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Sugar isomerases (SIs) catalyze the reversible conversion of aldoses to ketoses. A novel putative SI gene has been identified from the genome sequence information on the psychrophilic bacterium Paenibacillus sp. R4. Here, we report the crystal structure of the putative SI from Paenibacillus sp. R4 (PbSI) at 2.98 A resolution. It was found that the overall structure of PbSI adopts the triose-phosphate isomerase (TIM) barrel fold. PbSI was also identified to have two heterogeneous metal ions as its cofactors at the active site in the TIM barrel, one of which was confirmed as a Zn ion through X-ray anomalous scattering and inductively coupled plasma mass spectrometry analysis. Structural comparison with homologous SI proteins from mesophiles, hyperthermophiles, and a psychrophile revealed that key residues in the active site are well conserved and that dimeric PbSI is devoid of the extended C-terminal region, which tetrameric SIs commonly have. Our results provide novel structural information on the cold-adaptable SI, including information on the metal composition in the active site.

The CIDE domain was initially identified in apoptotic nucleases and now forms a highly conserved family with diverse functions ranging from cell death to lipid metabolism. Based on structural determination of the DREP3 domain, it is suggested that the head-to-tail helical filament structure might be a unified mechanism of CIDE-domain assembly and represents a critical higher-order scaffolding structure that is important for the function of CIDE-domain-containing proteins in DNA fragmentation and lipid-droplet fusion., The cell-death-inducing DFF45-like effector (CIDE) domain is a protein-interaction module comprising ∼80 amino acids and was initially identified in several apoptotic nucleases and their regulators. CIDE-domain-containing proteins were subsequently identified among proteins involved in lipid metabolism. Given the involvement of CIDE-domain-containing proteins in cell death and lipid homeostasis, their structure and function have been intensively studied. Here, the head-to-tail helical filament structure of the CIDE domain of DNA fragmentation factor-related protein 3 (DREP3) is presented. The helical filament structure was formed by opposing positively and negatively charged interfaces of the domain and was assembled depending on protein and salt concentrations. Although conserved filament structures are observed in CIDE family members, the structure elucidated in this study and its comparison with previous structures indicated that the size and the number of molecules used in one turn vary. These findings suggest that this charged-surface-based head-to-tail helical filament structure represents a unified mechanism of CIDE-domain assembly and provides insight into the function of various forms of the filament structure of the CIDE domain in higher-order assembly for apoptotic DNA fragmentation and control of lipid-droplet size.

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.

Conserved immune cell signaling in fish was recently highlighted by the identification of various immune cell signaling molecules. Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins are critical adaptor molecules in immune cell signaling and contain E3 ubiquitin ligase activity. Here, we report the first crystal structure of the TRAF5 TRAF domain from the black rockcod (Notothenia coriiceps; ncTRAF5). Our structure revealed both similarities and differences with mammalian TRAF5. Structural and biochemical analyses indicated that ncTRAF5 forms a functional trimer unit in solution, with a structural flexibility that might be critical for imparting resistance to cold temperature-induced stress. We also found conserved surface residues on ncTRAF5 that might be critical binding hot spots for interaction with various receptors.

Members of the NLR family pyrin domain containing (NLRPs) are pattern recognition receptors that participate in innate immunity. They form inflammasomes, which are platforms for caspase-1 recruitment and activation. The NLRP pyrin domain (PYD) is critical for the assembly of inflammasomes due to its ability to mediate protein interactions. Despite intensive structural studies on inflammasomes with PYDs, the structure of the PYD of NLRP9-the least studied member of the family-remains unknown. Herein, we report the crystal structure of the human NLRP9 PYD at 2.1 A resolution, which reveals a kinked N-terminal loop oriented toward the interior of the helical bundle. Based on our findings, we propose a regulatory role for the kinked N-terminal loop of NLRP9 PYD in inflammasome assembly.

Supramolecular organizing center (SMOC)-mediated signal transduction is an emerging concept in the field of signal transduction that is ushering in a new era. The formation of location-specific, higher-order SMOCs is particularly important for cell death and innate immune signaling processes. Several protein interaction domains, including the death domain (DD) superfamily and the CIDE domain, are representative mediators of SMOC assembly in cell death and innate immune signaling pathways. DD superfamily- and CIDE domain-containing proteins form SMOCs that activate various caspases and provide signaling scaffold platforms. These assemblies can lead to signal transduction and amplification during signaling events. In this review, we summarize recent findings on the molecular basis of DD superfamily- and CIDE domain-mediated SMOC formation., Nonspecific immunity: Super signaling complexes Improved understanding of large molecular signaling complexes that form during innate (nonspecific) immune responses could help develop treatments for multiple diseases including cancer. Correct cell signaling requires precise protein interactions and binding, which are mediated by specific sites on the surface of the protein molecules involved. Innate immune responses and cell death mechanisms rely on such protein interactions, and defects can cause signaling abnormalities and trigger disease. Hyun Ho Park and co-workers at Chung-Ang University in Seoul, South Korea, reviewed recent insights into the presence of supramolecular organizing centers (SMOCs), localized complexes of signaling proteins that form during immune responses. The researchers highlight existing understanding of SMOC assembly processes. A better understanding of SMOCs will help to explain enzyme activation, signal amplification and cell signaling control mechanisms.

Although the functions of CIDE domain-containing proteins, including DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, in apoptotic DNA fragmentation and lipid homeostasis have been studied extensively in mammals, the functions of four CIDE domain-containing proteins identified in the fly, namely DREP1, 2, 3, and 4, have not been explored much. Recent structural study of DREP4, a fly orthologue of mammalian DFF40 (an endonuclease involved in apoptotic DNA fragmentation), showed that the CIDE domain of DREP4 (and DFF40) forms filament-like assembly, which is critical for the corresponding function. The current study aimed to investigate the mechanism of filament formation of DREP4 CIDE and to characterize the same. DREP4 CIDE was shown to specifically bind to histones H1 and H2, an event important for the nuclease activity of DREP4. Based on the current experimental results, we proposed the mechanism underlying the process of apoptotic DNA fragmentation.

During the glyoxylate cycle, isocitrate lyases (ICLs) catalyze the lysis of isocitrate to glyoxylate and succinate. Itaconate has been reported to inhibit an ICL from Mycobacterium tuberculosis (tbICL). To elucidate the molecular mechanism of ICL inhibition, we determined the crystal structure of tbICL in complex with itaconate. Unexpectedly, succinate and itaconate were found to bind to the respective active sites in the dimeric form of tbICL. Our structure revealed the active site architecture as an open form, although the substrate and inhibitor were bound to the active sites. Our findings provide novel insights into the conformation of tbICL upon its binding to a substrate or inhibitor, along with molecular details of the inhibitory mechanism of itaconate.

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.

Soluble fumarate reductase is essential for survival under anaerobic conditions. This enzyme can maintain the redox balance in the cell by catalyzing the reduction of fumarate to succinate. Although the overall reaction mechanism of soluble fumarate reductase in yeast, Osm1, has been proposed by a previous structural study, the details of the underlying mechanism are not completely elucidated. The present study provides the structural information regarding the active site mutant form of Osm1 (R326A), thus, revealing that R326A mutation does not affect the substrate binding. Structural alterations of the residues surrounding the active site, and the missing 2nd flavin adenine dinucleotide (FAD) in the previously defined 2nd FAD binding site, were observed as characteristic features of the Osm1 R326A crystal structure. Based on these findings, we provided a clue that can explain the loss of activity of Osm1 R326A.

Transaminases are pyridoxal 5′-phosphate-dependent enzymes that reversibly catalyze transamination reactions from an amino group donor substrate to an amino group acceptor substrate. ω-Transaminases (ωTAs) utilize compounds with an amino group not at α-carbon position as their amino group donor substrates. Recently, a novel ωTA with broad substrate specificity and high thermostability from the thermophilic bacterium Sphaerobacter thermophilus (St-ωTA) has been reported. Although St-ωTA has been biochemically characterized, little is known about its determinants of substrate specificity. In the present study, we determined the crystal structure of St-ωTA at 1.9 A resolution to clarify in detail its mechanism of substrate recognition. The structure of St-ωTA revealed that it has a voluminous active site resulting from the unique spatial arrangement of residues comprising its active site. In addition, our molecular docking simulation results suggest that substrate compounds may bind to active site residues via electrostatic interactions or hydrophobic interactions that can be induced by subtle rearrangements of active site residues. On the basis of these structural analyses, we propose a plausible working model of the enzymatic mechanism of St-ωTA. Our results provide profound structural insights into the substrate specificity of St-ωTA and extend the boundaries of knowledge of TAs.

Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although our recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. In this study, we present the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE.

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.

The apoptosis repressor with caspase recruiting domain (ARC) is aberrantly overexpressed in various cancers. ARC contains a CARD domain that is the main mediator of protein-protein interactions. Mutation of Leu31 within the CARD of ARC to Phe (ARC_L31F) is widely used as a functionally defective mutant of ARC despite a lack of clear experimental evidence regarding how its functionality is lost. In this study, we show that L31 in helix 2 (H2) is critical for stabilization of the helix bundle fold in the CARD domain. In addition, the L31F mutation disrupts homodimer formation that is critical to ARC functions. Our current study reveals the molecular basis for the L31F mutation disrupting the ARC CARD functions. This article is protected by copyright. All rights reserved.

This study was conducted to investigate the effect of different steam condition and dehydration condition on physicochemical characteristics of Olbyeossal made from Waxy rice. All samples were steamed at 95-100 o C for 60 min or at 121 o C for 20 min and then were dehydrated using hot air 45, 60, 80 o C and sun-dried to achieve moisture contents 13-15%. Initial pasting temperature of Olbyeossal was the lower than that of non- treatment sample by parboiling. The combination in steaming 95- 100 o C and hot drying at 45-60 o C was showed appropriate pasting properties compared with other condition. The hardness of Olbyeossal was significantly affected by steaming and dehydration condition. The hardness of Olbyeossal showed the higher value for steaming at 121 o C than 95-100 o C. The water binding capacity (WBC) and swelling power (SP) were higher steaming at 121 o C than 95-100 o C. No significant differences on WBC and SP of Olbyeossal by dehydration condition were observed. As the sensory evaluation results, the combination in steaming at 121 o C and hot-air drying at 45 or 80 o C led to increase preference for appearance, color, taste and overall acceptability. These results indicate that steaming and dehydration condition were affecting factor on physicochemical characteristics of Olbyeossal.

We demonstrate parametric resonance in a magneto-optical trap. When we modulate the intensity of the cooling laser at about twice the resonant frequency of the trap, the atoms in the trap are divided into two parts and oscillate with 180 degree phase difference with the finite length due to nonlinearity of the trap potential. These are the effects of general nonlinear dynamics, called the Hopf bifurcation, or limit cycle motion. The amplitude and the phase of the oscillations are measured and compared with the theoretical calculations based on simple Doppler cooling theory. The experimental results are in excellent agreement with the simulation results based on the simple Doppler cooling theory.

Objective In October 2013, the South Korean government introduced an incentive programme to increase the availability of Saturday treatment at clinics, hoping to increase the role of primary care providers as gatekeepers to medical care. To the best of our knowledge, no one has yet investigated this programme9s effect on overall outpatient care. Our study aims to analyse the change in Saturday outpatient volume and billings in clinics that adopted the Saturday incentive programme. Setting Our study used 3 types of data from the period October 2012 to March 2014: National Health Insurance Service (NHIS) claims data, hospital evaluation data and medical institution data. Participants These data consisted of 66 825 881 outpatient cases from 2837 clinics. Interventions Introducing the Saturday incentive programme. Outcome measure We performed a multilevel analysis that adjusted for clinic-level and outpatient-level variables to examine the difference in the percentage of Saturday outpatient volume and billings after introducing the Saturday incentive programme. Results The percentages of Saturday outpatient volume and billings were higher after introducing the programme (outpatient volume: β=2.065, p Conclusions Our findings suggest that the Saturday incentive programme has affected clinic outpatient care and is a worthwhile health policy in terms of promoting primary care. Thus, it may improve healthcare accessibility and quality of care, and prevent inappropriate usage such as emergency room visits by providing patients with weekend clinic hours.

This paper investigates the application of resolution enhancement techniques for 140nm back-end via2 contact hole process with KrF system. On bare wafer, 120nm DoF (depth of focus) is secured for via2 process. However, different focal plane are formed due to the severe topography between cell and other region. And DOF of isolated contact hole is less than dense pattern. These factors lowered the overall DOF from 120nm to only about 80nm and raise many problems. Therefore, enhancement of aerial image is needed to improve DOF. In this study, mask type, mask layout, OAI (Off axis illumination) and SRAF (sub resolution assist feature) are applied all together in developed sequence. Because the isolated pattern and dense pattern are coexisted in via2 process, illumination and layout of mask are considered all at once. By using source mask optimization (SMO), optimum illumination and proper SRAF rule are developed and optimized. Through these processes, we obtain improvement of process window in comparison with existing condition. Furthermore coincidence of best focus is assured in real image on real conditioned wafer.

The double structure potential predicted in the atomic cloud of a magneto-optical trap by one- (Doppler part) and two-photon process (sub-Doppler part) is experimentally observed and studied through the parametric excitation with the modulation of cooling laser intensities. Modulating the intensity of the cooling laser at around twice its resonant frequency of Doppler part, the atoms in Doppler part are divided and they oscillate reciprocally with finite amplitude due to the nonlinearity of the trap, and the others in sub-Doppler part are not influenced. By the modulation, we can investigate the sub-Doppler part spatially without Doppler part and also measure the temperature of sub-Doppler part independently.