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YidC/Alb3/Oxa1 family proteins are involved in the insertion and assembly of membrane proteins. The core five transmembrane regions of YidC, which are conserved in the protein family, form a positively charged cavity open to the cytoplasmic side. The cavity plays an important role in membrane protein insertion. In all reported structural studies of YidC, the second cytoplasmic loop (C2 loop) was disordered, limiting the understanding of its role. Here, we determined the crystal structure of YidC including the C2 loop at 2.8 A resolution with R/Rfree = 21.8/27.5. This structure and subsequent molecular dynamics simulation indicated that the intrinsic flexible C2 loop covered the positively charged cavity. This crystal structure provides the coordinates of the complete core region including the C2 loop, which is valuable for further analyses of YidC.

Although gut microbiota and early life events are likely involved in the development of irritable bowel syndrome (IBS), it remains unclear how these factors interact in the pathophysiology of IBS. In the present study, using rats subjected to maternal separation (MS) as a model of IBS, we investigated interrelationships among gut microbiota, stress susceptibility and intestinal permeability, and examined the effect of the probiotic Bifidobacterium bifidum G9-1 (BBG9-1) on those interrelationships. When compared with the controls at postnatal day 20, MS rats showed hypercorticosteronemia, enhanced intestinal permeability and changes in gut microbiota structure. All of these changes in MS rats were prevented by treatment with BBG9-1. Although the gut microbiota profile and basal serum corticosterone level did not differ between MS and control rats at postnatal day 56, MS rats showed hypersensitivity to restraint stress in terms of serum corticosterone level and fecal frequency. However, such hypersensitivity was not observed in MS rats treated with BBG9-1. These findings suggest that MS initiates the link between gut microbiota alteration and hypersensitivity to stress and that the triggering of this process can be prevented by the treatment with the probiotic BBG9-1.

Dissolution dynamic nuclear polarization (DNP) has recently gained attention as a method to enhance the sensitivity of liquid NMR spectroscopy and MRI. We demonstrate dissolution of the sample hyperpolarized by DNP using photoexcited triplet electrons in 0.38 T at room temperature. The achieved polarization of 0.8% is 6100 times as high as that at thermal equilibrium under the condition. The result is an important step for DNP using photoexcited triplet electrons to become widely used in chemical and biomedical research.

In recent years, application of carbon-based nano material to electrode material has been paid attention, however, due to its higher cost, it would be difficult to put it into practical use. Then, we have proposed to make nano carbon fiber with lower production cost. The purpose of our research was, to apply our nano carbon fiber to electrical double-layer capacitor electrode. We used cotton candy method to make nano fiber, and applied microwave heating for carbonization. By applying nano carbon fiber to electrical double-layer capacitor electrode, we got results that thicker electrode containing nano carbon fiber leads to lower resistance value, compared with electrode without containing nano carbon fiber. From this result, it was indicated that by containing nano carbon fiber, the electric bypass was formed in the electrode.

Determination of the crystal structure of the zebrafish LPA6 receptor shows that the lipid ligand binds to an unusual ligand-binding pocket in the receptor that is laterally accessible through the membrane. The lysophosphatidic acid (LPA) receptors are a group of G-protein-coupled receptors (GPCRs) implicated in the development of cancer and fibrosis. The six LPA receptors consist of LPA1–LPA3 and the recently discovered LPA4–LPA6. LPA4–LPA6 are interesting potential therapeutic targets—the LPA6 gene deletion, for example, results in congenital hair loss—but lack of structural data has hampered research efforts in this area. Here, Osamu Nureki and colleagues report the crystal structure of the zebrafish LPA6 receptor, which contains an unusual ligand binding pocket that is open to the cell membrane. The authors propose that the lipid ligand binds to this lateral pocket. This work provides information on ligand recognition and could inform potential drug discovery efforts. Lysophosphatidic acid (LPA) is a bioactive lipid composed of a phosphate group, a glycerol backbone, and a single acyl chain that varies in length and saturation. LPA activates six class A G-protein-coupled receptors to provoke various cellular reactions1. Because LPA signalling has been implicated in cancer2 and fibrosis3, the LPA receptors are regarded as promising drug targets. The six LPA receptors are subdivided into the endothelial differentiation gene (EDG) family (LPA1–LPA3)1 and the phylogenetically distant non-EDG family (LPA4–LPA6)4. The structure of LPA1 has enhanced our understanding of the EDG family of LPA receptors5. By contrast, the functional and pharmacological characteristics of the non-EDG family of LPA receptors have remained unknown, owing to the lack of structural information. Although the non-EDG LPA receptors share sequence similarity with the P2Y family of nucleotide receptors4, the LPA recognition mechanism cannot be deduced from the P2Y1 and P2Y12 structures6,7,8 because of the large differences in the chemical structures of their ligands. Here we determine the 3.2 A crystal structure of LPA6, the gene deletion of which is responsible for congenital hair loss9,10, to clarify the ligand recognition mechanism of the non-EDG family of LPA receptors. Notably, the ligand-binding pocket of LPA6 is laterally open towards the membrane, and the acyl chain of the lipid used for the crystallization is bound within this pocket, indicating the binding mode of the LPA acyl chain. Docking and mutagenesis analyses also indicated that the conserved positively charged residues within the central cavity recognize the phosphate head group of LPA by inducing an inward shift of transmembrane helices 6 and 7, suggesting that the receptor activation is triggered by this conformational rearrangement.

Summary: Protein secretion mediated by SecYEG translocon and SecA ATPase is enhanced by membrane-embedded SecDF by using proton motive force. A previous structural study of SecDF indicated that it comprises 12 transmembrane helices that can conduct protons and three periplasmic domains, which form at least two characterized transition states, termed the F and I forms. We report the structures of full-length SecDF in I form at 2.6- to 2.8-Å resolution. The structures revealed that SecDF in I form can generate a tunnel that penetrates the transmembrane region and functions as a proton pathway regulated by a conserved Asp residue of the transmembrane region. In one crystal structure, periplasmic cavity interacts with a molecule, potentially polyethylene glycol, which may mimic a substrate peptide. This study provides structural insights into the Sec protein translocation that allows future analyses to develop a more detailed working model for SecDF. : SecDF, a motor protein, uses proton motive force to facilitate bacterial protein translocation mediated by the SecYEG translocon and SecA ATPase. Furukawa et al. describe high-resolution (2.6–2.8 Å) structures of SecDF in I forms, providing insight into a substrate binding site and a proton transport pathway through SecDF. Keywords: protein translocation, Sec proteins, membrane protein, crystal structure, SecYEG, SecDF

Physics letters / B 802, 135200 (2020). doi:10.1016/j.physletb.2020.135200, Published by North-Holland Publ., Amsterdam

The European physical journal / A 56(5), 143 (2020). doi:10.1140/epja/s10050-020-00153-5, Published by Springer, Heidelberg

Nitrogen-doped carbon quantum dots synthesized by one-pot, microwave-assisted pyrolysis of citric acid in the presence of a small number of N-doping precursors, 1,2-ethylenediamine, were found to be involved in the chemically initiated electron exchange luminescence enabling peroxyoxalate chemiluminescence assay of hydrogen peroxide in the concentration range of 10–1000 µM.

The Sec translocon provides the lipid bilayer entry for ribosome‐bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo‐electron microscopy‐based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α‐helices 2b, 7, and 8 tilt within the membrane core to “unzip” the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α‐helices of SecE subunit modulate the lateral gate conformation. Site‐specific cross‐linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.

Physical review / C covering nuclear physics 99(6), 064313 (2019). doi:10.1103/PhysRevC.99.064313, Published by Inst., Woodbury, NY

There are many marine resources in Japanese ocean, those have been surveyed by a underwater robot. The accuracy of the resources survey results using the vehicle depends on the accuracy of its self-localization. Thus, we propose a self-localization method by Kalman filter consdering the effect of vehicle's oscillation and the effect of the current. It is possible to reduce the positioning accuracy by 0.2 m by considering the effect of the vehicle's oscillation. In future work, we will develop an algorithm considering delay time for sensor fusion with acoustic positioning device.

ATP9A is localized to phosphatidylserine-positive early and recycling endosomes, but not late endosomes, in HeLa cells. ATP9A plays a crucial role in recycling of transferrin and glucose transporter 1 from endosomes to the plasma membrane., Type IV P-type ATPases (P4-ATPases) are phospholipid flippases that translocate phospholipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of lipid bilayers. In Saccharomyces cerevisiae, P4-ATPases are localized to specific subcellular compartments and play roles in compartment-mediated membrane trafficking; however, roles of mammalian P4-ATPases in membrane trafficking are poorly understood. We previously reported that ATP9A, one of 14 human P4-ATPases, is localized to endosomal compartments and the Golgi complex. In this study, we found that ATP9A is localized to phosphatidylserine (PS)-positive early and recycling endosomes, but not late endosomes, in HeLa cells. Depletion of ATP9A delayed the recycling of transferrin from endosomes to the plasma membrane, although it did not affect the morphology of endosomal structures. Moreover, depletion of ATP9A caused accumulation of glucose transporter 1 in endosomes, probably by inhibiting their recycling. By contrast, depletion of ATP9A affected neither the early/late endosomal transport and degradation of epidermal growth factor (EGF) nor the transport of Shiga toxin B fragment from early/recycling endosomes to the Golgi complex. Therefore ATP9A plays a crucial role in recycling from endosomes to the plasma membrane.

A V. cholerae MATE transporter was crystallized using the lipidic cubic phase (LCP) method. X-ray diffraction data sets were collected from single crystals obtained in a sandwich plate and a sitting-drop plate to resolutions of 2.5 and 2.2 Å, respectively., Multidrug and toxic compound extrusion (MATE) transporters, one of the multidrug exporter families, efflux xenobiotics towards the extracellular side of the membrane. Since MATE transporters expressed in bacterial pathogens contribute to multidrug resistance, they are important therapeutic targets. Here, a MATE-transporter homologue from Vibrio cholerae, VcmN, was overexpressed in Escherichia coli, purified and crystallized in lipidic cubic phase (LCP). X-ray diffraction data were collected to 2.5 Å resolution from a single crystal obtained in a sandwich plate. The crystal belonged to space group P212121, with unit-cell parameters a = 52.3, b = 93.7, c = 100.2 Å. As a result of further LCP crystallization trials, crystals of larger size were obtained using sitting-drop plates. X-ray diffraction data were collected to 2.2 Å resolution from a single crystal obtained in a sitting-drop plate. The crystal belonged to space group P212121, with unit-cell parameters a = 61.9, b = 91.8, c = 100.9 Å. The present work provides valuable insights into the atomic resolution structure determination of membrane transporters.

The delignification of Japanese beech (Fagus crenata) has been evaluated under conditions of subcritical phenol (230°C/1.2 MPa) and subcritical water (230°C/2.9 MPa). In the former, more than 90% of the original lignin was decomposed and removed, while in subcritical water, around half of the original lignin was left as insoluble residue. Ultraviolet (UV) microscopic images of the insoluble residues showed that the lignin in the secondary walls is decomposed and removed under both conditions. These images also revealed that the lignin in the compound middle lamella (CML) is resistant to subcritical water, but not to subcritical phenol. Results of alkaline nitrobenzene oxidation of the residual lignin confirmed these observations. Lignin in Japanese beech wood was phenolated by subcritical phenol, which was efficiently removed due to its high solubility in the reactant. It is obvious that CML is rich in condensed-type linkages facilitating rapid solvolysis by phenol. The topochemistry of the plant has a pronounced impact on its delignification behavior.

A novel method for simultaneous measurement of masses, Q-values, isomer excitation energies, half-lives and decay branching ratios of exotic nuclei has been demonstrated. The method includes first use of a stopping cell as an ion trap, combining containment of precursors and decay-recoils for variable durations in a cryogenic stopping cell (CSC), and afterwards the identification and counting of them by a multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS). Feasibility has been established by recording the decay and growth of $^{216}$Po and $^{212}$Pb (alpha decay) and of $^{119m2}$Sb (t$_{1/2}$ = 850$\pm$90 ms) and $^{119g}$Sb (isomer transition), obtaining half-lives and branching ratios consistent with literature values. Hardly any non-nuclear-decay losses have been observed in the CSC for up to $\sim$10 seconds, which exhibits its extraordinary cleanliness. For $^{119}$Sb, this is the first direct measurement of the ground and second isomeric state masses, resolving the discrepancies in previous excitation energy data. These results pave the way for the measurement of branching ratios of exotic nuclei with multiple decay channels., 11 pages, 9 figures, 3 tables. Submitted to European Physics Journal A

Sarco/endoplasmic reticulum (ER) Ca

Purpose The purpose of this study was to evaluate the prediction accuracy of effective lens position (ELP) after cataract surgery using a multiobjective evolutionary algorithm (MOEA). Methods Ninety-six eyes of 96 consecutive patients (aged 73.9 ± 8.6 years) who underwent cataract surgery were retrospectively studied; the eyes were randomly distributed to a prediction group (55 eyes) and a verification group (41 eyes). The procedure was repeated randomly 30 times to create 30 data sets for both groups. In the prediction group, based on the parameters of preoperative optical coherence tomography (OCT), biometry, and anterior segment (AS)-OCT, the prediction equation of ELP was created using MOEA and stepwise multiple regression analysis (SMR). Subsequently, the prediction accuracy of ELPs was evaluated and compared with conventional formulas, including SRK/T and the Haigis formula. Results The rate of mean absolute prediction error of 0.3 mm or higher was significantly lower in MOEA (mean 4.9% ± 3.2%, maximum 9.8%) than SMR (mean 7.3% ± 4.8%, maximum 24.4%) (P = 0.0323). The median of the correlation coefficient (R2 = 0.771) between the MOEA predicted and measured ELP was higher than the SRK/T (R2 = 0.412) and Haigis (R2 = 0.438) formulas. Conclusions The study demonstrated that ELP prediction by MOEA was more accurate and was a method of less fluctuation than that of SMR and conventional formulas. Translational Relevance MOEA is a promising method for solving clinical problems such as prediction of ocular biometry values by simultaneously optimizing several conditions for subjects affected by various complex factors.

PurposeTo investigate the clinical significance of color visual acuity (CVA) in preperimetric glaucoma (PPG) and open-angle glaucoma (OAG).MethodsA total of 123 eyes of 73 subjects (22 normal eyes, 14 PPG eyes, and 87 OAG eyes; mean age: 44.9 ± 10.1 years, age range: 21-64 years) were enrolled. CVA was tested for red, green-yellow, blue-green and blue-purple with a newly developed test.ResultsThere was no statistical difference in clinical background factors, including age, sex, intraocular pressure, or spherical equivalent between the three groups. Red VA and blue-green VA were significantly worse in the OAG eyes than in the normal eyes (P = 0.008 and P = 0.015, respectively), although green-yellow VA and blue-purple VA were not significantly worse. Furthermore, red VA and blue-green VA were significantly correlated with MD in a group of eyes with either PPG or OAG (r = -0.23, P = 0.023; r = -0.25, P = 0.012, respectively), but green-yellow VA and blue-purple VA were not.ConclusionRed VA and blue-green VA were detectably worse in eyes with OAG, in close association with the degree of functional loss. This suggests that measuring CVA with the new color test described here may be a promising supplement to existing methods of detecting glaucoma and evaluating its severity.

The proton-unbound argon and chlorine isotopes have been studied by measuring trajectories of their decay-in-flight products by using a tracking technique with micro-strip detectors. The proton ($1p$) and two-proton ($2p$) emission processes have been detected in the measured angular correlations "heavy-fragment"+$p$ and "heavy-fragment"+$p$+$p$, respectively. The ground states of the previously unknown isotopes $^{30}$Cl and $^{28}$Cl have been observed for the first time, providing the $1p$-separation energies $S_p$ of $-0.48(2)$ and $-1.60(8)$ MeV, respectively. The relevant systematics of $1p$ and $2p$ separations energies have been studied theoretically in the core+$p$ and core+$p$+$p$ cluster models. The first-time observed excited states of $^{31}$Ar allow to infer the $2p$-separation energy $S_{2p}$ of 6(34) keV for its ground state. The first-time observed state in $^{29}$Ar with $S_{2p} = -5.50(18)$ MeV can be identified either as a ground or an excited state according to different systematics., 13 pages, 14 figures. Name changed, some affiliations changed and figures corrections added

Multidrug and toxic compound extrusion (MATE) transporters efflux toxic compounds, using a Na(+) or H(+) gradient across the membrane. Although the structures of MATE transporters have been reported, the cation-coupled substrate transport mechanism remains controversial. Here we report crystal structures of VcmN, a Vibrio cholerae MATE transporter driven by the H(+) gradient. High-resolution structures in two distinct conformations associated with different pHs revealed that the rearrangement of the hydrogen-bonding network around the conserved Asp35 induces the bending of transmembrane helix 1, as in the case of the H(+)-coupled Pyrococcus furiosus MATE transporter. We also determined the crystal structure of the D35N mutant, which captured a unique conformation of TM1 facilitated by an altered hydrogen-bonding network. Based on the present results, we propose a common step in the transport cycle shared among prokaryotic H(+)-coupled MATE transporters.

Bacterial pathogens or cancer cells can acquire multidrug resistance, which causes serious clinical problems. In cells with multidrug resistance, various drugs or antibiotics are extruded across the cell membrane by multidrug transporters. The multidrug and toxic compound extrusion (MATE) transporter is one of the five families of multidrug transporters. MATE from Pyrococcus furiosus uses H(+) to transport a substrate from the cytoplasm to the outside of a cell. Crystal structures of MATE from P. furiosus provide essential information on the relevant H(+)-binding sites (D41 and D184). Hybrid quantum mechanical/molecular mechanical simulations and continuum electrostatic calculations on the crystal structures predict that D41 is protonated in one structure (Straight) and, both D41 and D184 protonated in another (Bent). All-atom molecular dynamics simulations suggest a dynamic equilibrium between the protonation states of the two aspartic acids and that the protonation state affects hydration in the substrate binding cavity and lipid intrusion in the cleft between the N- and C-lobes. This hypothesis is examined in more detail by quantum mechanical/molecular mechanical calculations on snapshots taken from the molecular dynamics trajectories. We find the possibility of two proton transfer (PT) reactions in Straight: the 1st PT takes place between side-chains D41 and D184 through a transient formation of low-barrier hydrogen bonds and the 2nd through another H(+) from the headgroup of a lipid that intrudes into the cleft resulting in a doubly protonated (both D41 and D184) state. The 1st PT affects the local hydrogen bond network and hydration in the N-lobe cavity, which would impinge on the substrate-binding affinity. The 2nd PT would drive the conformational change from Straight to Bent. This model may be applicable to several prokaryotic H(+)-coupled MATE multidrug transporters with the relevant aspartic acids.

Toll-like receptor (TLR) 4 mediates inflammation and is also known to trigger apoptosis in microglia. Our time-lapse observations showed that lipopolysaccharide (LPS) stimulation induced rapid death in primary cultures of rat microglia, while a portion of the microglia escaped from death and survived for much longer than 2 days, in which time, all of the control cells had died. However, it remains unclear how the LPS-stimulated microglia subpopulation could continue to survive in the absence of any supplied growth factors. In the present study, to clarify the mechanism underlying the LPS-stimulated survival, we investigated whether microglia could produce their own survival factors in response to LPS, focusing on macrophage colony-stimulating factor (M-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-34, which are mainly supplied by astrocytes or neurons. The LPS-stimulated microglia drastically induced the expression of the GM-CSF mRNA and protein, while M-CSF and IL-34 levels were unchanged. The surviving microglia also significantly upregulated the expression of GM-CSF receptor (GM-CSFR) mRNA without affecting M-CSFR. As for the GM-CSFR downstream signal, LPS resulted in the phosphorylation of STAT5 and its translocation to the nucleus in the surviving microglia. Moreover, a specific JAK2 inhibitor, NVP-BSK805, suppressed STAT5 phosphorylation and microglia survival in response to LPS, indicating a critical role of the JAK2/STAT5 pathway in this survival mechanism. Together, these results suggest that a subpopulation of TLR4-activated microglia may survive by producing GM-CSF and up-regulating GM-CSFR. This autocrine GM-CSF pathway may activate the JAK2/STAT5 signaling pathway, which controls the transcription of survival-related genes. Finally, these surviving microglia may have neuroprotective functions because the neurons remained viable in co-cultures with these microglia.

An experimental platform for serial femtosecond crystallography using an X-ray free-electron laser and its applications at SACLA are described., An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Å was obtained.

This work was supported in part by the Helmholtz International Center for FAIR (HIC for FAIR), the Helmholtz Association (grant IK-RU-002), the Russian Ministry of Education and Science (grant No. NSh- 932.2014.2), the Russian Science Foundation (grant No. 17-12-01367), the Polish National Science Center (Contract No. UMO-2011/01/B/ST2/01943), the Polish Ministry of Science and Higher Education (Grant No. 0079/DIA/2014/43,Grant Diamentowy), the Helmholtz- CAS Joint Research Group (grant HCJRG-108), the FPA2009-08848 contract (MICINN, Spain), the Justus- Liebig-Universit¨at Giesen (JLU) and GSI under the JLUGSI strategic Helmholtz partnership agreement. This article is a part of PhD thesis of X.-D. Xu. The authors acknowledge the help of D. Kostyleva in the preparation of the manuscript.

Endoscopic sphincterotomy (EST) dissects the sphincter of Oddi by cutting and/or causing the coagulation of the tissue using an electrosurgical current. Complications of EST include acute pancreatitis, hemorrhage, duodenal perforation, and sepsis.1–5 These complications occur in 5% to 10% of the population.1–5 Factors involved in the occurrence of complications of EST have been considered to be the Oddi dysfunction,6 types of electrosurgical current,7,8 specialist training, disinfection, drainage, and collaboration with surgical colleagues.4 The characteristics of the electrosurgical current may affect the frequency and extent of the complications of EST because the nature of the thermal tissue injury depends on the characteristics of the electrosurgical current used to perform EST.9,10 A pure cutting current achieves better cutting ability, whereas a low-voltage coagulating current that is not used solely gains better hemostasis in EST.1 A mixed current comprising mixed patterns of both pure cutting and coagulating currents is currently available for EST in 2 modes, which are the blended cut and the Endocut (E)-mode.1,11,12 The blended cut (C)-mode is comprised of cutting and coagulating currents delivered together in one waveform.13 The E-mode (ERBE, Marietta, GA) gains both the effects of cutting and coagulating because the cutting and coagulating currents are mixed together and applied in turn in short bursts with an intermittent pause.13 When compared with the conventional blended C-mode, the E-mode can give a well-defined and constant degree of coagulation of the incision margins for hemostasis by its automatic voltage regulation and controlled cutting speed, which prevents perforation of the superior part of the papilla of Vater based on an uncontrolled cutting speed, by its automatically fractionated cut.14 Thus, the E-mode may reduce complications of EST theoretically compared with the conventional blended C-mode. An EST with blended currents increases the rate of pancreatitis after EST compared with EST with a pure cutting current.7,8 There is, however, a contradictory report that EST with a pure cutting current has a similar pancreatitis rate, a higher rate of immediate hemorrhage, and poorer control of the incision than that with a blended current.15 A recent meta-analysis shows that the pure cutting current has a similar pancreatitis rate and more episodes of bleeding, primarily mild bleeding, than the mixed current.16 As for the comparison of mixed current modes, the results were controversial in the development of acute pancreatitis and clinically significant bleeding in multiple clinical trials comparing the E-mode with the blended C-mode.14,17–19 However, the problems in the previous studies, including small sample size and retrospective analysis, make it difficult to conclude the advantage of efficacy and safety of the E-mode. Our study aim was to evaluate whether the E-mode surpasses the conventional blended C-mode in safety and efficacy.

Molecular studies have led to postulation of a relationship between gut microbiota and certain diseases. However, because studies of hitherto uncultured species in vivo are essential for characterizing the biology and pathogenic properties of gut bacteria, techniques for culturing and isolating such bacteria must be developed. Here, a technique is described that partially overcomes the obstacles that prevent detection of interbacterial communication in vitro and are thus responsible for the failure to culture certain bacterial species. For this purpose, a ring with a membrane filter at the bottom was designed and a relatively simple nutrient medium was used instead of conventional media. Gut bacteria were cocultivated in soft agar separated by the membrane filter to simulate interbacterial communication in vitro. Use of this soft agar coculture technique led to the successful isolation of hitherto uncultured bacteria and the demonstration of multistage interbacterial communication among gut bacteria in vitro. Cultivation and isolation of single colonies of bacteria that require other bacteria for growth will enhance efforts to better understand the physiological and pathogenic roles of gut microbiota.

Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247-251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.

Multidrug exporters expressed in pathogens efflux substrate drugs such as antibiotics, and thus, the development of inhibitors against them has eagerly been anticipated. Furthermore, the crystal structures of multidrug exporters with their inhibitors provide novel insights into the inhibitory mechanism and the development of more specific and effective inhibitors. We previously reported the complex structures of the Multidrug And Toxic compound Extrusion (MATE)-type multidrug exporter with the macrocyclic peptides, which inhibit the efflux of substrates by the MATE-type multidrug exporter (Tanaka et al., Nature 496:247-251, 2013). In this chapter, we describe methodologies of the screening and synthesis of macrocyclic peptides as inhibitors, as well as the purification, crystallization, and structure determination of the complexes of the MATE-type multidrug exporter with its inhibitors.

Purpose: To evaluate the color visual acuity (CVA) of young healthy subjects using colored Landolt rings and the effect of background luminance level on the CVA.Materials and methods: We measured the CVA of 20 young healthy subjects (age: 23.8 ± 3.8 years) with different colors using a computer and a liquid crystal display, with 15 Landolt ring colors (30 cd/m2) with a background luminance of 30 cd/m2, and then 100 cd/m2. We then used different background luminance levels (15–50 cd/m2) using four Landolt ring colors (red, green–yellow, green, and blue–green) to evaluate the effect of the background luminance level on CVA.Results: The CVA significantly differed among the colors with a background luminance of 30 cd/m2 (p

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N-terminal protease domain and a C-terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone-like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β-barrel assembly machinery (BAM) driving integration of β-barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site-directed in vivo photo-cross-linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.

Purpose To compare the self-sealing features and dimensional stability between the femtosecond laser (FL) and manual knife corneal incision. Methods For the clinical study, 29 consecutive eyes from 29 patients and 28 eyes from 28 patients who underwent cataract surgery with FL corneal incision and manual knife incision, respectively, were enrolled. Immediately after cataract surgery, the self-sealing features of the corneal incisions were evaluated. Scanning electron microscopy (SEM) images were obtained. For the experimental study, clear corneal incisions with a knife or FL with different energy settings (3, 6 and 9 μJ) were created in fresh porcine eyes, followed by a stress test. The incision width was measured before and after the stress test. Results In the clinical study, the knife group had a higher self-sealing score (0.60 ± 0.49 points) than the FL group (0.17 ± 0.38 points). In the experimental study, the deformation rate in the knife incision (5.04 ± 1.93) was significantly lower than that in the FL with any energy. The deformation rate in the 9 μJ (12.98 ± 2.76) was significantly higher than in the 3 μJ (8.54 ± 2.38) and 6 μJ (8.82 ± 2.85) FL energies. Scanning electron microscopy (SEM) images revealed that the corneal stromal surface of the knife incision was smoother than that of the FL. Higher energy FL showed more irregular surfaces. Conclusion Higher FL energy tended to widen a clear corneal incision when mechanical stress was applied. The histological differences at the inner tunnel surface may cause differences in wound stability of the corneal incision.

Summary The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 A resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane.

Bifidobacterium, a major component of the intestinal microbiota, has been clinically used for the treatment of diarrhoea and constipation. 5-Fluorouracil (5-FU), widely used for cancer chemotherapy, is known to frequently induce intestinal mucositis accompanied by severe diarrhoea. The present study examined the effect of Bifidobacterium bifidum G9-1 (BBG9-1) on 5-FU-induced intestinal mucositis in mice. Intestinal mucositis was induced by repeated administration of 5-FU for 6 days. BBG9-1 was administered orally once daily for 9 days, beginning 3 days before the onset of 5-FU treatment. Repeated administration of 5-FU caused severe intestinal mucositis, characterised by shortening of villi and destruction of crypts, accompanied by increases in intestinal myeloperoxidase activity and inflammatory cytokine expression, body weight loss, and diarrhoea on day 6. Daily administration of BBG9-1 significantly reduced the severity of intestinal mucositis and inflammatory responses and tended to attenuate clinical symptoms. In contrast, BBG9-1 failed to prevent apoptosis induction on day 1 after the first 5-FU administration. The structure of the intestinal microbiota, as analysed by weighted UniFrac distance, was largely altered by 5-FU treatment, but this change was mitigated by daily administration of BBG9-1. Moreover, 5-FU treatment decreased the abundance of Firmicutes and increased the abundance of Bacteroidetes, but these responses were also significantly inhibited by daily administration of BBG9-1. These results suggest that BBG9-1 has an ameliorative effect against 5-FU-induced intestinal mucositis through the attenuation of inflammatory responses via improve dysbiosis. BBG9-1 could be useful for the prevention of intestinal mucositis during cancer chemotherapy. This article is protected by copyright. All rights reserved.

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Two bacterial strains, designated 180-3T and 214-4T, isolated from human faeces were characterized by using a polyphasic taxonomic approach that included analysis of their phenotypic and biochemical features, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these strains represented members of the genus Butyricimonas . These strains shared 97.9 % 16S rRNA gene sequence similarity with each other and were related to Butyricimonas virosa JCM 15149T (97 % sequence similarity) and Butyricimonas synergistica JCM 15148T (94–95 %). Although strain 180-3T was related to (but distinct from) B. virosa JCM 15149T and B. synergistica JCM 15148T, with hsp60 gene sequence similarities of 89.4 and 84.6 %, respectively, strain 214-4T exhibited high hsp60 gene sequence similarity (100 %) with B. virosa JCM 15149T and was different from B. synergistica JCM 15148T (83.5 %). DNA–DNA hybridization experiments demonstrated a genomic distinction of strains 180-3T and 214-4T from B. virosa JCM 15149T and B. synergistica JCM 15148T. The strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-stain-negative rods. Growth of the strains was inhibited on medium containing 20 % bile. The two strains produced butyric and isobutyric acids as the end products from glucose, as has been observed in the other two species of the genus Butyricimonas . The major cellular fatty acid of strains 180-3T and 214-4T was iso-C15 : 0. The major menaquinone of the isolates was MK-10 (>50 %). Strains 180-3T and 214-4T have DNA G+C contents of 45 mol%. On the basis of these data, strains 180-3T and 214-4T represent two novel species of the genus Butyricimonas , for which the names Butyricimonas faecihominis sp. nov. and Butyricimonas paravirosa sp. nov., respectively, are proposed. The type strains of B. faecihominis and B. paravirosa are 180-3T ( = JCM 18676T = CCUG 65562T) and 214-4T ( = JCM 18677T = CCUG 65563T), respectively.

YidC, a membrane-protein chaperone/insertase from B. halodurans, was expressed, purified and crystallized in the lipidic cubic phase. An X-ray diffraction data set was collected to 2.4 Å resolution., YidC, a member of the YidC/Oxa1/Alb3 family, inserts proteins into the membrane and facilitates membrane-protein folding in bacteria. YidC plays key roles in both Sec-mediated integration and Sec-independent insertion of membrane proteins. Here, Bacillus halodurans YidC2, which has five transmembrane helices conserved among the other family members, was identified as a target protein for structure determination by a fluorescent size-exclusion chromatography analysis. The protein was overexpressed, purified and crystallized in the lipidic cubic phase. The crystals diffracted X-rays to 2.4 Å resolution and belonged to space group P21, with unit-cell parameters a = 43.9, b = 60.6, c = 58.9 Å, β = 100.3°. The experimental phases were determined by the multiwavelength anomalous diffraction method using a mercury-derivatized crystal.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1, 2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3, 4, 5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane., [プレスリリース]バイオサイエンス研究科膜分子複合機能学研究室の塚崎智也准教授らの研究グループが、タンパク質を細胞膜に組み込むメカニズムを解明しました（2014/04/17）

Summary Sarco/endoplasmic reticulum (ER) Ca2+-ATPase 2b (SERCA2b) is a ubiquitously expressed membrane protein that facilitates Ca2+ uptake from the cytosol to the ER. SERCA2b includes a characteristic 11th transmembrane helix (TM11) followed by a luminal tail, but the structural basis of SERCA regulation by these C-terminal segments remains unclear. Here, we determined the crystal structures of SERCA2b and its C-terminal splicing variant SERCA2a, both in the E1-2Ca2+-adenylyl methylenediphosphonate (AMPPCP) state. Despite discrepancies with the previously reported structural model of SERCA2b, TM11 was found to be located adjacent to TM10 and to interact weakly with a part of the L8/9 loop and the N-terminal end of TM10, thereby inhibiting the SERCA2b catalytic cycle. Accordingly, mutational disruption of the interactions between TM11 and its neighboring residues caused SERCA2b to display SERCA2a-like ATPase activity. We propose that TM11 serves as a key modulator of SERCA2b activity by fine-tuning the intramolecular interactions with other transmembrane regions.