth helix (H6), which was detected at the typical death domain fold, was not detected in the structure of ARC, indicating that H6 may be dispensable for the function of the death domain superfamily.

The in vitro and in vivo activities of DW286, a novel fluoronaphthyridone with potent antibacterial activity, were compared with those of ciprofloxacin, gemifloxacin, sparfloxacin, and trovafloxacin. Against gram-positive bacteria, such as Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus pneumoniae , and Enterococcus faecalis , the in vitro activity of DW286 was stronger than that of any other reference antibiotic. Against gram-negative bacteria, the activity of DW286 was similar to those of trovafloxacin and gemifloxacin but was weaker than that of ciprofloxacin. In a mouse systemic infection caused by three S. aureus strains, including methicillin-resistant S. aureus and quinolone-resistant S. aureus (QRSA), DW286 demonstrated the most potent activity, as found in vitro. Specially, DW286 is ≥8-fold more active against QRSA than the other fluoroquinolones. And the 50% protective doses for DW286 were correspondent with the in vitro activities.

Penicillin-binding proteins (PBPs), which catalyze peptidoglycan synthesis, have been extensively studied as a well established target of antimicrobial agents, including β-lactam derivatives. However, remarkable resistance to β-lactams has developed among pathogenic bacteria since the clinical use of penicillin began. Recently, the glycosyltransferase (GT) domain of class A PBPs has been proposed as an attractive target for antibiotic development as moenomycin-bound GT-domain structures have been determined. In this study, a class A PBP4 from Listeria monocytogenes was overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected to 2.1 Å resolution using synchrotron radiation. The crystal belonged to the primitive orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 84.6, b = 127.8, c = 54.9 Å. The structural information will contribute to the further development of moenomycin-derived antibiotics possessing broad-spectrum activity.

Penicillin-binding proteins catalyze the biosynthesis of the peptidoglycan chains of the bacterial cell wall, which protects cells from osmotic pressure. Although Lmo0540 has been identified as a putative penicillin-binding protein that contributes to the virulence of Listeria monocytogenes, the biochemical role of Lmo0540 remains unclear. To provide insights into its biochemical function, Lmo0540 was overexpressed, purified and crystallized by the sitting-drop vapour-diffusion method. Diffraction data were collected to 1.5 A resolution using synchrotron radiation. The crystal belonged to the C-­centred monoclinic space group C2, with unit-cell parameters a = 82.5, b = 75.7, c = 75.9 A, α = γ = 90, β = 121.8°. A full structural determination is under way in order to elucidate the structure–function relationship of this protein.

YgjG is a putrescine aminotransferase enzyme that transfers amino groups from compounds with terminal primary amines to compounds with an aldehyde group using pyridoxal-5'-phosphate (PLP) as a cofactor. Previous biochemical data show that the enzyme prefers primary diamines, such as putrescine, over ornithine as a substrate. To better understand the enzyme's substrate specificity, crystal structures of YgjG from Escherichia coli were determined at 2.3 and 2.1 angstrom resolutions for the free and putrescine-bound enzymes, respectively. Sequence and structural analyses revealed that YgjG forms a dimer that adopts a class III PLP-dependent aminotransferase fold. A structural comparison between YgjG and other class III aminotransferases revealed that their structures are similar. However, YgjG has an additional N-terminal helical structure that partially contributes to a dimeric interaction with the other subunit via a helix-helix interaction. Interestingly, the YgjG substrate-binding site entrance size and charge distribution are smaller and more hydrophobic than other class III aminotransferases, which suggest that YgjG has a unique substrate binding site that could accommodate primary aliphatic diamine substrates, including putrescine. The YgjG crystal structures provide structural clues to putrescine aminotransferase substrate specificity and binding.

EgtD is an S-adenosyl-l-methionine (SAM)-dependent histidine N,N,N-methyltransferase that catalyzes the formation of hercynine from histidine in the ergothioneine biosynthetic process of Mycobacterium smegmatis. Ergothioneine is a secreted antioxidant that protects mycobacterium from oxidative stress. Here, we present three crystal structures of EgtD in the apo form, the histidine-bound form, and the S-adenosyl-l-homocysteine (SAH)/histidine-bound form. The study revealed that EgtD consists of two distinct domains: a typical methyltransferase domain and a unique substrate binding domain. The histidine binding pocket of the substrate binding domain primarily recognizes the imidazole ring and carboxylate group of histidine rather than the amino group, explaining the high selectivity for histidine and/or (mono-, di-) methylated histidine as substrates. In addition, SAM binding to the MTase domain induced a conformational change in EgtD to facilitate the methyl transfer reaction. The structural analysis provides insights into the putative catalytic mechanism of EgtD in a processive trimethylation reaction.

The 3D shape reconstruction of an object from its 2D cross-sections is important for reproducing it by NC machining or rapid prototyping, Although several different reconstruction methods have been proposed most of them have allowed only simple branching, or have had difficulty in handling complex branching structures. In this paper, a new method is presented for solving branching problems in surface reconstruction from a set of free-form contours in planar cross-sections. In this method, we decompose each multiple branching region into a set of single branching regions by providing a set of intermediary contours using modified distance maps. Then, each pair of contours in the single branching regions is linked with triangular facets to construct a piecewise triangular G 1 Bezier surface, An experimental result is given to show that our method gives reasonably good solutions for the representation of complex-shaped objects from planar contours.

The shape reconstruction of a 3D object from its 2D crosssections is important for reproducing it by NC machining or rapid prototyping. In this paper, we present a method of surface approximation to cross-sections with multiple branching problems. In this method, we first decompose each multiple branching problem into a set of single branching problems by providing a set of intermediate contours using distance maps. For each single branching region, a procedure then performs the skinning of contour curves represented by cubic B-spline curves on a common knot vector, each of which is fitted to its contour points within a given accuracy. In order to acquire a more compact representation for the surface, the method includes an algorithm for reducing the number of knots in the common knot vector. The approximation surface to the crosssections is represented by a set of bicubic B-spline surfaces. This method provides a smooth surface model, yet realises efficient data reduction.

In this paper, we present a new procedure for generating tool paths using discrete distance maps, especially for dealing with free-form shaped pockets with multiple islands. In this procedure, a discrete distance map is computed by shading a right circular cone having the height of a given offset distance, while moving its apex along the boundary curve segments. Using the discrete distance maps, the proposed algorithm effectively extracts the characteristic points or the valid self-intersection points of offset curve segments. For an offset distance, one or more offset profiles are constructed without the topological problems by offsetting the boundary curve segments within the parameter values specified by these characteristic points, and connecting them into closed profiles using the topological information contained in the distance map. The gouging-free tool path is constructed by connecting these offset profiles, starting from the innermost offset profile. In the proposed method, we do not need any artificial bridges for a pocket with multiple islands to merge the pocket profile and the island profiles into a single boundary profile.

Voronoi diagrams for closed shapes have many practical applications, ranging from numerical control machining to mesh generation. Curve offsetting based on Voronoi diagrams avoids the topological problems encountered in the traditional offsetting algorithms. In this paper, we propose a procedure using Voronoi diagram-based tool-path generation for machining pockets with free-form boundaries. A pocket can be bounded by 2D free-form curve elements that are differentiable twice. The procedure consists of three steps

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

The crystal structure of Ton1535, a hypothetical protein from Thermococcus onnurineus NA1, was determined at 2.3 Å resolution. With two antiparallel α-helices in a helix-turn-helix motif as a repeating unit, Ton1535 consists of right-handed coiled N- and C-terminal regions that are stacked together using helix bundles containing a left-handed helical turn. One left-handed helical turn in the right-handed coiled structure produces two unique structural properties. One is the presence of separated concave grooves rather than one continuous concave groove, and the other is the contribution of α-helices on the convex surfaces of the N-terminal region to the extended surface of the concave groove of the C-terminal region and vice versa.