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SshEstI, a carboxylesterase from the thermoacidophilic archaeon Saccharolobus shibatae, is a member of the hormone-sensitive lipase family that displays slightly alkaliphilic activity with an optimum activity at pH 8.0. In this study, three distinct strategies were explored to confer acidophilic properties to SshEstI. The first strategy involved engineering the oxyanion hole by replacing Gly81 with serine or aspartic acid. The G81S mutant showed optimum activity at pH 7.0, whereas the aspartic acid mutant (G81D) rendered the enzyme slightly acidophilic with optimum activity observed at pH 6.0; however, k cat and k cat /K m values were reduced by these substitutions. The second strategy involved examining the effects of surfactant additives on the pH-activity profiles of SshEstI. The results showed that cetyltrimethylammonium bromide (CTAB) enhanced wild-type enzyme (WT) activity at acidic pH values. In the presence of 0.1 mM CTAB, G81S and G81D were acidophilic enzymes with optimum activity at pH 6.0 and 4.0, respectively, although their enzyme activities were low. The third strategy involved engineering the active site to resemble that of kumamolisin-As (kuma-As), an acidophilic peptidase of the sedolisin family. The catalytic triad of kuma-As was exchanged into SshEstI using site-directed mutagenesis. X-ray crystallographic analysis of the mutants (H274D and H274E) revealed that the potential hydrogen donor-acceptor distances around the active site of WT were fully maintained in these mutants. However, these mutants were inactive at pH 4-8., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Neonatal compartment syndrome, although rare, has a classic presentation with sentinel skin findings and development of swelling, erythema, and tenderness of the affected extremity. Neonatal compartment syndrome requires prompt surgical intervention to preserve the affected limb and ensure its normal growth and development. Our patient was born at term via vaginal delivery complicated by a compound presentation involving the left upper extremity. No physical exam abnormalities were noted at birth, but she developed signs of neonatal compartment syndrome by 15 h of life. She was surgically treated at 22 h of life and recovered well. At one year of age, she has normal growth and function of the affected extremity. Our case adds to the growing literature associating neonatal compartment syndrome with a compound fetal presentation., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: The aim of this study was to evaluate the phase composition, phase transformation temperatures, bending property, and cyclic fatigue resistance of different heat-treated nickel-titanium (NiTi) rotary instruments with the same tip diameter and taper at room (RT; 25 ± 1 °C) and body (BT; 37 ± 1 °C) temperatures., Methods: Five heat-treated NiTi rotary instruments, HyFlex EDM (EDM), HyFlex CM (CM), Vortex Blue (VB), RE file CT (RE) and JIZAI, and a non-heat-treated NiTi rotary instrument (Mtwo) with a size 40, 0.04 taper were investigated. Temperature-dependent phase transformation was examined with differential scanning calorimetry (DSC). The bending loads of the instruments at RT and BT were evaluated using a cantilever-bending test. Cyclic fatigue resistance at RT and BT was measured using a dynamic test, during which the instruments were rotated in combination with a 2-mm back-and-forth motion in an artificial curved canal, and the number of cycles to failure (NCF) was determined. The results were analyzed using two-way repeated measures analysis of variance, a simple main effect test, and the Bonferroni test (α = 0.05)., Results: DSC results indicated that EDM and Mtwo were primarily composed of martensite/R-phase and austenite, respectively, while the other heat-treated instruments were composed of a mix of martensite/R-phase and austenite at the tested temperatures. Regardless of the temperature setting, the bending loads of heat-treated instruments were significantly lower than those of Mtwo (p < 0.05). EDM showed the lowest bending loads and highest NCF at both temperatures (p < 0.05). CM, VB, and JIZAI showed significantly higher bending loads at BT than at RT (p < 0.05). The NCF of all the heat-treated instruments, except VB, was lower at BT than at RT (p < 0.05). At BT, the NCF of CM, VB, RE, and JIZAI were not significantly higher than that of Mtwo (p > 0.05)., Conclusions: Heat-treated NiTi instruments exhibited lower bending loads and higher NCF values than Mtwo. However, this tendency was less pronounced at BT than at RT, especially in the NCF values of instruments with a mixture of martensite/R-phase and austenite phases at the tested temperatures., (© 2023. The Author(s).)

Due to their remarkable structural diversity, glycans play important roles as recognition molecules on cell surfaces of living organisms. Carbohydrates exist in numerous isomeric forms and can adopt diverse structures through various branching patterns. Despite their relatively small molecular weights, they exhibit extensive structural diversity. On the other hand, lectins, also known as carbohydrate-binding proteins, not only recognize and bind to the diverse structures of glycans but also induce various biological reactions based on structural differences. Initially discovered as hemagglutinins in plant seeds, lectins have been found to play significant roles in cell recognition processes in higher vertebrates. However, our understanding of lectins in marine animals, particularly marine invertebrates, remains limited. Recent studies have revealed that marine animals possess novel lectins with unique structures and glycan recognition mechanisms not observed in known lectins. Of particular interest is their role as pattern recognition molecules in the innate immune system, where they recognize the glycan structures of pathogens. Furthermore, lectins serve as toxins for self-defense against foreign enemies. Recent discoveries have identified various pore-forming proteins containing lectin domains in fish venoms and skins. These proteins utilize lectin domains to bind target cells, triggering oligomerization and pore formation in the cell membrane. These findings have spurred research into the new functions of lectins and lectin domains. In this review, we present recent findings on the diverse structures and functions of lectins in marine animals.

Background/purpose: Using conventional approach to examine stationary torque of nickel-titanium rotary instruments contradicts the clinical condition, and its validity for motions involving clockwise and counterclockwise rotations is questionable. This study aimed to examine the effect of different kinematics on the torsional behavior using a JIZAI instrument (#25/.04) under stationary/dynamic test conditions using clinical torque limit settings., Materials and Methods: In the stationary test, the 5-mm tip of JIZAI was fixed in a cylinder-shaped vise and rotated in continuous rotation (CR) with auto-torque-reverse, optimum-torque-reverse (OTR), or reciprocation (REC) until fracture (n = 10, each). In the dynamic test, straight and severe curved canals were instrumented with JIZAI using the single-length technique with CR, OTR, or REC (n = 10, each). The stationary torque at fracture, time to fracture (T f ), dynamic torque, and screw-in force were recorded using automated-shaping-device with torque/force measuring unit. One-way ANOVA or Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction were used for statistical analysis ( ⍺  = 0.05)., Results: The kinematics did not influence the stationary or dynamic torques ( P  > 0.05); however, did influence the screw-in force in straight canals ( P  < 0.05). REC had significantly longer T f , and severe curved canals yielded significantly greater torque and screw-in force in CR ( P  < 0.05)., Conclusion: Under the present experimental conditions, parameters other than torque showed significant effects on different kinematics. The dynamic torque and screw-in force of OTR were similar to the other rotational modes and not influenced by the canal curvature., (© 2022 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Selection of an appropriate nickel−titanium (NiTi) rotary system is important for minimally invasive endodontic treatment, which aims to preserve as much root canal dentin as possible. This study aimed to evaluate selected mechanical properties and the root canal shaping ability of TruNatomy (TRN), a NiTi rotary system designed for minimally invasive endodontic shaping, in comparison with existing instruments: HyFlex EDM (HEDM), ProTaper Next (PTN), and WaveOne Gold (WOG). Load values measured with a cantilever bending test were ranked as TRN < HEDM < WOG < PTN (p < 0.05). A dynamic cyclic fatigue test revealed that the number of cycles to fracture was ranked as HEDM > WOG > TRN > PTN (p < 0.05). Torque and vertical force generated during instrumentation of J-shaped artificial resin canals were measured using an automated instrumentation device connected to a torque and vertical force measuring system; TRN exhibited smaller torque and vertical force values in most comparisons with the other instruments. The canal centering ratio for TRN was smaller than or comparable to that for the other instruments except for WOG at the apex level. Under the present experimental conditions, TRN showed higher flexibility and lower torque and vertical force values than the other instruments.

This study aimed to evaluate how various rotational modes influence the torque/force production and shaping ability of ProTaper Universal (PTU; non-heat-treated) and ProTaper Gold (PTG; heat-treated) nickel−titanium instruments. J-shaped resin canals were instrumented with PTU or PTG using an automated instrumentation device operated with reciprocating rotation [150° clockwise and 30° counterclockwise (R150/30) or 240° clockwise and 120° counterclockwise (R240/120)], optimum torque reverse motion (OTR), or continuous rotation (CR) (n = 10 each). Maximum force and torque were recorded, and canal centering ratios were calculated. Statistical analysis was performed with two-way ANOVA and a Bonferroni test (α = 0.05). The results were considered with reference to previous studies on the microstructure of the instruments. The upward force generated by R240/120 and OTR was smaller than that generated by R150/30 and CR in PTG (p < 0.05). The clockwise torque produced by OTR was lower than that produced by R150/30 in PTU and R240/120 and CR in PTG (p < 0.05). R240/120 and OTR induced less canal deviation compared to CR in PTU at 0 mm from the apex (p < 0.05). In conclusion, R240/120 and OTR reduced the screw-in force in PTG and improved the canal centering ability in PTU, which may be associated with the heat treatment-induced microstructural difference of the two instruments.

This study aimed to examine how downward loads influence the torque/force and shaping outcome of ProTaper NEXT (PTN) rotary instrumentation. PTN X1, X2, and X3 were used to prepare J-shaped resin canals employing a load-controlled automated instrumentation and torque/force measuring device. Depending on the torque values, the handpiece was programmed to move as follows: up and down; downward at a preset downward load of 1 N, 2 N or 3 N (Group 1N, 2N, and 3N, respectively; each n = 10); or upward. The torque/force values and instrumentation time were recorded, and the canal centering ratio was calculated. The results were analyzed using a two-way or one-way analysis of variance and the Tukey test (α = 0.05). At the apex level, Group 3N exhibited the least canal deviation among the three groups (p < 0.05). The downward force was Group 3N > Group 2N > Group 1N (p < 0.05). The upward force, representing the screw-in force, was Group 3N > Group 1N (p < 0.05). The total instrumentation time was Group 1N > Group 3N (p < 0.05). In conclusion, increasing the downward load during PTN rotary instrumentation improved the canal centering ability, reduced the instrumentation time, and increased the upward force.

Objective: TNIIIA2 is a peptide of the extracellular matrix glycoprotein tenascin-C. We evaluated whether intra-articular injection of TNIIIA2 could prevent articular cartilage degeneration without inducing synovitis in an osteoarthritis mice model., Design: Ten micrograms per milliliter of TNIIIA2 were injected into the knee joint of mice (group II) to evaluate the induction of synovitis. The control group received an injection of phosphate buffered saline (group I). Synovitis was evaluated using synovitis score 2 and 4 weeks after injection. The ligaments of knee joints of mice were transected to make the osteoarthritis model. After transection, 10 µg/mL of TNIIIA2 was injected into the knee joint (group IV). The control group received an injection of phosphate buffered saline after transection (group III). Histologic examinations were made using hematoxylin and eosin and safranin-O staining at 2, 4, 8, and 12 weeks postoperatively. An in vitro study was also performed to determine the mechanism by which TNIIIA2 prevents cartilage degeneration. Human chondrocytes were isolated, cultured, and treated with TNIIIA2. The expressions of various mRNAs, including inflammatory cytokines, and anabolic and catabolic factors for cartilage were compared using real-time polymerase chain reaction., Results: There were no differences between groups in the study of intra-articular injection of mice (group I vs. group II). In the osteoarthritis model, we found development of osteoarthritis was suppressed in group IV at 4 and 8 weeks. TNIIIA2 upregulated the expressions of tumor necrosis factor-α, matrix metalloproteinase 3, and basic fibroblast growth factor., Conclusion: We demonstrated that TNIIIA2 could prevent cartilage degeneration without synovitis.

Recently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators' concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

Polyprenyl groups, products of isoprenoid metabolism, are utilized in peptidoglycan biosynthesis, protein N-glycosylation, and other processes. These groups are formed by cis-prenyltransferases, which use allylic prenyl pyrophosphates as prenyl-donors to catalyze the C-prenylation of the general acceptor substrate, isopentenyl pyrophosphate. Repetition of this reaction forms (Z,E-mixed)-polyprenyl pyrophosphates, which are converted later into glycosyl carrier lipids, such as undecaprenyl phosphate and dolichyl phosphate. MM&#95;0014 from the methanogenic archaeon Methanosarcina mazei is known as a versatile cis-prenyltransferase that accepts both isopentenyl pyrophosphate and dimethylallyl pyrophosphate as acceptor substrates. To learn more about this enzyme's catalytic activity, we determined the X-ray crystal structures of MM&#95;0014 in the presence or absence of these substrates. Surprisingly, one structure revealed a complex with O-prenylglycerol, suggesting that the enzyme catalyzed the prenylation of glycerol contained in the crystallization buffer. Further analyses confirmed that the enzyme could catalyze the O-prenylation of small alcohols, such as 2-propanol, expanding our understanding of the catalytic ability of cis-prenyltransferases., Competing Interests: Conflict of interest The authors declare no conflicts of interest in regard to this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Starch produced by plants is a stored form of energy and is an important dietary source of calories for humans and domestic animals. Disproportionating enzyme (D-enzyme) catalyzes intramolecular and intermolecular transglycosylation reactions of α-1, 4-glucan. D-enzyme is essential in starch metabolism in the potato. We present the crystal structures of potato D-enzyme, including two different types of complex structures: a primary Michaelis complex (substrate binding mode) for 26-meric cycloamylose (CA26) and a covalent intermediate for acarbose. Our study revealed that the acarbose and CA26 reactions catalyzed by potato D-enzyme involve the formation of a covalent intermediate with the donor substrate. HPAEC of reaction substrates and products revealed the activity of the potato D-enzyme on acarbose and CA26 as donor substrates. The structural and chromatography analyses provide insight into the mechanism of the coupling reaction of CA and glucose catalyzed by the potato D-enzyme. The enzymatic reaction mechanism does not involve residual hydrolysis. This could be particularly useful in preventing unnecessary starch degradation leading to reduced crop productivity. Optimization of this mechanism would be important for improvements of starch storage and productivity in crops., (© 2020 The Protein Society.)

The C-type lectins SPL-1 and SPL-2 from the bivalve Saxidomus purpuratus are composed of A and B chains and of two B chains, respectively. They bind specific carbohydrates containing acetamido groups, such as N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc), in a Ca 2+ -independent manner. Unlike ordinary C-type lectins, which require Ca 2+ ions for carbohydrate recognition, these lectins recognize specific carbohydrates mainly through interactions with the acetamido group without Ca 2+ ions, even though Ca 2+ enhances the binding affinity of these lectins, especially SPL-1. In the present study, the crystal structure of the SPL-1-GlcNAc complex in the presence of Ca 2+ revealed that the binding of SPL-1 to GlcNAc is stabilized by hydrogen bonds to the water molecule(s) coordinating Ca 2+ , whereas in ordinary C-type lectins Ca 2+ directly forms coordinate bonds to the hydroxy groups of carbohydrates. These differences may also allow SPL-1 and SPL-2 to recognize both GlcNAc and GalNAc, which have different orientations of the 4-hydroxy group.

Background: The effects of tenascin-C (TNC) on cartilage repair were examined in cartilage defect model mice. An in vitro study was also performed to determine the mechanism of cartilage repair with TNC., Methods: Full-thickness osteochondral defects were filled with TNC (group A: 100 μg/ml, group B: 10 μg/ml, group C: empty). Mice were sacrificed at 1, 2, 3, and 6 weeks postoperatively. Cartilage repair was histologically evaluated using the modified WAKITANI score. Chondrocytes were isolated and cultured, and they were treated with TNC. The expressions of various mRNAs including TNC, inflammatory cytokines, and anabolic and catabolic factors for cartilage were compared by real-time polymerase chain reaction., Results: The defects in group A were covered with hyaline-like cartilage after 3 weeks. Average modified WAKITANI scores were significantly better in group A than in groups B and C at 3 and 6 weeks. TNC upregulated the expressions of endogenous TNC, inflammatory cytokines, and anabolic and catabolic factors for cartilage. TNC downregulated the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5., Conclusions: Intra-articular injection of full-length TNC repaired cartilage in murine models of full-thickness osteochondral defects. TNC upregulated the expression of ADAMTS4, but downregulated the expression of ADAMTS5 that contributed to cartilage degradation., (Copyright © 2019 The Japanese Orthopaedic Association. Published by Elsevier B.V. All rights reserved.)

Novel Ca 2+ -independent C-type lectins, SPL-1 and SPL-2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL-2 composed of two B-chains) or distinct (SPL-1 composed of A- and B-chains) polypeptide chains, and show affinity for N-acetylglucosamine (GlcNAc)- and N-acetylgalactosamine (GalNAc)-containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C-type lectin family. X-ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate-recognition domain (CRD) of the C-type lectin family. Nevertheless, these lectins (especially SPL-2) showed Ca 2+ -independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL-2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate-recognition motifs among the C-type CRD (the QPD [Gln-Pro-Asp] and EPN [Glu-Pro-Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate-binding specificities of individual A- and B-chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α-glycosidic linkages with slightly different specificities., (© 2019 The Protein Society.)

Chitin, which is a major component of house dust mites (HDM), fungi, crustaceans, etc., can activate immune cells, suggesting that it contributes to development of allergic disorders such as asthma. Although the pathophysiological sensitization route of asthmatic patients to allergens is considered via the respiratory tract, the roles of intranasally-administered chitin in development of asthma remain unclear. After ovalbumin (OVA) challenge, development of airway inflammation was profoundly exacerbated in mice sensitized with OVA in the presence of chitin. The exacerbation was dependent on IL-33, but not IL-25, thymic stromal lymphopoietin or IL-17A. Chitin enhanced IL-33-dependent IL-1β production by dendritic cells (DCs). Furthermore, chitin- and IL-33-stimulated DC-derived IL-1β promoted OVA-specific Th2 cell activation, resulting in aggravation of OVA-induced airway inflammation. These findings indicate the adjuvant activity of chitin via a new mechanism and provide important clues for development of therapeutics for allergic disorders caused by HDM, fungi and crustaceans.

A novel galactose-specific lectin, AJLec (18.5 kDa), was isolated from the sea anemone, Anthopleura japonica. AJLec was characterized using the hemagglutination assay, isothermal titration calorimetry (ITC), and glycoconjugate microarray analysis and we found that AJLec has a specificity for galactose monomers and β-linked terminal galactose residues in complex carbohydrates, but not for N-acetylgalactosamine (GalNAc), which is commonly recognized by galactose-binding lectins. The primary structure of AJLec did not show homology with known lectins, and a crystal structural analysis also revealed a unique homodimeric structure. The crystal structure of AJLec complexed with lactose was solved by measuring the sulfur single-wavelength anomalous diffraction (S-SAD) phasing with an in-house Cu Kα source method. This analysis revealed that the galactose residue in lactose was recognized via its O2, O3, and O4 hydroxyl groups and ring oxygen by calcium coordination and two hydrogen bonds with residues in the carbohydrate-binding site, which demonstrated strict specificity for the β-linked terminal galactose in this lectin.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure., (© 2017 The Protein Society.)

The biosynthesis of isopentenyl diphosphate, a fundamental precursor for isoprenoids, via the mevalonate pathway is completed by diphosphomevalonate decarboxylase. This enzyme catalyzes the formation of isopentenyl diphosphate through the ATP-dependent phosphorylation of the 3-hydroxyl group of ( R )-5-diphosphomevalonate followed by decarboxylation coupled with the elimination of the 3-phosphate group. In this reaction, a conserved aspartate residue has been proposed to be involved in the phosphorylation step as the general base catalyst that abstracts a proton from the 3-hydroxyl group. In this study, the catalytic mechanism of this rare type of decarboxylase is re-investigated by structural and mutagenic studies on the enzyme from a thermoacidophilic archaeon Sulfolobus solfataricus The crystal structures of the archaeal enzyme in complex with ( R )-5-diphosphomevalonate and adenosine 5'- O -(3-thio)triphosphate or with ( R )-5-diphosphomevalonate and ADP are newly solved, and theoretical analysis based on the structure suggests the inability of proton abstraction by the conserved aspartate residue, Asp-281. Site-directed mutagenesis on Asp-281 creates mutants that only show diphosphomevalonate 3-kinase activity, demonstrating that the residue is required in the process of phosphate elimination/decarboxylation, rather than in the preceding phosphorylation step. These results enable discussion of the catalytic roles of the aspartate residue and provide clear proof of the involvement of a long predicted intermediate, ( R )-3-phospho-5-diphosphomevalonate, in the reaction of the enzyme., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A novel mannose-specific lectin, named CGL1 (15.5 kDa), was isolated from the oyster Crassostrea gigas. Characterization of CGL1 involved isothermal titration calorimetry (ITC), glycoconjugate microarray, and frontal affinity chromatography (FAC). This analysis revealed that CGL1 has strict specificity for the mannose monomer and for high mannose-type N-glycans (HMTGs). Primary structure of CGL1 did not show any homology with known lectins but did show homology with proteins of the natterin family. Crystal structure of the CGL1 revealed a unique homodimer in which each protomer was composed of 2 domains related by a pseudo two-fold axis. Complex structures of CGL1 with mannose molecules showed that residues have 8 hydrogen bond interactions with O1, O2, O3, O4, and O5 hydroxyl groups of mannose. The complex interactions that are not observed with other mannose-binding lectins revealed the structural basis for the strict specificity for mannose. These characteristics of CGL1 may be helpful as a research tool and for clinical applications.

Unlabelled: In the present study, the crystal structure of recombinant diphosphomevalonate decarboxylase from the hyperthermophilic archaeon Sulfolobus solfataricus was solved as the first example of an archaeal and thermophile-derived diphosphomevalonate decarboxylase. The enzyme forms a homodimer, as expected for most eukaryotic and bacterial orthologs. Interestingly, the subunits of the homodimer are connected via an intersubunit disulfide bond, which presumably formed during the purification process of the recombinant enzyme expressed in Escherichia coli. When mutagenesis replaced the disulfide-forming cysteine residue with serine, however, the thermostability of the enzyme was significantly lowered. In the presence of β-mercaptoethanol at a concentration where the disulfide bond was completely reduced, the wild-type enzyme was less stable to heat. Moreover, Western blot analysis combined with nonreducing SDS-PAGE of the whole cells of S. solfataricus proved that the disulfide bond was predominantly formed in the cells. These results suggest that the disulfide bond is required for the cytosolic enzyme to acquire further thermostability and to exert activity at the growth temperature of S. solfataricus., Importance: This study is the first report to describe the crystal structures of archaeal diphosphomevalonate decarboxylase, an enzyme involved in the classical mevalonate pathway. A stability-conferring intersubunit disulfide bond is a remarkable feature that is not found in eukaryotic and bacterial orthologs. The evidence that the disulfide bond also is formed in S. solfataricus cells suggests its physiological importance., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The purpose of this study is to report our clinical outcomes using intensity-modulated radiation therapy (IMRT) for adjuvant treatment of cervical cancer, compared with three-dimensional conformal radiation therapy (3DCRT), in terms of tumor control, complications and dose-volume histogram (DVH) parameters., Methods: Between March 2008 and February 2014, 62 patients were treated with concurrent nedaplatin-based chemotherapy and whole-pelvic external beam radiation therapy (RT). Of these patients, 32 (52%) received 3DCRT and 30 (48%) received IMRT., Results: The median follow-up periods were 40 months (range 2-74 months). The 3-year overall survival rate (OS), locoregional control rate (LRC) and progression-free survival rate (PFS) were 92, 95 and 92% in the IMRT group, and 85, 82 and 70% in the 3DCRT group, respectively. A comparison of OS, LRC and PFS showed no significant differences between IMRT and 3DCRT. The 3-year cumulative incidences of grade 2 or higher chronic gastrointestinal (GI) complications were significantly lower with IMRT compared to 3DCRT (3% vs. 45%, p < .02) and in patients with V40 of the small bowel loops of ≤340 mL compared to those with >340 mL (3% vs. 45%, p < .001). Patients treated with IMRT had a higher incidence of grade 3 acute hematologic complications (p < .05). V40 and V45 of the small bowel loops or bowel bag were predictive for development of both acute and chronic GI complications., Conclusions: Our results suggest that IMRT for adjuvant treatment of cervical cancer is useful for decreasing GI complications without worsening outcomes.

A rapid and efficient DNA extraction method was developed for detecting mastitis pathogens in milk. The first critical step involved cell wall disruption by bead-beating, as physical disruption using beads was more effective for DNA extraction from Gram-positive bacteria, such as Staphylococcus aureus, than enzymatic disruption using proteinase K. The second critical step involves the use of acetic acid and ammonium sulfate in the purification process, as these reagents effectively and efficiently remove the lipids and proteins in milk. Using these methods, DNA suitable for loop-mediated isothermal amplification was obtained within 30 min. Also, the rapid and sensitive detection of S. aureus in milk was possible at levels as low as 200 cfu/ml.

Background: IL-25, IL-33 and TSLP are produced predominantly by epithelial cells and are known to induce Th2-type cytokines. Th2-type cytokines are involved not only in host defense against nematodes, but also in the development of Th2-type allergic diseases. TSLP was reported to be crucial for development of allergic airway inflammation in mice after inhalation of allergens to which they had been sensitized epicutaneously (EC) beforehand. However, the roles of IL-25 and IL-33 in the setting remain unclear., Methods: Mice deficient in IL-25 and IL-33 were sensitized EC with ovalbumin (OVA) and then challenged intranasally with OVA. Airway inflammation, the number of inflammatory cells in bronchoalveolar lavage fluids (BALFs) and airway hyperresponsiveness (AHR) in the mice were determined, respectively, by histological analysis, with a hemocytometer, and by using plethysmograph chambers with a ventilator. Expression of mRNA in the skin and lungs was determined by quantitative PCR, while the BALF levels of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) and the serum levels of IgE were determined by ELISA., Results: Normal OVA-specific Th2- and Th17-cell responses of lymph nodes and spleens were observed in IL-25-deficient (IL-25-/-) and IL-33-/- mice after EC sensitization with OVA. Nevertheless, the number of eosinophils, but not neutrophils, in the BALFs, and the levels of Th2 cytokines, but not Th17 cytokines, in the lungs were significantly decreased in the IL-25-/- and IL-33-/- mice pre-sensitized EC with OVA, followed by inhalation of OVA, whereas their levels of AHR and OVA-specific serum IgE were normal., Conclusions: Both IL-25 and IL-33 are critical for induction of Th2-type cytokine-mediated allergic airway eosinophilia, but not Th17-type cytokine-mediated airway neutrophilia, at the local sites of lungs in the challenge phase of mice sensitized EC with OVA. They do not affect OVA-specific T-cell induction in the sensitization phase.

House dust mite-derived proteases contribute to allergic disorders in part by disrupting epithelial barrier function. Interleukin-33 (IL-33), produced by lung cells after exposure to protease allergens, can induce innate-type airway eosinophilia by activating natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2), to secrete Th2 type-cytokines. Because IL-33 also can induce mast cells (MCs) to secrete Th2 type-cytokines, MCs are thought to cooperate with NH cells in enhancing protease or IL-33-mediated innate-type airway eosinophilia. However, we found that MC-deficient Kit(W-sh/W-sh) mice exhibited exacerbated protease-induced lung inflammation associated with reduced numbers of regulatory T (Treg) cells. Moreover, IL-2 produced by IL-33-stimulated MCs promoted expansion of numbers of Treg cells, thereby suppressing development of papain- or IL-33-induced airway eosinophilia. We have thus identified a unique anti-inflammatory pathway that can limit induction of innate-type allergic airway inflammation mediated by NH cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Silica crystals (silica), which are the main mineral component of volcanic ash and desert dust, can activate the caspase-1-activating inflammasome in phagocytic cells to secrete IL-1β. Although inhalation of silica-containing dust is known to exacerbate chronic respiratory diseases, probably through inflammasome activation, its direct effects on bronchial epithelial cells remain unclear. Here, we show that silica and double-stranded RNA (dsRNA) synergistically induces caspase-9-dependent apoptosis, but not inflammasome activation, of bronchial epithelial cells. Intranasal administration of silica and dsRNA to mice synergistically enhanced neutrophil infiltration in the airway without IL-1β release in the bronchoalveolar lavage fluid. Histopathological analysis revealed that silica or dsRNA alone induced slight airway inflammation, whereas combined administration significantly enhanced airway inflammation and epithelial damage. These novel findings suggest that inhalation of silica-containing dust may cause inflammasome-independent airway inflammation, possibly by damaging the epithelial barrier, especially at the time of viral infection. These responses may also be involved in acute lung injury caused by inhaled silica-containing dust.

To investigate the mechanism for low pH adaptation by a carboxylesterase, structural and biochemical analyses of EstFa&#95;R (a recombinant, slightly acidophilic carboxylesterase from Ferroplasma acidiphilum) and SshEstI (an alkaliphilic carboxylesterase from Sulfolobus shibatae DSM5389) were performed. Although a previous proteomics study by another group showed that the enzyme purified from F. acidiphilum contained an iron atom, EstFa&#95;R did not bind to iron as analyzed by inductively coupled plasma MS and isothermal titration calorimetry. The crystal structures of EstFa&#95;R and SshEstI were determined at 1.6- and 1.5-Å resolutions, respectively. EstFa&#95;R had a catalytic triad with an extended hydrogen bond network that was not observed in SshEstI. Quadruple mutants of both proteins were created to remove or introduce the extended hydrogen bond network. The mutation on EstFa&#95;R enhanced its catalytic efficiency and gave it an alkaline pH optimum, whereas the mutation on SshEstI resulted in opposite effects (i.e. a decrease in the catalytic efficiency and a downward shift in the optimum pH). Our experimental results suggest that the low pH optimum of EstFa&#95;R activity was a result of the unique extended hydrogen bond network in the catalytic triad and the highly negatively charged surface around the active site. The change in the pH optimum of EstFa&#95;R happened simultaneously with a change in the catalytic efficiency, suggesting that the local flexibility of the active site in EstFa&#95;R could be modified by quadruple mutation. These observations may provide a novel strategy to elucidate the low pH adaptation of serine hydrolases., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata. This lectin is composed of two carbohydrate-binding domains (domains 1 and 2) and one oligomerization domain (domain 3). After binding to the cell surface carbohydrate chains through domains 1 and 2, domain 3 self-associates to form transmembrane pores, leading to cell lysis or death, which resembles other pore-forming toxins of diverse organisms. To elucidate the pore formation mechanism of CEL-III, the crystal structure of the CEL-III oligomer was determined. The CEL-III oligomer has a heptameric structure with a long β-barrel as a transmembrane pore. This β-barrel is composed of 14 β-strands resulting from a large structural transition of α-helices accommodated in the interface between domains 1 and 2 and domain 3 in the monomeric structure, suggesting that the dissociation of these α-helices triggered their structural transition into a β-barrel. After heptamerization, domains 1 and 2 form a flat ring, in which all carbohydrate-binding sites remain bound to cell surface carbohydrate chains, stabilizing the transmembrane β-barrel in a position perpendicular to the plane of the lipid bilayer.