DOCSLIB.ORG

Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Levin, Benjamin Joseph. 2019. Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41121264 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments A dissertation presented by Benjamin Joseph Levin to The Department of Chemistry and Chemical Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Chemistry Harvard University Cambridge, Massachusetts January 2019 © 2019 – Benjamin Joseph Levin All rights reserved. Dissertation advisor: Professor Emily P. Balskus Benjamin Joseph Levin Discovery and Characterization of Glycyl Radical Enzymes Found in the Human Gut Microbiota and Other Environments Abstract The human gastrointestinal tract is home to trillions of microorganisms, and these microbes have important impacts on human health. Despite the many known links between gut microbiota composition and host biology, the molecular mechanisms underlying these interactions are largely unknown and there are no general strategies to elucidate them. Glycyl radical enzymes (GREs) had been previously identified in the human gut microbiota, but their functions in this environment are largely unknown. This thesis presents our work towards characterizing the chemistry performed by GREs and their impacts on human health. Our results illustrate how integrating a biochemical and mechanistic understanding of enzymes and metagenomic sequencing data can lead to insight into host-gut microbial symbioses. Chapter 2 details the development and application of a metagenomic analysis workflow called chemically guided functional profiling to identify and quantify the GREs present in healthy human microbiomes. Our methodology combined biochemical knowledge of this protein superfamily with metagenomic sequencing data, and with this approach we computed abundances of specific GREs in these environments. We prioritized targets for biochemical characterization based on their abundance in the human gut and characterized two enzymes previously of unknown function, propanediol dehydratase (PD) and trans-4-hydroxy-L-proline dehydratase, demonstrating how chemically guided functional profiling can reveal novel biochemistry in complex microbial communities. iii Chapter 3 describes the mechanistic characterization of PD. Intriguingly, a B12-dependent propanediol dehydratase (B12-PD) catalyzes the same overall reaction as PD but requires a different cofactor. To probe the mechanisms of these enzymes, we synthesized all four possible 18O-labeled 1,2-propanediol stereoisomers in high enantiomeric excess and used these compounds as substrates for PD and B12-PD. The results suggest PD catalyzes direct elimination of the C2 hydroxyl group from an initial substrate-based radical, while B12-PD instead mediates a 1,2-hydroxyl group migration. These experiments clarify how PD and other GREs important to human health perform challenging transformations and reveal mechanistic differences between GREs and B12-dependent enzymes that have evolved identical functions. Chapter 4 details our efforts to further study protein families in metagenomic datasets. Complementing our work quantifying GREs in metagenomic reads, we searched for additional GREs in metagenomic assemblies and identified genes encoding for GREs not previously found in any sequenced microbial genome. We also applied chemically guided functional profiling to additional protein families. Our results highlight how existing metagenomics datasets can be mined to find both previously sequenced as well as novel enzymes. Chapter 5 presents our discovery and initial characterization of a new GRE catalyzing the decarboxylation of indole-3-acetate to skatole (3-methylindole). The production of skatole by boar and cattle gut microbes is important to the agricultural industry, but the enzymes involved in this pathway have not been characterized. Using comparative genomics and knowledge of GRE biochemistry, we discovered two GRE indole-3-acetate decarboxylases (IADs) and validated that they catalyze the expected reactions. These findings are important for developing methods to inhibit skatole biosynthesis and for further characterizing the scope of reactions performed by GREs. iv Table of Contents Abstract .......................................................................................................................................... iii Table of Contents ............................................................................................................................ v Acknowledgments.......................................................................................................................... xi List of Figures .............................................................................................................................. xiii List of Tables ................................................................................................................................ xv Chapter 1: Introduction to the human gut microbiota and to the biochemistry of glycyl radical enzymes............................................................................................................................... 1 1.1: General introduction to the human gut microbiota ...................................................... 1 1.2: Advances in sequencing technology have empowered studies of microbial communities ............................................................................................................ 3 1.3: Enzyme discovery in microbial genomes and metagenomes ...................................... 5 1.4: Introduction to glycyl radical enzymes (GREs): discovery and characterization of pyruvate formate-lyase............................................................................................ 7 1.5: Glycyl radical enzymes use similar mechanisms to catalyze reactions ..................... 11 1.6: Discovery and roles of glycyl radical enzymes in microbiotas and metagenomes ... 16 1.7: Microbial metabolism of L-fucose ............................................................................. 20 1.8: References .................................................................................................................. 22 Chapter 2: Quantification of glycyl radical enzymes in healthy human gut metagenomes and characterization of abundant dehydratases ....................................................................... 42 2.1: Introduction ................................................................................................................ 42 2.2: Results and discussion ............................................................................................... 45 2.2.1: GRE sequence similarity network construction.......................................... 45 2.2.2: Integrating the GRE SSN with quantitative metagenomics........................ 49 2.2.3: Abundances and distributions of characterized GREs in healthy human microbiomes .............................................................................................. 52 2.2.4: Previously uncharacterized GREs were profiled using CGFP ................... 54 v 2.2.5: Analysis of metatranscriptomes for GREs.................................................. 56 2.2.6: Biochemical characterization of propanediol dehydratase ......................... 57 2.2.7: Identification of conserved residues in dehydrating GREs ........................ 61 2.2.8: Comparing abundances of PD and B12-PD in the healthy human gut microbiome ............................................................................................... 64 2.2.9: Discovery and characterization of trans-4-hydroxy-L-proline dehydratase 65 2.2.10: Conclusions ............................................................................................... 70 2.3: Materials and methods ............................................................................................... 71 2.3.1: General materials and methods ................................................................... 71 2.3.2: Construction of GRE SSNs......................................................................... 72 2.3.3: Determination of enzyme abundances in metagenomes and metatranscriptomes ................................................................................... 74 2.3.4: Statistical analysis ....................................................................................... 78 2.3.5: Cloning of expression plasmids for PD and PD-AE ................................... 78 2.3.6: Site-directed mutagenesis of PD ................................................................. 80 2.3.7: Overexpression and purification of PD ...................................................... 81 2.3.8: Overexpression and purification of PD-AE ...............................................
Load more

Recommended publications
  • PP003: Large Scale Growth of M. Tuberculosis
    SOP: PP003.2 Modified: 6/10/2010 Large-scale growth of Mycobacterium tuberculosis Materials and Reagents: 1. Biosafety cabinet (BSC) 2. Three 100 ml frozen stocks of M. tuberculosis (see SOP PP002) 3. Eight 2.8 L glass fernbach flasks containing 900 ml sterile GAS medium, capped with cotton and cheesecloth plugs wrapped in aluminum foil 4. Orbital platform shaker 5. Four 50 ml disposable pipets 6. Electric pipetter 7. Bunsen burner 8. Pipet boat 9. 10% Lysol I. C. solution 10. Autoclave 11. Forty 1 liter rollerbottles containing 400 ml sterile GAS medium 12. Two 25 ml disposable pipets 13. Rollerbottle apparatus 14. Rubbermaid transport cart 15. Vacuum pump 16. Five 4 liter Winchester bottles, sterile 17. Five 0.2 um Zap Cap S plus bottle filtration units 18. 225 ml Falcon centrifuge bottle 19. 225 ml centrifuge bottle rack 20. Harvard trip balance 21. Sorvall benchtop centrifuge 22. Sorvall benchtop centrifuge rotor, containing 225 ml centrifuge bottle hanging buckets 23. Sterile Milli-Q water 24. 4oC cold room (BHRB Molecular Biology room 101) 25. –80oC freezer (option, note 17) Upscale protocol: (note 1) 1. _____ Inside a biosafety cabinet in a BSL-3 facility, thaw three “100 ml” stocks of M. tuberculosis. 2. _____ Carefully pour the contents of one stock into one 2.8 L fernbach flask containing GAS medium. 3. _____ Flame opening and aseptically replace foil and cotton plug in mouth of the fernbach flask. 4. _____ Repeat for two remaining “100 ml” stocks. 5. _____ Place the inoculated fernbach flasks on an orbital platform shaker at 37oC for two weeks (notes 2 and 3).
    [Show full text]
  • 7. Heating and Cooling Technology Contents
    GENERAL CATALOGUE EDITION 18 7. Heating and cooling technology Contents Cooling 730 Refrigerators and Freezers ex protected 730 Refrigerators and Freezers 733 Low and Ultra low temperature freezers 737 Ice machines 744 Cryogenic tubes 746 Cryogenic boxes 749 Cryogenic racks 756 Cryogenic accessories 757 Transport boxes 759 Dewar flasks 760 Cryogenic storage tanks 763 Liquid nitrogen-Accessories 766 Temperature regulators 767 Heating-Thermostats 767 Cooling-Thermostats 779 Circulator baths 787 Dynamic Temperature Control Systems 791 Temperature Control Accessories 794 Heating 798 Water baths 798 Shaking water baths 805 Heating baths 810 Microwaves 814 Universal, Heating and Drying incubators 815 Vacuum drying incubators 819 Incubators 821 Cooling incubators 825 CO2-Incubators 829 Shaking incubators 833 Test incubators 847 Ovens, Furnaces 854 Hotplates 858 Incubation hoods 863 Temperature controllers, thermostats 868 729 www.wenk-labtec.com 7. Heating and cooling technology GENERAL CATALOGUE EDITION 18 Cooling/Refrigerators and Freezers ex protected 1 Spark-free laboratory refrigerators The BGR 120 laboratory guidelines state that interiors of refrigerators in which AQUALYTIC dangerous, explosive atmospheres can develop, must be spark-free. Such explosive atmospheres can be created by stored flammable liquids, for example. The laboratory refrigerators by Aqualytic® meet these requirements. The interiors are free from sparking sources and are therefore explosion-proof. With glass shelves. Variable temperature control from +2°C to +10°C. Temperature is continuously controlled by thermostat. Capacity External Internal Weight Power Temp. PK Cat. No. dimensions dimensions range (W x D x H) (W x D x H) LmmmmkgW°C 150 602 x 600 x 885 515 x 443 x 700 37.00 120 +2 ..
    [Show full text]
  • July 26, 2017 Bruker Daltonik Gmbh Mr. David Cromwick Director Of
    DEPARTMENT OF HEALTH & HUMAN SERVICES Public Health Service __________________________________________________________________________________________________________________________ Food and Drug Administration 10903 New Hampshire Avenue Document Control Center – WO66-G609 Silver Spring, MD 20993-0002 July 26, 2017 Bruker Daltonik GmbH Mr. David Cromwick Director of Quality 40 Manning Rd Billerica, MA 01821 US Re: K163536 Trade/Device Name: MALDI Biotyper CA (MBT-CA) System, MBT smart CA System Regulation Number: 21 CFR 866.3361 Regulation Name: Mass spectrometer system for clinical use for the identification of microorganisms Regulatory Class: II Product Code: PEX Dated: December 16, 2016 Received: December 16, 2016 Dear Mr. Cromwick: We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading. If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls.
    [Show full text]
  • Structural and Functional Studies of Membrane Remodeling Machines
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE HHS Public Access provided by CDC Stacks Author manuscript Author ManuscriptAuthor Manuscript Author Methods Manuscript Author Cell Biol. Author Manuscript Author manuscript; available in PMC 2018 August 13. Published in final edited form as: Methods Cell Biol. 2015 ; 128: 165–200. doi:10.1016/bs.mcb.2015.02.007. Structural and Functional Studies of Membrane Remodeling Machines Raghav Kalia#1,2, Nathaniel Talledge#1,2, and Adam Frost1,2 1Department of Biochemistry, University of Utah, School of Medicine, Salt Lake City, UT 84112 2Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158 # These authors contributed equally to this work. Abstract Building cells from their component parts will hinge upon our ability to reconstitute biochemical compartmentalization and exchange between membrane-delimited organelles. By contrast with our understanding of other cellular events, the mechanisms that govern membrane trafficking has lagged because the presence of phospholipid bilayers complicates the use of standard methods. This chapter describes in vitro methods for purifying, reconstituting and visualizing membrane remodeling activities directly by electron cryomicroscopy (cryoEM). Keywords Membrane remodeling; electron cryomicroscopy; cryoEM; endocytosis; mitochondria; dynamin; endophilin; bilayer; lipid-binding Introduction to membrane remodeling Chemical compartmentalization was as critical in the evolution of life as was the realization of self-replicating molecules. Robert Hooke’s use of the metaphorical “cell” draws our attention to the inhabitants of the “small rooms” he observed with his microscope; but also to the walls that outline a room, distinguish it from its neighbors, and that segregate inside from outside activities (1).
    [Show full text]
  • New Brunswick Shakers Eppendorf and New Brunswick
    Eppendorf is your source for New Brunswick products New Brunswick Shakers Eppendorf and New Brunswick “In Touch with Life” Gets “A Culture of Innovation” New Brunswick’s world renowned Shakers, CO2 Incubators, Energy-efficient –86 °C ULT freezers are available in upright ULT Freezers and Fermentors/Bioreactors are now sold or chest configurations and feature two model ranges and through Eppendorf North America, in the US and Canada. 12 different sizes. Innova series freezers utilize space-saving vacuum insulation panels for increased internal capacity with The New Brunswick product line perfectly complements the a small footprint and Excella series utilize traditional insulation. Eppendorf product range of liquid handling, centrifugation, PCR, A standard feature found on all New Brunswick freezers is the sample prep, detection and cell biology instrumentation and built-in heated vent port to release vacuum after the door is shut consumables. to gain easy access to samples. New Brunswick Shakers With 3 product lines to choose from, Innova, I-Series or Excella series, there’s a shaker suited to your exact needs and budget. The world famous, reliable, triple-eccentric drive mechanism with cast iron top plate, found in the Innova and I-series, provides decades worth of durability. Shakers sold in the 1950’s are still going strong today. Every New Brunswick shaker drive is individually CAM’d for precise tolerances and dependability for years of trouble free service. State-of-the-art bioreactors and fermentors handle volumes from scale-up 1L, all the way up to 3000 L. Configurations are available with single-use vessels, autoclavable vessels, up to sterilize in place (SIP) systems.
    [Show full text]
  • Forensic Microbiology Reveals That Neisseria Animaloris Infections In
    www.nature.com/scientificreports OPEN Forensic microbiology reveals that Neisseria animaloris infections in harbour porpoises follow traumatic Received: 14 February 2019 Accepted: 20 September 2019 injuries by grey seals Published: xx xx xxxx Geofrey Foster 1, Adrian M. Whatmore2, Mark P. Dagleish3, Henry Malnick4, Maarten J. Gilbert5, Lineke Begeman6, Shaheed K. Macgregor7, Nicholas J. Davison1, Hendrik Jan Roest8, Paul Jepson7, Fiona Howie9, Jakub Muchowski2, Andrew C. Brownlow1, Jaap A. Wagenaar5,8, Marja J. L. Kik10, Rob Deaville7, Mariel T. I. ten Doeschate1, Jason Barley1,11, Laura Hunter1 & Lonneke L. IJsseldijk 10 Neisseria animaloris is considered to be a commensal of the canine and feline oral cavities. It is able to cause systemic infections in animals as well as humans, usually after a biting trauma has occurred. We recovered N. animaloris from chronically infamed bite wounds on pectoral fns and tailstocks, from lungs and other internal organs of eight harbour porpoises. Gross and histopathological evidence suggest that fatal disseminated N. animaloris infections had occurred due to traumatic injury from grey seals. We therefore conclude that these porpoises survived a grey seal predatory attack, with the bite lesions representing the subsequent portal of entry for bacteria to infect the animals causing abscesses in multiple tissues, and eventually death. We demonstrate that forensic microbiology provides a useful tool for linking a perpetrator to its victim. Moreover, N. animaloris should be added to the list of potential zoonotic bacteria following interactions with seals, as the fnding of systemic transfer to the lungs and other tissues of the harbour porpoises may suggest a potential to do likewise in humans.
    [Show full text]
  • Laboratory Supplies and Equipment
    Laboratory Supplies and Equipment Beakers: 9 - 12 • Beakers with Handles • Printed Square Ratio Beakers • Griffin Style Molded Beakers • Tapered PP, PMP & PTFE Beakers • Heatable PTFE Beakers Bottles: 17 - 32 • Plastic Laboratory Bottles • Rectangular & Square Bottles Heatable PTFE Beakers Page 12 • Tamper Evident Plastic Bottles • Concertina Collapsible Bottle • Plastic Dispensing Bottles NEW Straight-Side Containers • Plastic Wash Bottles PETE with White PP Closures • PTFE Bottle Pourers Page 39 Containers: 38 - 42 • Screw Cap Plastic Jars & Containers • Snap Cap Plastic Jars & Containers • Hinged Lid Plastic Containers • Dispensing Plastic Containers • Graduated Plastic Containers • Disposable Plastic Containers Cylinders: 45 - 48 • Clear Plastic Cylinder, PMP • Translucent Plastic Cylinder, PP • Short Form Plastic Cylinder, PP • Four Liter Plastic Cylinder, PP NEW Polycarbonate Graduated Bottles with PP Closures Page 21 • Certified Plastic Cylinder, PMP • Hydrometer Jar, PP • Conical Shape Plastic Cylinder, PP Disposal Boxes: 54 - 55 • Bio-bin Waste Disposal Containers • Glass Disposal Boxes • Burn-upTM Bins • Plastic Recycling Boxes • Non-Hazardous Disposal Boxes Printed Cylinders Page 47 Drying Racks: 55 - 56 • Kartell Plastic Drying Rack, High Impact PS • Dynalon Mega-Peg Plastic Drying Rack • Azlon Epoxy Coated Drying Rack • Plastic Draining Baskets • Custom Size Drying Racks Available Burn-upTM Bins Page 54 Dynalon® Labware Table of Contents and Introduction ® Dynalon Labware, a leading wholesaler of plastic lab supplies throughout
    [Show full text]
  • Growth in Motion
    Growth in Motion The next evolution in biological shakers – the Innova® S44i 2 Innova® S44i »Make your cells shine in the newest Innova shaker from Eppendorf. The Innova S44i combines the reliable performance of Innova shakers with the latest precision engineering from Eppendorf.« Innova® S44i 3 »The future is what you make it – Eppendorf makes it reliable.« Eppendorf has helped scientists achieve better results for over 70 years. Now, we want to move your lab forward by moving shaker technology forward; we want to grow your lab by growing better cultures. Eppendorf has been a trusted partner to the life science community, and the new Innova S44i Biological Shaker is the next great addition to our renowned portfolio of life science solutions. Expand the potential of your lab and reap the benefits of the exceptional performance and reliability that come with every Eppendorf product. Reliable shaking Exceeding demands Exceptionally convenient > The new Eppendorf X-Drive > Highest fl ask capacity in a > Optimize your space with provides smooth and uniform smaller footprint – grow more double- and triple-stacked agitation cells in less space confi gurations > Intelligent counterbalance > Ethernet connectivity for easy > Easily reach all your samples mechanism for vibration-free integration with building alarm using the slide-out platform motion and longevity systems or monitoring software > Glide-up door for convenient > Equally robust for shaking heavy > Intuitive touchscreen with user and eff ortless access and uneven loads management capabilities
    [Show full text]
  • Oral Microbiome in HIV-Associated Periodontitis
    Oral microbiome in HIV-associated periodontitis. Marc Noguera-Julian, IrsiCaixa AIDS Research Institute Yolanda Guillén, IrsiCaixa AIDS Research Institute Jessica Peterson, Emory University David Reznik, Emory University Erica V. Harris, Emory University Sandeep J. Joseph, Emory University Javier Rivera, IrsiCaixa AIDS Research Institute Sunil Kannanganat, Emory University Rama Amara, Emory University Minhly Nguyen, Emory University Only first 10 authors above; see publication for full author list. Journal Title: Medicine Volume: Volume 96, Number 12 Publisher: Wolters Kluwer Health | 2017-03, Pages e5821-e5821 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1097/MD.0000000000005821 Permanent URL: https://pid.emory.edu/ark:/25593/s0xtw Final published version: http://dx.doi.org/10.1097/MD.0000000000005821 Copyright information: © 2017 the Author(s). Published by Wolters Kluwer Health, Inc. This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/). Accessed September 29, 2021 5:15 AM EDT ® Observational Study Medicine OPEN Oral microbiome in HIV-associated periodontitis ∗ Marc Noguera-Julian, PhDa,b,c, , Yolanda Guillén, PhDa,b, Jessica Peterson, BsCd, David Reznik, DDSd,e, Erica V. Harris, BsCf, Sandeep J. Joseph, PhDd, Javier Rivera, MsCa,c, Sunil Kannanganat, PhDd,g, Rama Amara, PhDd,g, Minh Ly Nguyen, MDd, Simon Mutembo, MDh, Roger Paredes, MD, PhDa,b,c,i, ∗ Timothy D. Read, PhDd, Vincent C. Marconi, MDd,g, Abstract HIV-associated periodontal diseases (PD) could serve as a source of chronic inflammation. Here, we sought to characterize the oral microbial signatures of HIV+ and HIV– individuals at different levels of PD severity.
    [Show full text]
  • I. Designing Brighter Fluorophores: a Computational and Spectroscopic Approach to Predicting Photophysical Properties of Hydrazone-Based Dyes Ii
    University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2017 I. Designing Brighter Fluorophores: A Computational And Spectroscopic Approach To Predicting Photophysical Properties Of Hydrazone-Based Dyes Ii. Developing Spectroscopic Methods To Better Understand The Cofactors Of Metalloproteins Morgan Cousins University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Part of the Chemistry Commons Recommended Citation Cousins, Morgan, "I. Designing Brighter Fluorophores: A Computational And Spectroscopic Approach To Predicting Photophysical Properties Of Hydrazone-Based Dyes Ii. Developing Spectroscopic Methods To Better Understand The ofaC ctors Of Metalloproteins" (2017). Graduate College Dissertations and Theses. 787. https://scholarworks.uvm.edu/graddis/787 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. I. DESIGNING BRIGHTER FLUOROPHORES: A COMPUTATIONAL AND SPECTROSCOPIC APPROACH TO PREDICTING PHOTOPHYSICAL PROPERTIES OF HYDRAZONE-BASED DYES II. DEVELOPING SPECTROSCOPIC METHODS TO BETTER UNDERSTAND THE COFACTORS OF METALLOPROTEINS A Dissertation Presented by Morgan E. Cousins to The Faculty of the Graduate College of The University of Vermont In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Chemistry October, 2017 Defense Date: August 7, 2017 Dissertation Examination Committee: Matthew D. Liptak, Ph.D., Advisor Adrian Del Maestro, Ph.D., Chairperson Rory Waterman, Ph.D. Matthias Brewer, Ph.D. Cynthia J. Forehand, Ph.D., Dean of the Graduate College ABSTRACT Luminogens are molecules that emit light upon exposure to high-energy light, and fluorophores are one class of luminogens.
    [Show full text]
  • Maxq Shakers
    Thermo Scientific MaxQ Shakers Reliability. Simplicity. Flexibility. Thermo Scientific MaxQ Shaker Product Selector Guide MaxQ 2000 MaxQ 3000 MaxQ 4450 MaxQ 4000 Model Incubated temperatures in Spacious chamber holds Choice of 3 platform sizes Choice of 2 platform sizes a compact footprint 6 x 2L flasks Benchtop, floor or stackable Benchtop Benchtop Benchtop Benchtop Motion Orbital Orbital Orbital Orbital 18 x 18 24 x 18 13 x 11 30 x 18 36 x 24 13 x 11 18 x 18 Platform size inches (cm) (45.7 x (60.9 x (33 x 28) (76 x 45.7) (91 x 60.9) (33 x 28) (45.7 x 45.7) 45.7) 45.7)1 250 ml flask capacity dedicated* platform 12 25 N/A 40 70 10 25 250 ml flask capacity universal* platform 9 16 24 24 40 9 16 Maximum capacity largest flask on a Platform 1 x 6L 2 x 6L 3 x 6L 3 x 6L 6 x 6L 4 x 1L 6 x 2L Orbit diameter reciprocating 0.75 1.0 0.75 0.75 Motion Inches (cm) (1.9) (2.54) (1.9) (1.9) MaxQ Orbital Shakers RPM range Digital: 15–500 Digital: 15–500 Digital: 15–500 Digital: 15–500 Temperature range °C TA +10°C TA +10°C TA -15°C N/A N/A TA +5°C to 80°C (TA=ambient temperature) to 60°C to 80°C to 60°C 35 50 35 50 Maximum load lb. (kg) (15.9) (22.7) (15.9) (22.7) Refrigeration • Gassing manifold • Page 8–10 8–10 13–14 15–16 *See page 7 for a complete description of platform types 1Universal platform only; 2Fixed reciprocating stroke length and orbital motion; 3Adjustable reciprocating stroke length: 4Achievable maximum speeds may be dependent on load 1 MaxQ 5000 MaxQ 6000 MaxQ 8000 MaxQ 7000 Model Maximum flexibility, Large capacity applications Stackable,
    [Show full text]
  • Bacterial Diversity and Functional Analysis of Severe Early Childhood
    www.nature.com/scientificreports OPEN Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India Balakrishnan Kalpana1,3, Puniethaa Prabhu3, Ashaq Hussain Bhat3, Arunsaikiran Senthilkumar3, Raj Pranap Arun1, Sharath Asokan4, Sachin S. Gunthe2 & Rama S. Verma1,5* Dental caries is the most prevalent oral disease afecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidifcation due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3–V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were signifcantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with signifcant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of bioflm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound bioflm formation and augmented the growth and enhanced bioflm formation in S. mutans in both dual and multispecies cultures. Interaction among more than 700 species of microbiota under diferent micro-ecological niches of the human oral cavity1,2 acts as a primary defense against various pathogens. Tis has been observed to play a signifcant role in child’s oral and general health.
    [Show full text]