News Article | April 10, 2015
Cheryl Snapp Conner, founder and managing partner of Snapp Conner PR, has more than 22 years of experience in public relations. Snapp Conner has led four successful tech PR agencies since 1989, and has been recognized as one of Utah's 30 Women to Watch. She is a trustee of the Utah Technology Council (UTC) and had directed the UTC Communications Committee since 2004. Snapp Conner has directed strategic PR programs for companies ranging from Novell to Lexmark to Altiris (now a division of Symantec). Snapp Conner PR was named one of Utah's Top 25 Under 5 companies in 2007. Snapp Conner has also been named to the vSpring v100 list (executives most likely to launch a successful start-up within the next 5-7 years, as elected by their peers) for 2008. She can be reached at email@example.com.
News Article | March 12, 2016
Harvard University researchers have announced the development of a new 3D material capable of bending and folding without input from human controllers. When the new material lays flat, it is strong enough to withstand the weight of a fully grown elephant. When needed, it can alter its size, shape and volume, folding open into any desired shape Further development could lead to entire portable houses that can fit into backpacks, as well as walls that can form windows or open ceilings with just the flick of a switch. "This structural system has fascinating implications for dynamic architecture, including portable shelters, adaptive building facades, and retractable roofs. Whereas current approaches to these applications rely on standard mechanics, this technology offers unique advantages such as how it integrates surface and structure, its inherent simplicity of manufacture, and its ability to fold flat," said Chuck Hoberman of the Graduate School of Design at Harvard University. The new material was inspired by snapology, a technique utilized in origami. Cubes within the structure possess a total of 24 faces and 36 edges. The structure can be folded along hinge-like edges, resulting in a wide range of possible final forms. The unfolding process can be activated by electricity, water or heat, providing additional flexibility in real-world applications. As the structure unfolds, it is capable of altering its density. Because of this, flexible or stiff structures can be created from identical material. The research team was able to determine exactly what actuation approaches are required for tasks. Investigators developed a structure measuring four cubes on each side, but they believe their new technology could easily be scaled up to larger units. This means it may be possible to develop smaller versions of the 3D material that could be manufactured for use on the nanoscale. This technology could also be used in the design of everything from small devices such as a new generation of surgical stents to larger structures, including surgical tents. Each use of the new 3D material could prove invaluable in disaster areas, where space is at a minimum and transportation resources are scarce. Development of the new self-actuating material was profiled in the journal Nature Communications.
Home > Press > Unraveling the crystal structure of a -70° Celsius superconductor, a world first: Significant advancement in the realization of room-temperature superconductors Abstract: For the first time in the world, a research group led by Osaka University, Japan, clarified the crystal structure of hydrogen sulfide in its superconducting phase at the high temperature of -70°C. This was achieved by conducting a combination of experiments at one of the world's largest synchrotron radiation facilities, SPring-8 in Japan. These results mark a huge step towards developing room-temperature superconductors, which may provide promising solutions to energy problems. Superconductivity is a phenomenon that occurs when the electrical resistance of materials reaches zero as they are cooled down to a certain temperature. While the possible scenarios for its use are manifold, such as using superconductors as energy transmission lines without energy loss, widespread use is difficult as costs for cooling are high. Last year, hydrogen sulfide set a new record for highest superconducting transition temperature under high pressure. However, its crystal structure, necessary for understanding its superconductivity mechanism, was not understood. A research group led by Prof. Katsuya Shimizu and Dr. Mari Einaga at the Center for Science and Technology Under Extreme Conditions, Graduate School of Engineering Science, Osaka University, together with Dr. Mikhail Eremets at the Max Planck Institute for Chemistry, and Dr. Yasuo Ohishi at the Japan Synchrotron Radiation Research Institute, has now succeeded in clarifying this structure by simultaneously conducting measurements of high pressure electrical resistance and X-ray diffraction. Since hydrogen sulfide consists of light elements, measurements required a special setup. Therefore, these measurements were conducted at the synchrotron radiation facility SPring-8, and consisted of using a diamond anvil cell to conduct measurement under high-pressure and low temperature, and the high-pressure beam line BL10XU with which high-intensity, high-energy and micro-diameter X-ray beams for X-ray diffraction can be used, in order to examine the material's crystal structure. The researchers clarified that under high pressure, H2S molecules underwent a structural change to H3S and that this H3S structure exhibited superconductivity. Furthermore, from simultaneously measuring changes in pressure of superconducting transition temperature, they discovered that H3S displayed two superconducting phases: one with a cubic structure, the other with a hexagonal structure. They thereby managed to prove previous predictions from theoretical calculations. The results of this study will contribute to clarifying the mechanisms of the high-temperature superconductivity observed in hydrogen sulfide. They also mark a considerable step in developing room-temperature superconductors and provide new insights that will be useful in the development of new materials that spread under high pressure. For more information, please click If you have a comment, please us. Issuers of news releases, not 7th Wave, Inc. or Nanotechnology Now, are solely responsible for the accuracy of the content.
News Article | April 22, 2016
A research team led by Associate Professor Miyake Chikahiro and PhD student Takagi Daisuke from the Kobe University Graduate School of Agricultural Science have reproduced the reaction in which harmful reactive oxygen species are created during plant photosynthesis, and clarified a mechanism behind plant withering. This discovery could help to ensure stable food supplies by cultivating plants that can withstand environmental stresses such as global warming. The findings were published on March 2 in the online version of Plant Physiology. The majority of plants depend on photosynthesis as an essential energy source. However, when the light energy necessary for photosynthesis is absorbed in excess, harmful reactive oxygen species (ROS) are produced. In most cases plants use enzymes to deal with these reactive oxygen species. If plants are exposed to environmental stresses such as lack of water or excess minerals, their ability to photosynthesize is reduced, the ROS removal mechanism cannot keep up with the ROS produced from excess light energy, and plants wither and die. Researchers already knew that ROS are produced within chloroplasts in plant cells, but the exact location and the mechanism behind this were unclear. Professor Miyake's research group extracted chloroplasts and thylakoid membranes from leaves, and exposed them to excess light using repetitive short-pulse illumination. As a result of this treatment, a particle known as "P700" which absorbs light energy within photosystem I¹ stopped functioning, and three types of reactive oxygen species were produced: superoxide radicals (O2 -), hydroxyl radicals (OH?) and singlet oxygen (1O2). They further confirmed that by limiting the flow of electrons to photosystem I the production of reactive oxygen species was suppressed. Due to factors such as global warming, Earth's natural environment is becoming increasingly inhospitable for plant life. "By revealing the mechanism for the production of ROS and part of its regulatory mechanism, there are future possibilities for ensuring a stable food supply despite global warming," said Professor Miyake. "The next step is to reveal the regulatory mechanism for ROS on a molecular level."
C57BL/6J mice were purchased from Japan SLC (Shizuoka), and ICR mice and germ-free (IQI/Jic[Gf] ICR) mice were from CLEA Japan (Tokyo). Myd88−/− and Asc−/− mice backcrossed onto C57BL/6 mice for eight or more generations were used19, 20, 21. All mice were kept under SPF conditions at the Experimental Animal Facility, Graduate School of Medicine, Osaka University. All animal experiments were performed according to guidelines of the Animal Research Committee of the Graduate School of Medicine at Osaka University. RNA samples were prepared from indicated tissues using TRIzol reagent (Invitrogen). Total RNA was reverse transcribed using Moloney murine leukaemia virus reverse transcriptase (Promega) and random primers (Toyobo) after treatment with RQ1 DNase I (Promega). cDNA was analysed by real-time RT–PCR using GoTaq qPCR Master Mix (Promega) in ABI 7300 (Applied Biosystems). Values were normalized to the expression of Gapdh, and the fold difference in expression relative to that of Gapdh is shown. The following primer sets were used: Lypd8, 5′-GCCTTCACTGTCCATCTATTT-3′ and 5′-GTGACCATAGCAAGACATGCA-3′; Villin, 5′-CTATGCAGATGGTACCTGTTC-3′ and 5′-CCTGGGACGAGTCCTGGCCAA-3′; Muc2, 5′-ACATCACCTGTCCCGACTTC-3′ and 5′-GAGCAAGGGACTCTGGTCTG-3′; Lgr5, 5′-CATCACACTGTCACTGTGAGC-3′ and 5′-GGTAGCTGACTGATGTTGTTC-3′; Tnfa, 5′-TCCAGGCGGTGCCTATGT-3′ and 5′-CACCCCGAAGTTCAGTAGACAGA-3′; Il1b, 5′-TCAGGCAGGCAGTATCACTCA-3′ and 5′-GGAAGGTCCACGGGAAAGAC-3′; Ifng, 5′-TCAAGTGGCATAGATGTGGAAGAA-3′ and 5′-TGGCTCTGCAGGATTTTCATG-3′; Il6, 5′-CTGCAAGAGACTTCCATCCAGTT-3′ and 5′-AAGTAGGGAAGGCCGTGGTT-3′; Cx3cl1, 5′-GGCCGCGTTCTTCCATTTGT-3′ and 5′-TGATAGCGGATGAGCAAAGC-3′; Cxcl2, 5′-CTCAGTGCTGCACTGGTCCTG-3′ and 5′-CTGGGGGCGTCACACTCAAGC-3′; Ccl17, 5′-CTGCAGCATGCCAGAGCT-3′ and 5′-GGTCTTATACCAGCTCAC-3′; Ccl28, 5′-CAGCCTCACCTGAGTCATTGC-3′ and 5′-CAGTGCAACAGCTGGAGGCCA-3′; Gapdh, 5′-CCTCGTCCCGTAGACAAAATG-3′ and 5′-TCTCCACTTTGCCACTGCAA-3′. Plasmids (pCRII; Invitrogen) containing a cDNA fragment of Lypd8 were used as templates of RNA probes. Digoxigenin (DIG)-labelled antisense or sense probes were prepared with T7 and SP6 RNA polymerase (Ambion), respectively, using the DIG RNA Labelling Mix (Roche Diagnostics). Colonic tissues of C57BL/6J were fixed with 4% paraformaldehyde (PFA). Serial frozen sections were fixed in 4% PFA for 20 min, incubated in cold 0.1% H O , and permeabilized with 50 μg ml−1 proteinase K for 5 min. After an additional fixation with PFA, the sections were treated with acetic anhydrite in triethanolamine for 5 min. The sections were then pre-hybridized with 50% formamide, 5 × saline sodium citrate, 1 mg ml−1 yeast tRNA (Roche Diagnostics), 100 μg ml−1 heparin, 1 × Denhardt’s solution, 0.1% Tween 20 at 60 °C for 3 h, and hybridized at 60 °C for 16 h. After washing, sections were incubated with horseradish peroxide (HRP)-anti-DIG Fab fragment (clone 1.71.256: Roche Diagnostics), followed by biotin-labelled tyramide (TSA Biotin System; Perkin Elmer) for signal amplification. Hybridized probes were detected by ABC-Alkaline phosphatase (Vector Laboratories) and NBT/BCIP (Roche Diagnostics). Images were obtained using BZ-9000 (Keyence). Targeting vectors were constructed by replacement of genomic fragment containing the third and fourth exons of Lypd8 with a Cre recombinase internal ribosome entry site Venus neomycin-resistance gene cassette (Lypd8venus mice) or a neomycin-resistance gene cassette (Lypd8−/− mice), and a gene encoding HSV thymidine kinase driven by a phosphoglycerate kinase promoter inserted into the genomic fragment. After the targeting vector was transfected into V6.5 embryonic stem cells, G418 and ganciclovir double-resistant colonies were selected and screened by PCR and Southern blot analysis. Homologous recombinants were used for generation of Lypd8venus mice and Lypd8−/− mice. Lypd8venus mice and Lypd8−/− mice were backcrossed onto C57BL/6 mice for at least six generations, and Lypd8venus mice, Lypd8−/− mice and their wild-type littermates from intercrosses of heterozygous mice were kept in the same cages and used for experiments. There was no randomization, but stratification was used to achieve the similar ages and sex rations among experimental groups. The experiments were not blinded. Flow cytometry of isolated colonic epithelial cells Intestinal epithelial cells were isolated from Lypd8venus mice by shaking intestinal tissues in 5 mM EDTA/HBSS solution at 37 °C for 20 min and then washed with phosphate buffered saline (PBS). The intestinal epithelial cells were treated with PE-Cy7-conjugated anti-CD3ε Ab (clone 145-2C11: BD Biosciences) in PBS containing 2% FBS to block nonspecific binding. Flow cytometric analysis was performed using a FACSCanto II flow cytometer (BD Biosciences) with FlowJo software (Tree Star). For cell isolation, cells were sorted using a FACSAria (BD Biosciences). A sequence for Flag-tagged Lypd8 was constructed by inserting Flag-tag sequence into total Lypd8 coding sequence immediately downstream of predicted N-terminal signal sequence. CMT-93 cells and Caco-2 cells, which were originally obtained from ATCC and free of mycoplasma, were transfected with linearized pcDNA3.1 (+) vector (Invitrogen) inserted the sequence for Flag-tagged Lypd8 using Lipofectamine2000 (Invitrogen). These cells were cultured in G418-containing medium. The surviving cells were stained with anti-Flag M2 monoclonal antibody (cat F3165: Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG antibody (cat A11001: Molecular Probes) and cells expressing Flag-tagged Lypd8 were sorted using FACSAria (BD Biosciences). Caco-2 cells stably expressing Flag-tagged Lypd8 (1 × 105 cells) were cultured on transwell filters of 3.0-μm pore size (BD Biosciences) for 2 weeks. After confirming full confluency, Caco-2 cells were washed with PBS, fixed with 4% PFA and then blocked by 1% bovine serum albumin (BSA) in PBS. Cells were stained with mouse anti-Flag M2 mAb (Sigma-Aldrich) plus Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) or rabbit anti-human Claudin-1 Ab (cat ab15098; Abcam) plus Alexa Fluor 594 goat anti-rabbit IgG (cat A11012; Invitrogen), and counterstained with DAPI (Vector Laboratories). Transwell filters with these cells were cut, placed on slide glasses and analysed using a confocal microscope (FV1000-D; Olympus). The supernatants on Caco-2 cells with or without Lypd8 expression were incubated with anti-Flag M2 affinity gel (Sigma-Aldrich) at 4 °C for 3 h. The resin was collected, washed three times with Tris-buffered saline and then suspended in sample buffer for immunoblot analysis using anti-Flag M2 mAb (Sigma-Aldrich) and HRP-conjugated goat anti-mouse IgG (cat NA931: GE Healthcare). CMT93 cells stably expressing Lypd8 (1 × 106 cells) were rinsed twice with cold PBS and incubated with 0.5 ml of the same buffer containing 0.5 units of Bacillus cereus phosphatidylinositol-specific phospholipase C (Invitrogen) at 4 °C for 20 min. These cells were stained with anti-Flag M2 mAb (Sigma-Aldrich) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). The surface expression of Lypd8 was analysed using a FACSCanto II (BD Biosciences). Lypd8−/− mice were immunized with P3U1 cells with retroviral overexpression of Lypd8. Lymph node cells from immunized Lypd8−/− mice were fused with Sp2/0 mouse myeloma cells. Hybridomas were screened by analysing reactivity to RBL1 cells overexpressing Flag-tagged Lypd8 with flow cytometry. Positive clones were selected for further subcloning. Culture supernatants of subcloned hybridoma cells were screened by immunostaining of 4% PFA- or Carnoy’s-fixed colon sections from wild-type and Lypd8−/− mice. Culture supernatants of hybridoma clones (clone number 4F8 and 5A4), which stained Carnoy’s-fixed colon sections of wild-type mice, but not Lypd8−/− mice, were used as mouse anti-mouse Lypd8 mAb. The specificity of mAb was also confirmed by flow cytometry analysis of CMT93 cells with or without Lypd8 expression. Anti-mouse Lypd8 mAb was purified from the supernatant by Ex-pure Spin ProG (Kyoto monotech) and labelled with CF633 using Mix-n-Stain CF633 antibody labelling kit (Biotium). Colons from 8- to 12-week-old mice without washing were fixed in methanol–Carnoy’s fixative composed of 60% methanol, 30% chloroform and 10% acetic acid. Paraffin-embedded sections were dewaxed and hydrated. Sections were blocked with 1% BSA in PBS and stained with CF633-conjugated anti-mouse Lypd8 mAb or anti-Mucin2 Ab (clone H-300: Santa Cruz Biotechnology) and Alexa Fluor 594 goat anti-rabbit IgG (cat A11012: Invitrogen). Sections were incubated with 1 μg Cy3- or Cy5-conjugated EUB338 (5′-GCTGCCTCCCGTAGGAGT-3′) for detection of all bacteria or Cy3-conjugated pB-02110 (5′-ATGGGTTCATCCCATAGTGC-3′)14 for detection of Proteus spp. in 200 μl of hybridization buffer (750 mM NaCl, 100 mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.01% BSA, 10% dextran sulfate) at 40 °C for 16 h. Sections were rinsed in wash buffer (50 mM NaCl, 4 mM Tris-HCl (pH 7.4), 0.02 mM EDTA), washed at 45 °C for 20 min and counterstained with DAPI (Vector Laboratories). Human colons from the normal mucosa of resected colon tissues from colon cancer patients with no history of a diagnosis of inflammatory bowel diseases or the inflamed mucosa of resected colon tissues from ulcerative colitis patients were fixed in 4% PFA. The experiment was approved by the Ethical Committee of Osaka University School of Medicine. All participants provided informed consent. All ulcerative patients had a confirmed diagnosis by a gastroenterologist. Paraffin-embedded sections were dewaxed and hydrated. Sections were blocked with 1% BSA in PBS and stained with anti-human Lypd8 Ab (clone V-16: Santa Cruz Biotechnology) and Alexa Fluor 568 donkey anti-goat IgG (cat A11057: Invitrogen). Normal goat IgG (cat AB-108-C: R&D systems) was used as an isotype control. Sections were analysed using a confocal microscope (FV1000-D; Olympus). The distance between bacterial populations and the epithelial surface was measured at four points of the proximal, middle and distal colon in each mouse. Recombinant Lypd8 or LOC69864 (an uncharacterized protein that is structurally most similar to Lypd8) was purified from CMT93 cells stably expressing FLAG-tagged Lypd8 or LOC69864 using FLAG M Purification Kit (Sigma-Aldrich). As a negative control, cells transfected with empty vector (pcDNA3.1 (+) (Invitrogen)) were used. Recombinant Lypd8 proteins (1 μg) were incubated with PNGase F, sialidase A and O-glycanase (Prozyme) at 37 °C for 3 h. Recombinant Lypd8 proteins treated with glycanase were separated with SDS–PAGE and transferred to polyvinylidene fluoride membranes (Millipore) that were incubated with anti-Flag M2 mAb (Sigma-Aldrich) and then HRP-conjugated goat anti-mouse IgG (cat NA931: GE Healthcare). Immunoreactivity was detected using SuperSignal (Thermo Scientific). The mutant Lypd8 N–D sequence was designed so that thirteen Asp (N) residues were converted to Asn (D). 293T cells were transfected with the mutant Lypd8 (N–D) expression vector using Lipofectamine 2000 (Invitrogen) and cell lysates of 293T cells expressing mutant Lypd8 (N–D) protein were separated with SDS–PAGE and analysed by western blot. Faeces, luminal contents of the colon or colonic tissues were collected in tubes containing RNAlater (Ambion). After weights were measured, RNAlater was added to make tenfold dilutions of homogenates. Homogenates (200 μl) of faeces, luminal contents or colonic tissues, or bacterial suspension containing sorted bacteria were washed twice with 1 ml PBS, 0.3 g glass beads (diameter, 0.1 mm) (BioSpec Products), 300 μl Tris-SDS solution and 500 μl TE-saturated phenol were added to the suspension, and the mixture was vortexed vigorously using a FastPrep-24 (M.P. Biomedicals) at 5.0 power level for 30 s. After centrifugation at 20,000 g for 5 min at 4 °C, 400 μl of supernatants were collected. Subsequently, phenol–chloroform extraction was performed and 250 μl of supernatants were subjected to isopropanol precipitation. Finally, DNAs were suspended in 200 μl TE buffer and stored at −20 °C. PCR was performed using a primer set (784F, 5′-AGGATTAGATACCCTGGTA-3′; and 1061R, 5′-CRRCACGAGCTGACGAC-3′) targeting the V5–V6 region of the 16S rRNA genes with KAPA HiFi HotStart Ready Mix (KAPA Biosystems). Products were purified using DNA clean and Concentrator-5 (Zymo Research). Adaptor and barcode sequences were attached to the products by 10 cycles of PCR with 1 ng of each of initial PCR product as the template and primer sets and other PCR conditions were unchanged. Sequencing was performed using a 316 chip and Ion PGM Sequencing 400 Kit (Life Technologies) on the Ion PGM sequencer (Life Technologies). Raw sequences were demultiplexed and quality-trimmed by the following procedures: (1) trimming bases with quality below Q15 from 3′ end of each read, (2) removing reads with average quality below Q20, (3) removing reads without primer sequences on both ends, and (4) removing reads with length shorter than 260 basepairs, using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html) and BBtrim (http://bbmap.sourceforge.net/). The processed sequences were then clustered into operational taxonomic units (OTU) defined at 94% similarity cutoff using UCLUST version 1.2.22q. Representative sequences for each OTU were classified taxonomically using RDP Classifier version 2.2 with the SILVA 111 database. Quantitative PCR was performed in ABI7300 using GoTaq qPCR Master Mix (Promega). The following primer sets were used: ‘all bacteria’, 5′-CGGTGAATACGTTCCCGG-3′ and 5′-TACGGCTACCTTGTTACGACTT-3′; Bacteroides, 5′-GAGAGGAAGGTCCCCCAC-3′ and 5′-CGCTACTTGGCTGGTTCAG-3′; Prevotella, 5′-CACRGTAAACGATGGATGCC-3′ and 5′-GGTCGGGTTGCAGACC-3′; Lactobacillus, 5′-TGGAAACAGRTGCTAATACCG-3′ and 5′-GTCCATTGTGGAAGATTCCC-3′; Bifidobacterium, 5′-CTCCTGGAAACGGGTGG-3′ and 5′-GGTGTTCTTCCCGATATCTACA-3′; Escherichia/Shigella, 5′-GAGTAAAGTTAATACCTTTGCTCATTG-3′ and 5′-GAGACTCAAGCTKRCCAGTATCAG-3′; Helicobacter, 5′-CTATGACGGGTATCCGGCC-3′ and 5′-TCGCCTTCGCAATGAGTATT-3′; Staphylococcus, 5′-TTTGGGCTACACACGTGCTACAATGGACAA-3′ and 5′-AACAACTTTATGGGATTTGCWTGA-3′, Enterococcus, 5′-ATCAGAGGGGGATAACACTT-3′ and 5′-ACTCTCATCCTTGTTCTTCTC-3′; and Proteus, 5′-GTTATTCGTGATGGTATGGG-3′ and 5′-ATAAAGGTGGTTACGCCAGA-3′. In the analysis of Lypd8-bound and unbound bacteria, all samples were normalized to the 16S rRNA gene level of ‘all bacteria’. Colonic tissues were thoroughly washed twice and collected in tubes containing 5 ml PBS. After colonic tissues were homogenized in PBS using a homogenizer, 100 μl of the homogenates were incubated on lysogeny broth (LB) or MacConkey agar plates at 37 °C in Ruskinn Bugbox Plus anaerobic chamber (The Baker Company) for 24 h. The number of swarming colonies on LB agar plates or white colonies on MacConkey agar plates was counted at 16 h after the start of incubation and colony-forming units were calculated. P. mirabilis was cultured in LB medium at 37 °C until OD reached 0.6. The bacterial culture was centrifuged at 8,000g for 5 min and the pellet was resuspended in 1 ml PBS. The bacterial suspension was re-centrifuged and the pellet was labelled with CFSE Fluorescent Cell Labelling Kit (Abcam). After confirming CFSE labelling of P. mirabilis using a FACSCanto II (BD Biosciences), CFSE-labelled P. mirabilis was suspended with PBS (5 × 108 per ml) and the bacterial suspension (200 μl) was inoculated into anaesthetized mice via the transanal route with a catheter. At 1, 2, 4, and 8 h after inoculation, colons without washing were fixed in 4% PFA. Colon sections were counterstained with DAPI (Vector Laboratories), and analysed using a confocal microscope (FV1000-D; Olympus). Eight- to ten-week-old Lypd8−/− mice and their littermate wild-type mice were used for DSS-induced colitis experiments. Acute colitis was induced by administration of 2% DSS (36–50 kDa; MP Biomedicals) in the drinking water for 5 days. Mice were analysed for changes in weight, survival rates and histological changes. Mice were treated with gentamicin (2 g l−1; Nacalai Tesque) or vancomycin (500 mg l−1; Duchefa Biochemie B.V.) dissolved in autoclaved drinking water and provided for 2 weeks. Fluid intake was monitored. Faeces were collected in tubes containing PBS. After weights were measured, PBS was added to make tenfold diluted suspensions. PBS-diluted faeces were mixed well and centrifuged at 400g for 5 min to remove larger particles from bacteria. Supernatants (200 μl) were centrifuged at 8,000g for 10 min to remove non-bound immunoglobulins. Pellets were resuspended in 1 ml PBS containing 2% FBS, and used as a bacterial suspension. Bacterial suspensions from wild-type mice and Lypd8−/− mice were incubated with anti-Lypd8 antibody. After washing with PBS containing 2% FBS, bacterial pellets were stained with Alexa Fluor 647 goat anti-mouse IgG1 (Invitrogen). Bacterial suspensions from C57BL/6J mice were incubated with recombinant Lypd8 protein (1 μg) on ice for 30 min and washed with PBS. Next, bacterial pellets were treated with anti-Flag M2 mAb (Sigma-Aldrich). Finally, bacterial pellets were stained with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). Bacterial suspensions were analysed using a FACSCanto II (BD Biosciences) with FlowJo software (Tree Star) or a confocal microscope (FV1000-D; Olympus). Lypd8-bound and unbound bacteria were sorted using a FACSAria (BD Biosciences). Bacterial strains, Bacteroides sartrii JCM 17136T, Lactobacillus acidophilus JCM 1132T, Bifidobacterium breve JCM 1192T and Enterococcus gallinarum JCM 8728T, were obtained from Japan Collection of Microorganisms. P. mirabilis was isolated from the colonic tissue of Lypd8−/− mice. All bacteria were cultured in Gifu anaerobic medium (Nissui) under anaerobic conditions. The pure preparation of each bacteria was centrifuged at 8,000g for 5 min and the pellet was resuspended in 1 ml PBS containing 2% FBS. Bacteria were incubated with recombinant Lypd8 protein (1 μg) on ice for 1 h and washed with PBS. Next, the bacterial pellet was treated with anti-Flag M2 mAb (Sigma-Aldrich). Finally, the bacterial pellet was stained with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) and analysed using a FACSCanto II (BD Biosciences). Bacterial suspension was mixed with FLAG-tagged recombinant Lypd8 (rLypd8; 20 ng μl−1), and then incubated for 1 h at 4 °C. After the incubation, bacterial suspensions were centrifuged, and pellet was resuspended in PBS. A drop of bacterial suspension was placed on an 3-aminopropyltriethoxysilane (APS)-coated cover slip (Matsunami Glass IND), and blocked with bovine serum albumin and normal goat serum. The mounted bacteria were reacted with anti-FLAG M2 mAb (Sigma-Aldrich), washed with PBS, and reacted with 5-nm-gold-labelled anti-mouse IgG goat antibody (EY Laboratories). The sample was fixed with 2% glutaraldehyde and 1% OsO , dehydrated in a graded ethanol series and 3-methylbutyl acetate, and dried in a critical-point drying chamber (HCP-1, Hitachi High-Technologies). It was then coated with a platinum layer approximately 1 nm thick in an ion sputter coater (E-1030, Hitachi High-Technologies), and examined under a scanning electron microscope (S-5000, Hitachi High-Technologies). Bacterial suspension of P. mirabilis or E. coli JCM 1649T (Japan Collection of Microorganisms) in PBS was shaken 300 times per min for 60 min to remove flagella from bacterial bodies. Bacterial bodies were pelleted by centrifuging at 4,000g for 20 min. The supernatants were ultracentrifuged at 80,000g for 60 min to obtain flagella. Bacterial bodies or flagella were mixed with the solution of FLAG-tagged recombinant Lypd8 (10 ng μl−1) or LOC69864 (10 ng μl−1), and incubated for 3 h at 4 °C. After the incubation, the bacterial bodies or flagella were pelleted by centrifugation or ultracentrifugation, respectively. Then, the supernatant was collected and the pellet was resuspended in PBS. The supernatant and pellet suspension were separated with SDS–PAGE and transferred to polyvinylidene fluoride membranes (Millipore) that were incubated with anti-Flag M2 mAb (Sigma-Aldrich) and then HRP-conjugated goat anti-mouse IgG (cat NA931: GE Healthcare). Immunoreactivity was detected using SuperSignal (Thermo Scientific). Bacterial DNA of P. mirabilis was extracted from the culture of P. mirabilis using a previously described protocol (see above). Using the extracted bacterial DNA as template, PCR was performed to isolate the flagellin gene with a primer set (5′-GGATCCGCACAAGTTATTAATACTAATTAT-3′ and 5′-GCGGCCGCTTAACGTAACAGAGACAGAACAGT-3′) targeting the full-length flagellin gene of P. mirabilis. The isolated DNA was cloned in frame into EcoRI- and NotI-digested pGEX-6P-2 (GE Healthcare) to generate a glutathione S-transferase (GST)–flagellin fusion construct. A recombinant flagellin protein was prepared by growing E. coli harbouring the pGEX-flagellin plasmids in LB medium containing 100 μg ml−1 of ampicillin at 37 °C until OD reached 0.6. After growth, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the medium, and bacterial culture was continued for 4 h at 37 °C. Cells were centrifuged and the cell pellet was suspended in PBS with 1% Triton X-100. The cell suspension was sonicated 5 times for 30 s at 4 °C, and cleared by centrifugation. The resulting solution was bound to Glutathione-Sepharose 4 Fast Flow (GE Healthcare). The beads were washed 3 times. The GST fusion proteins were then eluted with elution buffer (5 mM glutathione, 50 mM Tris-HCl, pH 9.6). Elution buffer was replaced with PBS with Amicon Ultra-15 (Millipore). The amounts and purity of the protein were estimated by SDS–PAGE. 96-well plates (Corning) were coated with 5 μg ml−1 GST peptides, 5 μg ml−1 GST-tagged flagellin protein of P. mirabilis, 100 μg ml−1 flagella of P. mirabilis or PBS 1% BSA for 16 h at 4 °C. Plates were then washed, and increasing concentrations of mouse TLR5 Fc protein (R&D systems) or FLAG-tagged Lypd8 protein diluted in PBS 1% BSA were added and incubated for 2 h at room temperature. Plates were then washed, and 0.5 μg ml−1 of HRP-conjugated goat anti-mouse IgG (cat NA931: GE Healthcare) or HRP-conjugated anti-FLAG M2 mAb (Sigma-Aldrich) diluted in PBS 1% BSA was added and incubated for 2 h at room temperature. Plates were then washed, and TMB substrate was added. Plates were then read at 450 nm with a spectrometer. P. mirabilis (1 × 105) were incubated in LB medium (1 ml) with the indicated concentrations of recombinant Lypd8 protein for 6 h at 37 °C. The bacterial cultures were applied to MacConkey agar plates and incubated at 37 °C for 16 h, and the number of colonies was counted and colony-forming units were calculated. Human colon epithelia were obtained from the normal mucosa of resected colon tissues from colon cancer patients. Colonic epithelial layers were first stripped by shaking sections of colon in 5 mM EDTA/HBSS. Total RNA was isolated from Caco-2 cells and human colon epithelia and reverse transcribed. cDNA was analysed by real-time RT–PCR. Values were normalized to the expression of GAPDH, and the fold difference in expression relative to that of GAPDH is shown. The following primer sets were used: LYPD8, 5′-GAACACTTTCATTTTGTAAGC-3′ and 5′-ACGACAGGAAGTTCCATTAGA-3′; GAPDH, 5′-TGGATATTGTTGCCATCAATG-3′ and 5′-TGATGGGATTTCCATTGATGA-3′. The experiment was approved by the Ethical Committee of Osaka University School of Medicine and informed consent for specimen use was obtained from all patients. E. gallinarum or P. mirabilis (1 × 105) was added to a culture of polarized Caco-2 cells. For PNGase F treatment, cells were irradiated with 30 Gy to stop cell growth and then incubated in culture medium containing PNGaseF (Sigma-Aldrich) (2 unit/ml) for 2 h. At 8 h after the addition of P. mirabilis, each well was washed with PBS three times and then Caco-2 cells were removed from transwell filters with trypsin/EDTA solutions and suspended in PBS. PBS containing Caco-2 cells were applied to LB or MacConkey agar plates and incubated at 37 °C for 16 h, and the number of colonies was counted and colony-forming units were calculated. P. mirabilis was cultured in LB medium at 37 °C until OD was 0.6. LB agar plates (1.5%) were centrally inoculated with 5 μl of bacterial culture and incubated at 37 °C. After 1 h, polarized Caco-2 cells with or without Flag-tagged Lypd8 expression, cultured on 75 mm diameter transwell filters of 3.0 μm pore size (BD Biosciences) for approximately 2 weeks, were added to P. mirabilis-inoculated agar plate so that the apical side of cells adhered to the surface of the agar plate. Motility was assessed by examining circular swarms formed by growing motile bacterial cells. The length from the centre to four points of the edge of circular swarms was measured at 5, 9 and 13 h time points following P. mirabilis inoculation. CMT-93 cells (5 × 107 cells) with or without Flag-tagged Lypd8 were lysed with 1 ml of CelLytic M Cell Lysis Reagent (Sigma-Aldrich). After removal of any insoluble material by centrifugation and filtration with a 0.45 μm filter, the cell lysates were incubated with 50 μl of anti-Flag M2 affinity gel (Sigma-Aldrich) for 3 h. The resin was centrifuged and washed with Tris-buffered saline three times. The resin containing Flag-tagged Lypd8 was mixed with 1 ml of LB medium containing 0.3% agar. The final concentration of Lypd8 in LB agar was estimated by SDS–PAGE and was approximately 1.25 μg ml−1. P. mirabilis and E. coli were cultured in LB medium at 37 °C until OD was 0.6. Semisolid LB agar (0.3%) containing Flag-tagged Lypd8 were centrally inoculated with 0.5 μl of bacterial culture and incubated at 37 °C. Motility was assessed by examining circular migration. The radius of circles formed by bacterial migration was measured at 4 h after the bacterial inoculation. Data are presented as mean ± s.d., as indicated in the figure legends. Differences between control and experimental groups were evaluated using a two-tailed unpaired Student’s t-test. For the swarming motility assay, a paired Student’s t-test was performed. A P value of <0.05 was considered significant. For the determination of microbiota by deep sequencing, principal component analysis was used to visualize data sets by statistical programming language R 2.1.5. No statistical methods were used to predetermine sample size. No animal or sample was excluded from the analysis.