News Article | May 10, 2017
To generate CRISPR–Cas9 plasmids targeting the last exon of LGR5 (exon 18) or KRT20 (exon 8), 20-bp target sequences were cloned into a pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid (Addgene 42230) to obtain single vectors bicistronically expressing sgRNA and human codon-optimized Cas9 nuclease as previously described36. The sgRNA sequences targeting LGR5 or KRT20 are available in Supplementary Table 1. To construct donor vectors for LGR5–GFP- and KRT20–GFP-knock-in, 5′ and 3′ homology arms (1 kbp each) were amplified by PCR and cloned into an Ires-GFP-loxp-pEF1α-RFP-T2A-puro-loxp plasmid (HR180PA-1, SBI) using the In-Fusion HD Cloning kit (Clontech). For CreER or iCaspase9-T2A-tdTomato knock-in, we replaced GFP of the LGR5–GFP or KRT20–GFP construct with CreER or iCaspase9-T2A-tdTomato, respectivley. The final plasmid sequences were verified by DNA sequencing. To obtain a rainbow reporter, the rainbow cassette was excised from a CMV-Brainbow-2.1R plasmid (Addgene 18723) and cloned into a PiggyBac vector (PB510B-1, SBI). For bioluminescent imaging, we cloned optimized firefly luciferase luc2 into a GFP-expressing PiggyBac vector (PB513B-1, SBI). Knock-in efficiency and diver mutation profiles for each CCO line are available in Supplementary Table 2. All organoids were established as previously reported16 from patients who had given informed consent under the ethical committee of Keio University School of Medicine. The organoids were embedded in Matrigel and cultured with previously described basal culture medium37, specifically Advanced Dulbecco’s modified Eagle’s medium/F12 supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1× B27 (Life Technologies), 10 nM gastrin I (Sigma) and 1 mM N-acetylcysteine (Sigma). The following niche factors were added to the basal culture medium depending on the niche requirements of CRC organoid lines: 50 ng ml−1 mouse recombinant EGF, 100 ng ml−1 mouse recombinant noggin (PeproTech) and 500 nM A83-01 (Tocris). We electroporated the vectors under previously reported conditions37. Three days after electroporation, the organoids were selected with puromycin (2 μg ml−1) treatment for two days. For in vitro ablation experiments, we treated the organoids with 1 nM dimerizer (AP20187, Clontech). Drug-resistant organoid clones were manually selected and expanded individually. Genomic DNA was isolated using the QIAamp DNA blood mini kit (Qiagen). Legitimate knock-in was determined by PCR. Southern blotting was performed based on the standard procedure using 1 μg of genomic DNA. The sequences of PCR primers and Southern blot probes are shown in Supplementary Table 1. The puromycin-RFP selection cassette flanked by loxP sequences was excised by transient infection of Cre-expressing adenovirus (TaKaRa) at multiplicity of infection of 5–10. After the infection, we manually selected and cloned RFP− organoids. Deletion of the puromycin cassette was validated by PCR diagnostics. Percentage of successful knock-in for each line is shown in Supplementary Table 2. Once a knock-in reporter CCO was cloned, we used the same clone for further experiments. Organoids were dissociated into single cells with TrypLE Express (Life Technology), and large clusters were removed with a CellTrics 20-μm cell strainer (Partec). The cells were washed with cold PBS and stained with 7-amino-actinomycin D (7-AAD) staining solution (BD Biosciences) to exclude dead cells. Single cells were gated based on the SSC-H versus SSC-W profile. The cells were subsequently analysed using a flow cytometer with a 70-μm nozzle (FACS JAZZ, BD Biosciences). Then, 1,000 sorted cells were embedded in 25 μl of Matrigel and cultured in a 48-well plate for 7–10 days. We added 10 μM Y27632 for the first two days of culture, and the organoid colony formation was assessed using a BZX-700 fluorescence microscope (Keyence). RNA was extracted from 1 × 106 sorted cells using the RNeasy Plus mini kit (Qiagen). The RNA quality was determined by the RNA integrity number (RIN) value with the RNA6000 assay (Agilent). Only specimens with RIN > 7.0 were used in this study. Gene expression was determined by microarray (GeneChip PrimeView Human Gene Expression Array, Affymetrix) according to the manufacturer’s instructions. The data were normalized using the robust multi-array analysis implemented in the R package affy. The probes were summarized into genes by selecting probes with the highest median absolute deviation value per gene. GSEA was performed using gsea (in the R package phenoTest) with 1,000 permutations. Two independent intestinal stem cell signature gene sets from refs 17, 38 were used. All animal procedures were approved by the Keio University School of Medicine Animal Care Committee. NOD/Shi-scid,IL-2Rγnull (NOG) mice39 (7–12 weeks of age, male) were obtained from the Central Institute for Experimental Animals (CIEA, Japan). Organoids with the indicated genetic reporter and with or without GFP-luc2-reporter, equivalent to 1 × 105 cells, were xenotransplanted subcutaneously or into the renal subcapsules as previously described40. We monitored the tumour size with a calliper or through bioluminescence imaging. Tumour volumes were measured according to the formula (length × width2) / 2. Once any individual tumour reached 2 cm in size, the mouse was euthanized. For bioluminescence imaging, we intraperitoneally administered 3 mg of D-luciferin (SPI, Tokyo) to tumour-bearing mice 10–20 min before imaging and anaesthetized the animals with isoflurane. The bioluminescence signal was measured with an IVIS imaging system (Xenogen), and the specific signal was calculated as the ratio of photon counts from the region of interest to counts from a background region. The grafts were fixed for subsequent histological analyses. An investigator blinded to the experimental conditions measured the tumour sizes. For the lineage-tracing experiments, each mouse received a single intraperitoneal injection of 0.25 mg (clonal dose) or 1 mg of tamoxifen (Sigma-Aldrich) diluted in corn oil. For the ablation studies, 40 μg of dimerizer was administered for five days daily for short term ablation and on alternate days for long term ablation. To label the proliferating cells, we intraperitoneally administered BrdU (40 mg kg−1, BD Biosciences) and EdU (10 mg kg−1, Life Technologies) at the indicated times. For chemotherapeutic studies, CTX (40 mg kg−1, Merck Serono) or oxaliplatin (15 mg kg−1, AdooQ Bioscience) was administered intraperitoneally at the indicated times. We isolated tumours from xenografted mice and immediately fixed them with 4% paraformaldehyde. Eight-micrometre OCT frozen tissue sections or 5-μm paraffin-embedded tissue sections were processed using a standard histological protocol. For rainbow fluorescent imaging, the frozen sections were visualized using an SP8 confocal microscope (Leica) with the following settings: mCFP was excited at 405 nm and collected using a 480–485-nm filter, nuclear GFP was excited at 488 nm and collected using a 494–507-nm filter, EYFP was excited at 514 nm and collected using a 560–566-nm filter, and RFP was excited at 552 nm and collected using a 601–665-nm filter. Nuclei were counterstained with the near-infrared nuclear dye DRAQ5 (BioStatus). For DLS 3D imaging, the whole tumours were cut into 1–2 mm3 pieces, fixed and embedded in agarose gel. 3D images were acquired using the Leica SP8 DLS system. The proportion of surviving clones was determined by counting the number of RFP+ cells at day 3 and day 31 after tamoxifen administration. Clone identification and raw volume measurement were carried out automatically using the ImageJ 3D-image processing package ‘3D object counter’41, 42. False identification rate of this automatic measurement was determined manually by random sampling. Raw volume was adjusted by randomly subtracting a proportion of clones according to the false-rate. The threshold volume for total colonies on day 3 was set as <2 × 105 μm3 and for large colonies on day 31 as >2 × 105 μm3 (equivalent to 20 cells). Colony-formation efficiency was defined as the ratio of the number of large colonies on day 31 to the number of clones on day 3. For immunostaining, the following primary antibodies were used: mouse anti-cytokeratin-20 (M7019, clone K 20.8, Dako, 1:50), goat anti-GFP (ab6673, Abcam, 1:200), rabbit anti-Ki67 (ab16667, Abcam, 1:100), mouse anti-α smooth muscle actin ab-1 (MS-113-P, Thermo Scientific, 1:800), mouse anti-BrdU (347580, BD, 1:100), anti-cleaved caspase-3 (9661, Cell Signaling, 1:100) and anti-tdTomato (600-401-379, ROCKLAND, 1:500). Alexa Fluor 488-, 568- or 647-conjugated secondary antibodies (Life Technologies, donkey anti-mouse, rabbit, rat or goat antibodies) were used at 1:200 dilution. For EdU staining, we used the Click-IT Plus EdU Imaging kit (Life Technologies) according to the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33342 or DAPI. Images were captured with a Leica SP8 confocal microscope or a BZX-700 fluorescence microscope (Keyence). To count the number of BrdU/EdU-stained cells, we used Imaris (Bitplane). In situ hybridization was performed using an RNAscope 2.5HD kit (Advanced Cell Diagnostics) according to the manufacturer’s instructions. For each experiment, we used PPIB and DapB genes as positive and negative controls, respectively. Tumour tissues were homogenized using TissueLyser LT (Qiagen) and RNA was extracted with the RNAeasy mini kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the Omniscript RT kit (Qiagen). Quantitative real-time PCR was performed on LightCycler 96 (Roche Diagnostics) using FastStart Essential DNA Probes Master (Roche Diagnostics) and the cDNAs as templates. Relative LGR5 expression to ACTB was calculated based on the comparative C method. Primers and probes for LGR5 and ACTB are available in Supplementary Table 1. The sample size was determined by previous experience and preliminary experiments. The vehicle/dimerizer/chemotherapy-treated group was randomly assigned on the basis of tumour size at the time of injection. Appropriate statistical analyses were performed dependent on the comparisons referenced in the figure legends. The n values represent biological replicates. All graphs show mean and error bars represent the standard error of the mean (s.e.m.). Genetic mutation data of organoids are summarized in Supplementary Table 2 and described in ref. 16. The microarray dataset generated in this study is available in the Gene Expression Omnibus (accession number: GSE83513). All other data are available from the corresponding author upon reasonable request.
News Article | May 10, 2017
— Raising demand for usage of flow cytometry in clinical trials, new technological improvements in flow cytometers, advancements in intuitive softwares are some factors fostering the market growth. However, lack of availability of technical expertise, huge investments in flow cytometry instruments are hindering the growth of the market. Based on the Technology segment, Cell-based flow cytometry leads the market globally as they are widely used in the study of tumor stem cells and in the study of disease mechanism. Among applications, the demand for clinical diagnosis is increasing owing to growing demand for disease diagnostic tools. North America is dominating the flow cytometry market globally due to the support of government involved in developing new technologies for molecular diagnostics and also due to the presence of skilled professionals int he region. Some of the key players in Flow Cytometry Market are Affymetrix Inc., Beckman Coulter, Inc., Miltenyi Biotec GmbH, Sony Biotechnology Inc., Becton, Dickinson and Company, Thermo Fisher Scientific, Inc., Bio-Rad Laboratories Inc., Luminex Corporation, Sysmex Partec GmbH, Merck KGAA, Advanced Cell Diagnostics, Bangs Laboratory Inc. , Pointcare Technologies Inc., Inivai Technologies, Cytek Development Inc, and Agilent Technologies. Applications Covered: • Clinical Applications o Immunodeficiency Diseases o Cancer and solid tumor o Organ Transplantation o Hematology o Oncology o Blood Transfusion o HIV o Other clinical applications • Research Applications o Pharmaceutical and Biotechnology o Cell Viability and Transfection o Cell Sorting and screening o Apoptosis o Intracellular Calcium flux o Immunology o Cell Cycle Analysis o Other research applications • Industrial Applications Products & Services Covered: • Flow Cytometry Instruments o Cell Sorters o Cell Analyzers o Replaceable components • Reagents and consumables o Beads o Antibodies o Dyes o Other reagents and consumables • Accessories • Services • Software Regions Covered: • North America o US o Canada o Mexico • Europe o Germany o France o Italy o UK o Spain o Rest of Europe • Asia Pacific o Japan o China o India o Australia o New Zealand o Rest of Asia Pacific • Rest of the World o Middle East o Brazil o Argentina o South Africa o Egypt What our report offers: - Market share assessments for the regional and country level segments - Market share analysis of the top industry players - Strategic recommendations for the new entrants - Market forecasts for a minimum of 7 years of all the mentioned segments, sub segments and the regional markets - Market Trends (Drivers, Constraints, Opportunities, Threats, Challenges, Investment Opportunities, and recommendations) - Strategic recommendations in key business segments based on the market estimations - Competitive landscaping mapping the key common trends - Company profiling with detailed strategies, financials, and recent developments - Supply chain trends mapping the latest technological advancements About Stratistics MRC We offer wide spectrum of research and consulting services with in-depth knowledge of different industries. We are known for customized research services, consulting services and Full Time Equivalent (FTE) services in the research world. We explore the market trends and draw our insights with valid assessments and analytical views. We use advanced techniques and tools among the quantitative and qualitative methodologies to identify the market trends. Our research reports and publications are routed to help our clients to design their business models and enhance their business growth in the competitive market scenario. We have a strong team with hand-picked consultants including project managers, implementers, industry experts, researchers, research evaluators and analysts with years of experience in delivering the complex projects. For more information, please visit: http://www.strategymrc.com For more information, please visit http://www.strategymrc.com/
News Article | February 16, 2017
Research and Markets has announced the addition of the "The Nanocoatings Global Opportunity Report" report to their offering. 'The Nanocoatings Global Opportunity Report' examines a market that is already providing significant economic, hygiene and environmental benefit for sectors such as consumer electronics, construction, medicine & healthcare, textiles, oil & gas, infrastructure and aviation. Research and development in nanotechnology and nanomaterials is now translating into tangible consumer products, providing new functionalities and opportunities in industries such as electronics, sporting goods, wearable electronics, textiles, construction etc. A recent example is quantum dot TVs, a multi-billion dollar boon for the High-definition TV market. Countless other opportunities exist for exploiting the exceptional properties of nanomaterials and these will increase as costs come down and production technologies improve. The incorporation of nanomaterials into thin films, coatings and surfaces leads to new functionalities, completely innovative characteristics and the possibility to achieve multi-functional coatings and smart coatings. The use of nanomaterials also results in performance enhancements in wear, corrosion-wear, fatigue and corrosion resistant coatings. Nanocoatings demonstrate significant enhancement in outdoor durability and vastly improved hardness and flexibility compared to traditional coatings. - Oil and gas - - Corrosion and scaling chemical inhibitors. - - Self-healing coatings. - - Smart coatings. - - Coatings for hydraulic fracturing. - Aerospace & aviation - - Shape memory coatings. - - Corrosion resistant coatings for aircraft parts. - - Thermal protection. - - Novel functional coatings for prevention of ice-accretion and insect-contamination. - Renewable energy - - Anti-fouling protective coatings for offshore marine structures. - - Anti-reflective solar module coatings. - - Ice-phobic wind turbines. - - Coatings for solar heating and cooling. - Automotive - - Anti-fogging nanocoatings and surface treatments. - - Improved mar and scratch resistance. - - Flexible glass. - - Corrosion prevention. - - Multi-functional glazing. - - Smart surfaces. - - Surface texturing technologies with enhanced gloss. - - New decorative and optical films. - - Self-healing. - Textiles & Apparel - - Sustainable coatings. - - High UV protection. - - Smart textiles. - - Electrically conductive textiles. - - Enhanced durability and protection. - - Anti-bacterial and self-cleaning. - - Water repellent while maintaining breathability.. - Medical - - Hydrophilic lubricious, hemocompatible, and drug delivery coatings. - - Anti-bacterial coatings to prevent bacterial adhesion and biofilm formation. - - Hydrophobic and super-hydrophobic coatings. - - Lubricant coatings. - - Protective implant coatings. - - High hardness coatings for medical implants. - - Infection control. - - Antimicrobial protection or biocidic activity. - Marine - - Anti-fouling and corrosion control coatings systems. - - Reduced friction coatings. - - Underwater hull coatings. - Buildings - - Thermochromic smart windows. - - Anti-reflection glazing. - - Self-cleaning surfaces. - - Passive cooling surfaces. - - Air-purifying. - Consumer electronics - - Waterproof electronic devices. - - Anti-fingerprint touchscreens. - Global market size for target markets - Addressable markets for nanocoatings, by nanocoatings type and industry - Estimated market revenues for nanocoatings to 2025 - 300 company profiles including products and target markets 1 Executive Summary 1.1 High performance coatings 1.2 Nanocoatings 1.3 Market drivers and trends 1.4 Market size and opportunity 1.5 Market and technical challenges 2 Introduction 2.1 Properties of nanomaterials 2.2 Categorization 2.3 Nanocoatings 2.4 Hydrophobic coatings and surfaces 2.5 Superhydrophobic coatings and surfaces 2.6 Oleophobic and omniphobic coatings and surfaces 6 Market Segment Analysis, By Coatings Type 6.1 Anti-Fingerprint Nanocoatings 6.2 Anti-Microbial Nanocoatings 6.3 Anti-Corrosion Nanocoatings 6.4 Abrasion & Wear-Resistant Nanocoatings 6.5 Barrier Nanocoatings 6.6 Anti-Fouling And Easy-To-Clean Nanocoatings 6.7 Self-Cleaning (Bionic) Nanocoatings 6.8 Self-Cleaning (Photocatalytic) Nanocoatings 6.9 Uv-Resistant Nanocoatings 6.10 Thermal Barrier And Flame Retardant Nanocoatings 6.11 Anti-Icing And De-Icing 6.12 Anti-Reflective Nanocoatings 6.13 Other Nanocoatings Types 7 Market Segment Analysis, By End User Market 7.1 Aerospace 7.2 Automotive 7.3 Construction, Architecture And Exterior Protection 7.4 Electronics 7.5 Household Care, Sanitary And Indoor Air Quality 7.6 Marine 7.7 Medical & Healthcare 7.8 Military And Defence 7.9 Packaging 7.10 Textiles And Apparel 7.11 Renewable Energy 7.12 Oil And Gas Exploration 7.13 Tools And Manufacturing 7.14 Anti-Counterfeiting - 3M - Abrisa Technologies - Accucoat, inc - Aculon, Inc - Acreo Engineering - ACTNano, inc - Advanced Materials-JTJ S.R.O - Advanced Silicon Group - Advenira Enterprises, Inc - Aeonclad Coatings - agPolymer S.r.l - Agienic Antimicrobials - Agion Technologies, Inc - AkzoNobel - Albert Rechtenbacher GmbH - ALD Nanosolutions, Inc - Alexium, Inc - AM Coatings - Analytical Services & Materials, Inc - Ancatt - Applied Nanocoatings, Inc - Applied Nano Surfaces - Applied Sciences, Inc - Applied Thin Films, Inc - ARA-Authentic GmbH - Asahi Glass Co., Ltd - Autonomic Materials - Aurolab - Avaluxe International GmbH - Bactiguard AB - BASF Corporation - Battelle - Beijing ChamGo Nano-Tech Co., Ltd., - Beneq OY - BigSky Technologies LLC - Biocote Ltd - Bio-Gate AG - Bioni CS GmbH - Bionic Technology Holding BV - Boral Limited - Buhler Partec - BYK-Chemie GmbH - California Nanotechnologies Corporation - Cambridge Nanotherm Limited - Cambrios Technologies Corporation - Canatu Oy - Carbodeon Ltd. Oy - Ceko Co., Ltd - Cellutech AB - CeNano GmbH & Co. KG - Cellmat Technologies S.L - Centrosolar Glas GmbH Co. KG - Cetelon Nanotechhnik GmbH - CG2 Nanocoatings, Inc - Cima Nanotech - Clarcor Industrial Air - Clariant Produkte (Deutschland) GmbH - Cleancorp Nanocoatings - Clearbridge Technologies Pte. Ltd - Clearjet Ltd - Clou - CMR Coatings GmbH - CNM Technologies GmbH - Coating Suisse GmbH - Corning, Incorporated - Cotec GmbH - Coval Molecular Coatings - Crossroads Coatings - CSD Nano, Inc - CTC Nanotechnology GmbH - C3 Nano - Cytonix CLLC - Daicel FineChem Limited - Daikin Industries, ltd - Diamon-Fusion International, Inc - Diarc-Technology Oy - DFE Chemie GmbH - Dow Corning - Dropwise Technologies Corporation - DryWired - Dry Surface Technologies LLC - DSP Co., Ltd - Duralar Technologies - Duraseal Coatings - Eeonyx Corporation - Eikos, Inc - Engineered Nanoproducts Germany AG - Enki Technology - Envaerospace, Inc - Eurama Corporation - Europlasma NV - Excel Coatings - Evonik Hanse - Few Chemicals GmbH - FN Nano, Inc - ForgeNano - Formacoat - Fujifilm - Fumin - FutureCarbon GmbH - Future Nanocoatings - General Paints - Green Earth nano Science, Inc - Green Millenium, Inc - Grenoble INP-Pagora - Grupo Repol - GSI Creos - GVD Corporation - GXC Coatings - Hanita Coatings - Hardide Coatings - HeiQ Materials AG - Hemoteq GmbH - Henkel AG & Co. KGaA - Hexis S.A - Hiab Products - Hitachi Chemical - Honeywell International, Inc - Hy-Power Nano, Inc - HzO, Inc - Hygratek, LLC - iFyber, LLC - Imbed Biosciences, Inc - Imerys - Industrial Nanotech, Inc - Inframat Corporation - INM - Leibniz Institute for New Materials - InMat, Inc - InMold Biosystems - Innovcoat Nanocoatings and Surface Technologies Inc - Inno-X - Innventia AB - Inspiraz Technology pte LTd - Instrumental Polymer Technologies LLC - Ishihara Sangyo Kaisha, Ltd - Integrated Surface Technologies, Inc - Integran Technologies, Inc - Integricote - Interlotus Nanotechnologie GmbH - Intumescents Associates Group - ISTN, Inc - ISurTech - ITN Nanovation AG - Izovac Ltd - JNC Corporation - Joma International AS - Jotun Protective Coatings - Kaneka Corporation - Klockner Pentaplast Europe GmbH & Co. KG - Kon Corporation - Kriya Materials B.V - Laiyang Zixilai Environment Protection Technology Co., Ltd - Life Air Iaq Ltd - Lintec of America, Inc., - Liquiglide, Inc - Liquipel, LLC - Lofec Nanocoatings - Lotus Applied Technology - Lotus Leaf Coatings - Luna Innovtions - Magnolia Solar - MDS Coating Technologies Corporation - Melodea - Merck Performance Materials - Mesocoat, Inc - Metal Estalki - Millidyne Oy - MMT Textiles Limited - Modumetal, Inc - Molecular Rebar - Muschert - N2 Biomedical - Naco Technologies, Inc - Nadico Technologie GmbH - Nagase & Co - Nanohygienix LLC - Namos GmbH - Nanobiomatters S.I - Nano-care AG - NanoCover A/S - Nanocure GmbH - Nanocyl - Nanofilm, Ltd - Nano Frontier Technology - Nanoex Company - Nanogate AG - Nanohmics - Nanohorizons, Inc - Nanokote Pty Ltd - Nanomate Technology - Nano Labs Corporation - NanoLotus Scandanavia Aps - Nanomembrane - NanoPack, Inc - NanoPhos SA - Nanopool GmbH - Nanops - Nanoservices BV - Nanoshell Ltd - Nanosol AG - Nanosonic, Inc - The NanoSteel Company, Inc - Nano Surface Solutions - NanoSys GmbH - Nanotech Security Corporation - Nano-Tex, Inc - NanoTouch Materials, LLC - Nanovere Technologies, LLC - Nanovis Incorporated - Nanoveu Pte. LTD - Nanowave Co., Ltd - Nano-X GmbH - Nanoyo Group Pte Ltd - Nanto Protective Coating - NBD Nano - NEI Corporation - Nelum Sciences LLC - Nelumbo - Neverwet LLC - NGimat - NIL Technology ApS - Nissan Chemical Industries Ltd - NOF Corporation - NTC Nanotech Coatings GmbH - n-tec GmbH - NTT Advanced Technology Corporation - Oceanit - Opticote Inc - Optics Balzers Ag - Optitune International Pte - Organiclick AB - Oxford Advanced SUrfaces - P2i Ltd - Panahome Corporation - Percenta AG - Perpetual Technologies, Inc - Philippi-Hagenbuch, Inc - Picosun Oy - Pioneer Medical Devices GmbH - Pneumaticicoat Technologies - PJI Contract Pte Ltd - Polymerplus, LLC - Powdermet, Inc - PPG Industries - Promimic AB - Pureti, Inc - Quantiam Technologies, Inc, - RBNano - Reactive Surfaces, LLP - Resodyn Corporation - Rochling Engineering Plastics - Royal DSM N.V - Saint-Gobain Glass - Sandvik Materials Technology - Sarastro GmbH - Schott AG - Seashell Technology LLC-Hydrobead - Semblant - Shandong Huimin Science & Technology Co., Ltd - Sharklet Technologies, Inc - Shin-Etsu Silicones - SHM - Sioen Industries NV - SiO2 Nanotech, LLC - Sketch Co., Ltd - Slips Technology - Sono-Tek Corporation - Spartan Nano Ltd - Starfire Systems, inc - Sub-One Technology, INc - Sumitomo Electric Hard-Metal Ltd - Suncoat GmbH - SupraPolix BV - SurfaceSolutions GmbH - Surfactis Technologies SAS - Surfatek LLC - Surfix BV - Suzhou Super Nano-Textile Teco Co - Takenake Seisakusho Co., Ltd - Tesla Nanocoatings - Theta Coatings - TNO - TopChim NV - Topasol LLC - Toray Advanced Film Co., Ltd - Toto - TripleO Performance Solution - Ultratech International, Inc - Vadlau GmbH - Valentis Nanotech - Vestagen Protective Technologies, Inc - Viriflex - VTT Technical Research Center - Wacker Chemie AG - Wattglass, LLC - Well Shield LLC - Zschimmer & Schwarz For more information about this report visit http://www.researchandmarkets.com/research/4ktr5t/the_nanocoatings Research and Markets is the world's leading source for international market research reports and market data. 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News Article | December 16, 2015
A. thaliana plants were either harvested from natural populations or grown in different natural soils and used for bacterial isolations by colony picking, limiting dilution or bacterial cell sorting as well as 16S rRNA gene-based community profiling. To obtain a library of representative root colonizing bacteria, A. thaliana plants were grown in different soils (50.958 N, 6.856 E, Cologne, Germany; 52.416 N, 12.968 E, Golm, Germany; 50.982 N, 6.827 E, Widdersdorf, Germany; 47.941 N, 04.012 W, Saint-Evarzec, France; 48.725 N, 3.989 W, Roscoff, France) and harvested before bolting. Briefly, Arabidopsis roots were washed twice in washing buffers (10 mM MgCl for limiting dilution and PBS for colony picking6) on a shaking platform for 20 min at 180 rpm and then homogenized twice by Precellys24 tissue lyser (Bertin Technologies) using 3 mM metal beads at 5,600 rpm for 30 s. Homogenates were diluted and used for isolation approaches on several bacterial growth media (Supplementary Data 7). For isolations based on colony picking, diluted cell suspensions were plated on solidified media and incubated, before isolates of plates containing less than 20 colony-forming units (CFUs) were picked after a maximum of two weeks of incubation. For limiting dilution, homogenized roots from each root pool were sedimented for 15 min and the supernatant was empirically diluted, distributed and cultivated in 96-well microtitre plates20. In parallel to the isolation of root-derived bacteria, roots of plants grown in Cologne soil were harvested and used to assess bacterial diversity by culture-independent 16S rRNA gene sequencing. Additionally, soil-derived bacteria were extracted from unplanted Cologne soil by washing soil with PBS buffer, supplemented with 0.02% Silwet L-77 and subjected to bacterial isolation as well as 16S rRNA gene community profiling. For the isolation of representative phyllosphere strains, naturally grown Arabidopsis plants were collected at eight different sites in southern Germany and Switzerland (six main sampling sites used for bacterial isolations and community profiling: 47.4090306 N, 8.470169444 E, Hoengg, Switzerland; 47.474825 N, 8.305008333 E, Baden, Switzerland; 47.4816806 N, 8.217547222 E, Brugg, Switzerland; 48.5560194 N, 9.134944444 E, Farm, Tuebingen, Germany; 48.5989861 N, 9.201655556 E, Haeslach, Germany; 48.602682 N, 9.213247258 E, Haeslach, Germany; and two additional sites only used for bacterial isolation: 47.4074722 N, 8.50825 E, Zurich, Switzerland; 47.4227222 N, 8.548666667 E, Seebach, Switzerland) during spring and autumn of 2013 and used for bacterial isolations as well as 16S rRNA gene profiling. Leaf-colonizing bacteria of individual leaves were washed off by alternating steps of intense mixing and sonication. The suspension was subsequently filtered (CellTrics filters, 10 μM, Partec GmbH, Görlitz, Germany) in order to remove remaining plant or debris particles as well as cell aggregates and applied to cell sorting on a BD FACS Aria III (BD Biosciences) as well as to plating on different media (Supplementary Data 1 and 7). All isolates were subsequently stored in 30% or 40% glycerol at −80 °C. Parts of A. thaliana leaves, roots and corresponding unplanted soil samples used for bacterial isolation were also processed for bacterial 16S rRNA gene community profiling using 454 pyrosequencing. Frozen root and corresponding soil samples were homogenized, DNA was extracted with Lysing Matrix E (MP Biomedicals) at 5,600 rpm for 30 s, and DNA was extracted from all samples using the FastDNA SPIN Kit for soil (MP Biomedicals) according to the manufacturer’s instructions. Lyophilized leaf samples were transferred into 2 ml microcentrifuge tubes containing one metal bead and subsequently homogenized twice for 2 min at 25 Hz using a Retsch tissue lyser (Retsch, Haan, Germany). Homogenized leaf material was resuspended in lysis buffer of the MO BIO PowerSoil DNA isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA), transferred into lysis tubes, provided by the supplier, and DNA extraction was performed following the manufacturer’s protocol. DNA concentrations were measured by PicoGreen dsDNA Assay Kit (Life technologies), and subsequently diluted to 3.5 ng μl−1. Bacterial16S rRNA genes were subsequently amplified6 using primers targeting the variable regions V5-V7 (799F26 and 1193R6, Supplementary Data 7). Each sample was amplified in triplicate by two independent PCR mixtures (a total of 6 replicates per sample plus respective no template controls). PCR products of triplicate were subsequently combined, purified and subjected to 454 sequencing. Obtained sequences were demultiplexed as well as quality and length filtered (average quality score ≥25, minimum length 319 bp with no ambiguous bases and no errors in the barcode sequences allowed)27. High-quality sequences were subsequently processed using the UPARSE24 pipeline and OTUs were taxonomically classified using the Greengenes database28 and the PyNAST29 method. We adopted a two-step barcoded PCR protocol20 in combination with 454 pyrosequencing to define V5-V8 sequences of bacterial 16S rRNA genes of all leaf, root- and soil-derived bacterial (Supplementary Fig. 1). DNA of isolates was extracted by lysis of 6 μl of bacterial cultures in 10 μl of buffer I containing 25 mM NaOH, 0.2 mM EDTA, pH 12 at 95 °C for 30 min, before the pH value was lowered by addition of 10 μl of buffer II containing 40 mM Tris-HCl at pH 7.5. Position and taxonomy of isolates in 96-well microtitre plates were indexed by a two-step PCR protocol using the degenerate primers 799F and 1392R containing well- and plate-specific barcodes (Supplementary Data 7) to amplify the variable regions V5 to V8. During the first step of PCR amplification, DNA from 1.5 μl of lysed cells was amplified using 2 U DSF-Taq DNA polymerase, 1× complete buffer (both Bioron GmbH), 0.2 mM dNTPs (Life technologies), 0.2 μM of 1 of 96 barcoded forward primer with a 18-bp linker sequence (for example, A1_454_799F1_PCR1_wells; Supplementary Data 7) and 0.2 μM reverse primer (454B_1392R) in a 25 μl reaction. PCR amplification was performed under the following conditions: DNA was initially denaturised at 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 50 °C for 30 s and 72 °C for 45 s, and a final elongation step at 72 °C for 10 min. PCR products of each 96-well microtitre plate were combined and subsequently purified in a two-step procedure using the Agencourt AMPure XP Kit (Beckman Coulter GmbH, Krefeld, Germany) first, then DNA fragments were excised from a 1% agarose gel using the QIAquick Gel Extraction Kit (Qiagen). DNA concentration was measured by Nanodrop and diluted to 1 ng μl−1. During the second PCR step, 1 ng of pooled DNA (each pool represents one 96-well microtitre plate) was amplified by 1.25 U PrimeSTAR HS DNA Polymerase, 1× PrimeSTAR Buffer (both TaKaRa Bio S.A.S, Saint-Germain-en-Laye, France), 0.2 mM dNTPs (Thermo Fisher Scientific Inc.), 0.2 μM of 1 of 96 barcoded forward primer targeting the 18-bp linker sequence (for example, P1_454_PCR2; Supplementary Data 7) and 0.2 μM reverse primer (454B_1392R) in a 50 μl reaction. The PCR cycling conditions were as follows. First, denaturation at 98 °C for 30 s, followed by 25 cycles of 98 °C for 10 s, 58 °C for 15 s and 72 °C for 30 s, and a final elongation at 72 °C for 5 min. PCR products were purified using the Agencourt AMPure XP Kit (Beckman Coulter GmbH) and QIAquick Gel Extraction Kit (Qiagen) as described for the purification of first step PCR amplicons. DNA concentration was determined by PicoGreen dsDNA Assay Kit (Life technologies) and samples were pooled in equal amounts. The final PCR product libraries were sequenced on the Roche 454 Genome Sequencer GS FLX +. Each sequence contained a plate-barcode, a well-barcode and V5-V8 sequences. The sequences were quality filtered, demultiplexed according to well and plate identifiers27. OTUs were clustered at 97% similarity by UPARSE algorithum24. A nucleotide-based blast (v. 2.2.29) was used to align representative sequences of isolated OTUs to culture-independent OTUs and only hits ≥97% sequence identity covering at least 99% of the length of the sequences were considered. Based on representative sequences of OTUs from this as well as previously published culture-independent community analysis, bacterial CFUs in the culture collections with ≥97% 16S rRNA gene identity to root-, leaf- and soil-derived OTUs were purified by three consecutive platings on the respective solidified media before an individual colony was used to inoculate liquid cultures. These liquid cultures were used for validation by Sanger sequencing with both 799F and 1392R primers as well as for the preparation of glycerol stocks for the culture collections and for the extraction of genomic DNA for whole-genome sequencing. A total of 21 leaf-derived strains, previously described as phyllosphere bacteria8, 9, were added to the At-LSPHERE collection although these were undetectable in the present culture-independent leaf community profiling. To obtain high molecular weight genomic DNA of bacterial isolates in our culture collections, we used a modified DNA precipitation protocol and the Agencourt AMPure XP Kit (Beckman Coulter GmbH). For each bacterial liquid culture, cells were collected by centrifugation at 3,220g for 15 min, the supernatant removed and cells were resuspended in 5 ml SET buffer containing 75 mM NaCl, 25 mM EDTA, 20 mM Tris/HCl at pH 7.5. A total of 20 μl lysozyme solution (50 mg ml−1, Sigma) was added before the mixture was incubated for 30 min at 37 °C. Subsequently, 100 μl 20 mg ml−1 proteinase K (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) and 10% SDS (Sigma-Aldrich Chemie GmbH) were added, mixed, and incubated by shaking every 15 min at 55 °C for 1 h. If bacterial cells were insufficiently lysed, remaining cells were collected at 3,220g for 10 min and homogenized using the Precellys24 tissuelyser in combination with lysing matrix E tubes (MP Biomedicals) at 6,300 rpm for 30 s. After cell lysis, 2 ml 5 M NaCl and 5 ml chloroform were added and mixed by inversion for 30 min at room temperature. After centrifugation at 3,220 g for 15 min, 6 ml supernatant were transferred into fresh falcon tubes and 3.6 ml isopropanol were added and gently mixed. After precipitation at 4 °C for 30 min, genomic DNA was collected at 3,220g for 5 min, washed once with 1 ml 70% (v/v) ethanol, dried for 15 min at room temperature and finally dissolved in 250 μl elution buffer (Qiagen). 2 μl 4 mg ml−1 RNase A (Sigma-Aldrich Chemie GmbH) was added to bacterial genomic DNA solution and incubated over night at 4 °C. The genomic DNA was subsequently purified using the Agencourt AMPure XP Kit (Beckman Coulter GmbH) and analysed by agarose gel (1% (w/v)) electrophoresis. Concentrations were estimated based on loaded Lambda DNA Marker (GeneRuler 1kb Plus, Thermo Scientific) and approximately 1 μg of genomic DNA was transferred into micro TUBE Snap-Cap AFA Fibre vials (Covaris Inc., Woburn, MA, USA). DNA was sheared into 350 bp fragments by two consecutive cycles of 30 s (duty cycle: 10%, intensity: 4, cycle/burst: 200) on a Covaris S2 machine (Covaris, Inc.). The Illumina sequencing libraries were prepared according to the manual of NEBNext Ultra UltraTM DNA Library Prep Kit for Illumina (New England Biolabs, USA). Quality and quantity was assessed at all steps by capillary electrophoresis (Agilent Bioanalyser and Agilent Tapestation). Finally libraries were quantified by fluorometry, immobilized and processed onto a flow cell with a cBot (Illumina Inc., USA) followed by sequencing-by-synthesis with TruSeq v3 chemistry on a HiSeq2500 (Illumina Inc., USA). Paired-end Illumina reads were subjected to quality and length trimming using Trimmomatic v. 0.3330 and assembled using two independent methods (A531 and SOAPdenovo32 v. 20.1). In each case, the assembly with the smaller number of scaffolds was selected. Detailed assembly statistics for each sequenced isolate can be found in Supplementary Data 3 and 4. Identification of putative protein-encoding genes and annotation of the genomes were performed using GLIMMER v. 3.0233. Functional annotation of genes was conducted using Prokka v. 1.1134 and the SEED subsystems approach using the RAST server API35. Additionally, annotation of KEGG Orthologue (KO) groups was performed by first generating HMM models for each KO in the database36, 37 the HMMER toolkit (v. 3.1b2)38. Next, we employed the HMM models to search all predicted ORFs using the hmmsearch tool, with an E value threshold of 10 × 10−5. Only hits covering at least 70% of the protein sequence were retained and for each gene and the match with the lowest E value was selected. Each proteome was searched for the presence of the 31 well-conserved, single-copy, bacterial AMPHORA genes39, designed for the purpose of high-resolution phylogeny reconstruction of genomes. Subsequently, a concatenated alignment of these marker genes was performed using Clustal Omega40 v. 1.2.1. Based on this multiple sequence alignment, a species tree was inferred using FastTree41 v. 2.1, a maximum likelihood tool for phylogeny inference. Whole-genome taxonomic classification of sequenced isolates was conducting using taxator-tk42, a homology/based tool for accurate classification of sequences. Analyses of phylogenetic diversity were performed independently for each cluster based on pairwise tree distances between all isolates (Supplementary Data 5). Analyses of functional diversity between sequenced isolates were conducted by generating, for each genome in the data set, a profile of presence/absence of each KO group (or phyletic pattern). Subsequently, a distance measure based on the Pearson correlation of each pair of phyletic patterns was calculated, which allowed us to embed each genome as a data point in a metric space. PCoA was performed on this space of functional distances using custom scripts written in R. Pairwise functional distances within each family-level cluster was performed by calculating the average distance between all pairs of genomes belonging to each cluster. Finally, we calculated RAs of each functional category based on the percentage of annotated KO terms assigned to each category. Enrichment tests were performed to identify differentially abundant categories between groups of genomes based on their origin (root versus leaf and root versus soil) using the non-parametric Mann–Whitney Test (MWT). P values were corrected for multiple testing using the Bonferroni method, with a significance threshold α = 0.05. Calcined clay16, an inert soil substitute, was washed with water, sterilized twice by autoclaving and heat-incubated until being completely dehydrated. A. thaliana Col-0 seeds were surface-sterilized with ethanol and stratified overnight at 4 °C. Leaf-, root- and soil-derived bacteria of the culture collections were cultivated in 96-deep-well plates and subsequently pooled (in equal or unequal ratios) in order to prepare synthetic bacterial communities (SynComs) for inoculations below the carrying capacity of leaves and roots43, 44. To inoculate SynComs into the calcined clay matrix, OD was adjusted to 0.5 and 1 ml (~2.75 × 108 cells) was added to 70 ml 0.5× MS media (pH 7; including vitamins, without sucrose), and mixed with 100 g calcined clay in Magenta boxes (~2.75 × 106 cells per gr calcined clay), directly before sowing of surface-sterilized seeds. Plants were grown at 22 °C, 11 h light, and 54% humidity. Alive cell counts (CFUs) of root-associated bacteria by serial dilutions of root homogenates after seven weeks of co-incubation were 1.4 × 108 ± 8.4 × 107 cells per gram root tissue. For leaf spray-inoculation of A. thaliana plants, bacterial SynComs were prepared as described above and adjusted to OD 0.2, before the solution was diluted tenfold and 170 μl (~1.87 × 106 cells) were sprayed into each magenta box containing four three-week-old plants using a TLC chromatographic reagent sprayer (BS124.000, Biostep GmbH, Jahnsdorf, Germany). The average volume per spraying event was determined by spraying repeatedly into 50 ml tubes and weighing before and after. All plants and corresponding unplanted clay samples were harvested under sterile conditions after a total incubation period of seven weeks. All plants and corresponding unplanted clay samples were harvested under sterile conditions after a total incubation period of seven weeks. During harvest, leaves and roots of individual plants were carefully separated using sterilized tweezers and scissors to avoid cross-contamination and processed separately thereafter. All leaves being obviously contaminated with clay particles or touching the ground were carefully removed and omitted from further processing. Remaining aerial parts of four plants collected from one magenta box were combined and transferred into lysing matrix E tubes (MP Biomedicals), frozen in liquid nitrogen and stored at −80 °C until used for DNA extraction. Roots from one Magenta box were pooled, washed twice in 5 ml PBS at 180 rpm for 20 min, dried on sterilized Whatman glass microfibre filters (GE Healthcare Life Sciences), transferred into lysing matrix E tubes (MP Biomedicals), frozen in liquid nitrogen and stored at −80 °C until further processing. The corresponding unplanted clay samples were washed in 100 ml PBS supplemented with 0.02% Silwet L-77 at 180 rpm for 10 min, before particles were allowed to settle down for 5 min. The supernatant was collected by centrifugation at 3,220g for 15 min. The pellet was subsequently resuspended in 1 ml water, transferred into lysing matrix E tubes (MP Biomedicals), frozen in liquid nitrogen and stored at −80 °C. To prepare DNA for bacterial 16S rRNA gene-based community analysis, all samples were homogenized twice by Precellys24 tissue lyser (Bertin Technologies), DNA was extracted and concentrations were measured by PicoGreen dsDNA Assay Kit (Life technologies), before bacterial 16S rRNA genes were amplified by degenerate PCR primers (799F and 1193R) targeting the variable regions V5-V7 (Supplementary Data 7). Each sample was amplified in triplicate (plus respective no template control) in 25 μl reaction volume containing 2 U DFS-Taq DNA polymerase, 1× incomplete buffer (both Bioron GmbH, Ludwigshafen, Germany), 2 mM MgCl , 0.3% BSA, 0.2 mM dNTPs (Life technologies GmbH, Darmstadt, Germany), 0.3 μM forward and reverse primer and 10 ng of template DNA. After an initial denaturation step at 94 °C for 2 min, the targeted region was amplified by 25 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s, followed by a final elongation step of 5 min at 72 °C. The three independent PCR reactions were pooled and the remaining primers and nucleotides were removed by addition of 20 U exonuclease I and 5 U Antarctic phosphatase (both New England BioLabs GmbH, Frankfurt, Germany) and incubated for 30 min at 37 °C in the corresponding 1× Antarctic phosphatase buffer. Enzymes were heat-inactivated and the digested mixture was used as template for the 2nd step PCR using the Illumina compatible primers B5-F and 1 of 96 differentially barcoded reverse primers (B5-1 to B5-96, Supplementary Data 7). All samples were amplified in triplicate for 10 cycles using identical conditions of the first-step PCR. Technical replicates of each sample were combined, run on a 1.5% (w/v) agarose gel and the bacterial 16S rRNA gene amplicons were extracted using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer’s instructions. DNA concentration was subsequently measured using the PicoGreen dsDNA Assay Kit (Life technologies) and 100 ng of each sample were combined. Final amplicon libraries were cleaned twice using the Agencourt AMPure XP Kit (Beckman Coulter GmbH) and subjected to sequencing on the Illumina MiSeq platform using an MiSeq Reagent kit v3 following the 2 × 350 bp paired-end sequencing protocol (Illumina Inc. USA). Forward and reverse reads were joined, demultiplexed and subjected to quality controls using scripts from the QIIME toolkit27, v. 180 (Phred ≥ 20). The resulting high quality sequences were further clustered at 97% sequence identity together with Sanger sequences of leaf, root and soil isolates using the UPARSE24 pipeline as described above. Taxonomic assignments of representative sequences were performed as explained in the previous sections. OTUs only corresponding to one or more Sanger 16S rRNA gene sequence(s) of purified strains in the At-RSPHERE, At-LSPHERE or soil collection were selected and designated ‘indicator OTUs’. The heat maps were generated using the ggplot2 R package. Sequencing reads (454 16S rRNA, MiSeq 16S rRNA and WGS HiSeq reads) have been deposited in the European Nucleotide Archive (ENA) under accession numbers PRJEB11545, PRJEB11583 and PRJEB11584. Genome assemblies and annotations corresponding to the leaf, root and soil culture collections have been deposited in the National Center for Biotechnology Information (NCBI) BioProject database under accession numbers PRJNA297956, PRJNA297942 and PRJNA298127, respectively. All scripts for computational analysis and corresponding raw data are available at http://www.mpipz.mpg.de/R_scripts. The sequenced bacterial genomes as well as any future updates are available at http://www.at-sphere.com.
Bennert H.W.,Ruhr University Bochum |
Horn K.,Bro fur Angewandte Geobotanik und Landschaftskologie BaGL |
Kauth M.,Bergmannsheil Universittsklinikum |
Fuchs J.,Leibniz Institute of Plant Genetics and Crop Plant Research |
And 5 more authors.
Annals of Botany | Year: 2011
Background and AimsInterspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.MethodsFlow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.Key ResultsA considerable variation of DNA 2C values (5·267·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·4810·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.ConclusionsDiploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns. © The Author 2011.