Publications by authors named "Grant Mills"

25 Publications

  • Page 1 of 1

Evolution of Colistin Resistance in the Klebsiella pneumoniae Complex Follows Multiple Evolutionary Trajectories with Variable Effects on Fitness and Virulence Characteristics.

Antimicrob Agents Chemother 2020 12 16;65(1). Epub 2020 Dec 16.

Department of Medical Microbiology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

The increasing prevalence of multidrug-resistant has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the evolutionary trajectories toward colistin resistance in four clinical complex strains and their impact on fitness and virulence characteristics. Through population sequencing during evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one strain, but not those of the other three complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. strains exhibited cross-resistance to LL-37, in contrast to the subsp. strain. Virulence, as determined in a survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant .
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http://dx.doi.org/10.1128/AAC.01958-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927857PMC
December 2020

Emergence of anomalous dynamics in soft matter probed at the European XFEL.

Proc Natl Acad Sci U S A 2020 09 15;117(39):24110-24116. Epub 2020 Sep 15.

Photon Science, Deutsches Elektronen-Synchrotron DESY, 22607 Hamburg, Germany.

Dynamics and kinetics in soft matter physics, biology, and nanoscience frequently occur on fast (sub)microsecond but not ultrafast timescales which are difficult to probe experimentally. The European X-ray Free-Electron Laser (European XFEL), a megahertz hard X-ray Free-Electron Laser source, enables such experiments via taking series of diffraction patterns at repetition rates of up to 4.5 MHz. Here, we demonstrate X-ray photon correlation spectroscopy (XPCS) with submicrosecond time resolution of soft matter samples at the European XFEL. We show that the XFEL driven by a superconducting accelerator provides unprecedented beam stability within a pulse train. We performed microsecond sequential XPCS experiments probing equilibrium and nonequilibrium diffusion dynamics in water. We find nonlinear heating on microsecond timescales with dynamics beyond hot Brownian motion and superheated water states persisting up to 100 μs at high fluences. At short times up to 20 μs we observe that the dynamics do not obey the Stokes-Einstein predictions.
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http://dx.doi.org/10.1073/pnas.2003337117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533660PMC
September 2020

Segmented flow generator for serial crystallography at the European X-ray free electron laser.

Nat Commun 2020 09 9;11(1):4511. Epub 2020 Sep 9.

School of Molecular Sciences, Arizona State University, Tempe, AZ, 85287-1604, USA.

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.
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http://dx.doi.org/10.1038/s41467-020-18156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7481229PMC
September 2020

A Case of Cheilitis Granulomatosa/Orofacial Granulomatosis.

Clin Cosmet Investig Dent 2020 23;12:219-224. Epub 2020 Jun 23.

Department of Restorative Dentistry, Howard University College of Dentistry, Washington, DC, USA.

A case of a 19-year-old female patient is presented to a private practice dental clinician with swelling of the lower lip and inflammation of the anterior dorsal tongue. The patient presented with moderate oral pain as well as abdominal pain. The lesions were biopsied and noted for a granulomatous histopathologic appearance. The patient reported a history of using cinnamon as a flavoring agent. The lesions resolved within two weeks after the biopsy procedures and topical steroid therapy. The lesions were diagnosed as cheilitis granulomatosa/orofacial granulomatosis. The patient has remained lesion free as of the three-year follow-up. Etiologic, diagnostic and therapeutic issues related to this relatively rare condition of cheilitis granulomatosa/orofacial granulomatosis are discussed.
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http://dx.doi.org/10.2147/CCIDE.S254899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7320889PMC
June 2020

An Approach to Engaging Schools In Oral Health Initiatives: The Howard Meharry Adolescent Caries Study (HMACS).

J Health Care Poor Underserved 2020 ;31(1):35-42

Use of community-based participatory research (CBPR) principles can help identify strategies for development and implementation of studies that can address oral health disparities disfavoring African American youth. This paper summarizes approaches of the Howard Meharry Adolescent Caries Study (HMACS) to provide sustained oral health services beyond the life of a research study.
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http://dx.doi.org/10.1353/hpu.2020.0006DOI Listing
January 2020

Author Correction: Membrane protein megahertz crystallography at the European XFEL.

Nat Commun 2020 Jan 30;11(1):703. Epub 2020 Jan 30.

Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ, 85287-5001, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-14436-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992783PMC
January 2020

Membrane protein megahertz crystallography at the European XFEL.

Nat Commun 2019 11 4;10(1):5021. Epub 2019 Nov 4.

Biodesign Center for Applied Structural Discovery, Arizona State University, Tempe, AZ, 85287-5001, USA.

The world's first superconducting megahertz repetition rate hard X-ray free-electron laser (XFEL), the European XFEL, began operation in 2017, featuring a unique pulse train structure with 886 ns between pulses. With its rapid pulse rate, the European XFEL may alleviate some of the increasing demand for XFEL beamtime, particularly for membrane protein serial femtosecond crystallography (SFX), leveraging orders-of-magnitude faster data collection. Here, we report the first membrane protein megahertz SFX experiment, where we determined a 2.9 Å-resolution SFX structure of the large membrane protein complex, Photosystem I, a > 1 MDa complex containing 36 protein subunits and 381 cofactors. We address challenges to megahertz SFX for membrane protein complexes, including growth of large quantities of crystals and the large molecular and unit cell size that influence data collection and analysis. The results imply that megahertz crystallography could have an important impact on structure determination of large protein complexes with XFELs.
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http://dx.doi.org/10.1038/s41467-019-12955-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828683PMC
November 2019

Reducing Dental Plaque Scores in Long-term Care Facilities Using a Checklist and Random Inspections: A Pilot Study.

Bull Tokyo Dent Coll 2019 Sep 20;60(3):177-184. Epub 2019 Jun 20.

Howard University College of Dentistry.

The development and use of tailored interventions in overcoming barriers to optimum health in long-term care facility residents is of the highest importance. One successful approach to improving health outcomes is the use of a checklist by health care providers. Despite the evidence of the success of such checklists in nursing and medicine, there is little evidence on their use in improving dental outcomes. This study investigated whether an intervention comprising the daily use of a checklist for oral care by nursing staff supplemented by random inspections by a charge nurse resulted in lower dental plaque scores in patient participants at a long-term care facility (n=19) as compared with in those at another long-term care facility that did not receive the intervention (n=13). All participants received a dental cleaning at baseline. At a follow-up examination at 6 to 8 weeks post-baseline, significant differences were observed in the plaque scores between the participants at each location, with the median plaque scores in those undergoing the intervention being less than half of those in the patients that did not (20.8% vs. 52.8%, p<0.001). After adjusting for age in a linear regression model, this difference remained significant. The use of a daily checklist for oral care supplemented by random inspections by a charge nurse was associated with lower plaque scores (p<0.001). These results warrant further research, including prospective studies aimed at establishing how use of both clinical supervision and a checklist for oral health may influence plaque scores in geriatric patients in long-term care facilities over time.
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http://dx.doi.org/10.2209/tdcpublication.2018-0056DOI Listing
September 2019

The Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography instrument of the European XFEL: initial installation.

J Synchrotron Radiat 2019 May 12;26(Pt 3):660-676. Epub 2019 Apr 12.

European XFEL, Holzkoppel 4, 22869 Schenefeld, Germany.

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation - in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described.
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http://dx.doi.org/10.1107/S1600577519003308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6510195PMC
May 2019

MHz data collection of a microcrystalline mixture of different jack bean proteins.

Sci Data 2019 04 3;6(1):18. Epub 2019 Apr 3.

Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.

We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (<1 µs) between pulses requires fast flowing microjets for sample injection and high frame rate detectors. A data set was recorded of a microcrystalline mixture of at least three different jack bean proteins (urease, concanavalin A, concanavalin B). A one megapixel Adaptive Gain Integrating Pixel Detector (AGIPD) was used which has not only a high frame rate but also a large dynamic range. This dataset is publicly available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development and for data analysis training for prospective XFEL users.
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http://dx.doi.org/10.1038/s41597-019-0010-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472352PMC
April 2019

Initial observations of the femtosecond timing jitter at the European XFEL.

Opt Lett 2019 Apr;44(7):1650-1653

Intense, ultrashort, and high-repetition-rate X-ray pulses, combined with a femtosecond optical laser, allow pump-probe experiments with fast data acquisition and femtosecond time resolution. However, the relative timing of the X-ray pulses and the optical laser pulses can be controlled only to a level of the intrinsic error of the instrument which, without characterization, limits the time resolution of experiments. This limitation inevitably calls for a precise determination of the relative arrival time, which can be used after measurement for sorting and tagging the experimental data to a much finer resolution than it can be controlled to. The observed root-mean-square timing jitter between the X-ray and the optical laser at the SPB/SFX instrument at European XFEL was 308 fs. This first measurement of timing jitter at the European XFEL provides an important step in realizing ultrafast experiments at this novel X-ray source. A method for determining the change in the complex refractive index of samples is also presented.
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http://dx.doi.org/10.1364/OL.44.001650DOI Listing
April 2019

Megahertz serial crystallography.

Authors:
Max O Wiedorn Dominik Oberthür Richard Bean Robin Schubert Nadine Werner Brian Abbey Martin Aepfelbacher Luigi Adriano Aschkan Allahgholi Nasser Al-Qudami Jakob Andreasson Steve Aplin Salah Awel Kartik Ayyer Saša Bajt Imrich Barák Sadia Bari Johan Bielecki Sabine Botha Djelloul Boukhelef Wolfgang Brehm Sandor Brockhauser Igor Cheviakov Matthew A Coleman Francisco Cruz-Mazo Cyril Danilevski Connie Darmanin R Bruce Doak Martin Domaracky Katerina Dörner Yang Du Hans Fangohr Holger Fleckenstein Matthias Frank Petra Fromme Alfonso M Gañán-Calvo Yaroslav Gevorkov Klaus Giewekemeyer Helen Mary Ginn Heinz Graafsma Rita Graceffa Dominic Greiffenberg Lars Gumprecht Peter Göttlicher Janos Hajdu Steffen Hauf Michael Heymann Susannah Holmes Daniel A Horke Mark S Hunter Siegfried Imlau Alexander Kaukher Yoonhee Kim Alexander Klyuev Juraj Knoška Bostjan Kobe Manuela Kuhn Christopher Kupitz Jochen Küpper Janine Mia Lahey-Rudolph Torsten Laurus Karoline Le Cong Romain Letrun P Lourdu Xavier Luis Maia Filipe R N C Maia Valerio Mariani Marc Messerschmidt Markus Metz Davide Mezza Thomas Michelat Grant Mills Diana C F Monteiro Andrew Morgan Kerstin Mühlig Anna Munke Astrid Münnich Julia Nette Keith A Nugent Theresa Nuguid Allen M Orville Suraj Pandey Gisel Pena Pablo Villanueva-Perez Jennifer Poehlsen Gianpietro Previtali Lars Redecke Winnie Maria Riekehr Holger Rohde Adam Round Tatiana Safenreiter Iosifina Sarrou Tokushi Sato Marius Schmidt Bernd Schmitt Robert Schönherr Joachim Schulz Jonas A Sellberg M Marvin Seibert Carolin Seuring Megan L Shelby Robert L Shoeman Marcin Sikorski Alessandro Silenzi Claudiu A Stan Xintian Shi Stephan Stern Jola Sztuk-Dambietz Janusz Szuba Aleksandra Tolstikova Martin Trebbin Ulrich Trunk Patrik Vagovic Thomas Ve Britta Weinhausen Thomas A White Krzysztof Wrona Chen Xu Oleksandr Yefanov Nadia Zatsepin Jiaguo Zhang Markus Perbandt Adrian P Mancuso Christian Betzel Henry Chapman Anton Barty

Nat Commun 2018 10 2;9(1):4025. Epub 2018 Oct 2.

Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestrasse 85, 22607, Hamburg, Germany.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.
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http://dx.doi.org/10.1038/s41467-018-06156-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168542PMC
October 2018

Megahertz data collection from protein microcrystals at an X-ray free-electron laser.

Nat Commun 2018 08 28;9(1):3487. Epub 2018 Aug 28.

Max Planck Institute for Medical Research, Jahnstrasse 29, 69120, Heidelberg, Germany.

X-ray free-electron lasers (XFELs) enable novel experiments because of their high peak brilliance and femtosecond pulse duration. However, non-superconducting XFELs offer repetition rates of only 10-120 Hz, placing significant demands on beam time and sample consumption. We describe serial femtosecond crystallography experiments performed at the European XFEL, the first MHz repetition rate XFEL, delivering 1.128 MHz X-ray pulse trains at 10 Hz. Given the short spacing between pulses, damage caused by shock waves launched by one XFEL pulse on sample probed by subsequent pulses is a concern. To investigate this issue, we collected data from lysozyme microcrystals, exposed to a ~15 μm XFEL beam. Under these conditions, data quality is independent of whether the first or subsequent pulses of the train were used for data collection. We also analyzed a mixture of microcrystals of jack bean proteins, from which the structure of native, magnesium-containing concanavalin A was determined.
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http://dx.doi.org/10.1038/s41467-018-05953-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6113309PMC
August 2018

Identification and Characterization of Two Klebsiella pneumoniae Lipid A Late Acyltransferases and Their Role in Virulence.

Infect Immun 2017 09 18;85(9). Epub 2017 Aug 18.

Wellcome-Wolfson Institute for Experimental Medicine, Queen's University Belfast, Belfast, United Kingdom

causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of , which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available genomes revealed that this pathogen's genome encodes two orthologues of LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated lipid A, whereas only LpxL2 mediated lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of and expression. LpxL2-dependent lipid A acylation protects from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti- drugs.
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http://dx.doi.org/10.1128/IAI.00068-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563558PMC
September 2017

A antibiotic resistance mechanism that subdues host defences and promotes virulence.

EMBO Mol Med 2017 04;9(4):430-447

Centre for Experimental Medicine, Queen's University Belfast, Belfast, UK

is an important cause of multidrug-resistant infections worldwide. Recent studies highlight the emergence of multidrug-resistant strains which show resistance to colistin, a last-line antibiotic, arising from mutational inactivation of the regulatory gene. However, the precise molecular resistance mechanisms of -associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an gene mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mutation induces PhoPQ-governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.
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http://dx.doi.org/10.15252/emmm.201607336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5376759PMC
April 2017

The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium.

PeerJ 2016 22;4:e2130. Epub 2016 Jun 22.

Bioluminescent Superbugs Lab, University of Auckland, Auckland, New Zealand; Department of Molecular Medicine and Pathology, University of Auckland, Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery, New Zealand.

Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.
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http://dx.doi.org/10.7717/peerj.2130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924136PMC
July 2016

Phosphoinositide-mediated oligomerization of a defensin induces cell lysis.

Elife 2014 Apr 1;3:e01808. Epub 2014 Apr 1.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia.

Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3968744PMC
http://dx.doi.org/10.7554/eLife.01808DOI Listing
April 2014

In vivo correlates of molecularly inferred virulence among extraintestinal pathogenic Escherichia coli (ExPEC) in the wax moth Galleria mellonella model system.

Virulence 2014 Apr 11;5(3):388-93. Epub 2014 Feb 11.

Faculty of Medical and Health Sciences; University of Auckland; Auckland, New Zealand; Maurice Wilkins Centre for Molecular Biodiscovery; University of Auckland; Auckland, New Zealand.

In contrast to commensal Escherichia coli, extraintestinal pathogenic E. coli (ExPEC) strains possess an array of virulence-associated genes. We sought to establish the feasibility of using the invertebrate Galleria mellonella (greater wax moth) for assessing ExPEC virulence and to investigate the correlation between genotypic determinants of virulence and in vivo pathogenicity. We observed a correlation between the number of virulence genes and larval survival, such that ExPEC isolates with higher virulence scores killed larvae significantly faster than isolates with lower virulence scores. By correlating genotypic and phenotypic virulence, we provide preliminary validation of this model for future studies investigating ExPEC virulence.
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http://dx.doi.org/10.4161/viru.27912DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979865PMC
April 2014

Crystallization reports are the backbone of Acta Cryst. F, but do they have any spine?

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Jul 30;69(Pt 7):712-8. Epub 2013 Jun 30.

Materials, Science and Engineering Division, CSIRO, 343 Royal Parade, Parkville, VIC 3052, Australia.

Crystallization of macromolecules is famously difficult. By knowing what has worked for others, researchers can ease the process, both in the case where the protein has already been crystallized and in the situation where more general guidelines are needed. The 264 crystallization communications published in Acta Crystallographica Section F in 2012 have been reviewed, and from this analysis some information about trends in crystallization has been gleaned. More importantly, it was found that there are several ways in which the utility of these communications could be increased: to make each individual paper a more complete crystallization record; and to provide a means for taking a snapshot of what the current `best practices' are in the field.
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http://dx.doi.org/10.1107/S1744309113014152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3702311PMC
July 2013

Dimerization of plant defensin NaD1 enhances its antifungal activity.

J Biol Chem 2012 Jun 17;287(24):19961-72. Epub 2012 Apr 17.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.

The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys(4) as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys(4) that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys(4) mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.
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http://dx.doi.org/10.1074/jbc.M111.331009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370180PMC
June 2012

Crystallization and preliminary X-ray crystallographic analysis of the plant defensin NaD1.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Jan 24;68(Pt 1):85-8. Epub 2011 Dec 24.

Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia.

Plant defensins are small (~5 kDa) basic cysteine-rich proteins that are being explored in important agricultural crops for their ability to confer enhanced disease resistance against fungal pathogens. NaD1, isolated from the flowers of the ornamental tobacco (Nicotiana alata), is a particularly well characterized antifungal defensin. Here, the crystallization and preliminary X-ray crystallographic analysis of NaD1 is reported. Crystals of NaD1 were crystallized using the sitting-drop vapour-diffusion method at 291 K. Data were collected from two crystal forms to 1.4 and 1.6 Å resolution, respectively. The crystals of form A belonged to the monoclinic space group P2(1), with unit-cell parameters a = 32.697, b = 32.685, c = 41.977 Å, α = 90, β = 100.828, γ = 90°, whereas crystals of form B belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 33.091, c = 128.77 Å, α = β = 90, γ = 120°.
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http://dx.doi.org/10.1107/S1744309111049530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3253843PMC
January 2012

Effective admissions practices to achieve greater student diversity in dental schools.

J Dent Educ 2010 Oct;74(10 Suppl):S87-97

School of Dentistry, West Virginia University, 1 Medical Center Drive, P.O. Box 9407, HSN, Morgantown, WV 26505, USA.

In this chapter we describe the institutional and policy-level strategies that dental schools in the Pipeline, Profession, and Practice: Community-Based Dental Education program used to modify their admissions practices to increase the diversity of their student bodies. Schools developed and used clear statements recognizing the value of diversity. They incorporated recent U.S. Supreme Court rulings regarding educational diversity into their revised admissions practices; these rulings cited diversity as both a "compelling interest" and its use in only "narrowly tailored" circumstances. We make a case for admissions decisions based on a comprehensive evaluation that balances the quantitative and qualitative qualities of a candidate. It refutes the practice of overreliance on standardized tests by detailing the whole-file review process to measure merit and professional promise. Also described is a range of noncognitive variables (e.g., leadership, ability to sustain academic achievement with competing priorities, volunteerism, communication, social background, and disadvantaged status) that schools can take into consideration in admissions decisions. Admissions committees can tie this comprehensive review of candidates into the case for promoting cross-cultural understanding and enhanced competence to provide care to patients from diverse backgrounds. In addition, the chapter reviews the challenges schools face in developing admissions policies and procedures that reflect the university's mission for diversity. It addresses the importance of a diverse composition of the admissions committee. It also describes how tailored workshops and technical assistance for admissions committees can help schools improve their student diversity and how admissions committees can engage in a process of periodic review of their diversity objectives in relationship to the school's mission.
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October 2010

The Pipeline program at Howard University College of Dentistry.

J Dent Educ 2009 Feb;73(2 Suppl):S70-81; discussion S81-2

UCLA School of Public Health, Los Angeles, CA 90095-1772, USA.

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February 2009

Familial and cultural perceptions and beliefs of oral hygiene and dietary practices among ethnically and socio-economicall diverse groups.

Community Dent Health 2004 Mar;21(1 Suppl):102-11

Department of Clinical Psychology, The Royal Hospitals, Belfast, Northern Ireland.

Unlabelled: OBJECTIVE; The aim of this international study was to develop a valid and reliable psychometric measure to examine the extent to which parents' attitudes about engaging in twice-daily tooth brushing and controlling sugar snacking predict these respective behaviours in their children. A supplementary objective was to assess whether ethnic group, culture, level of deprivation or children's caries experience impact upon the relationships between oral health related behaviours, attitudes to these respective behaviours and to dental caries.

Clinical Setting: Nurseries, health centres and dental clinics in 17 countries.

Participants: 2822 children aged 3 to 4 years and their parents.

Main Outcome Measures: Dental examination of children and questionnaire to parents.

Results: Factor analysis identified 8 coherent attitudes towards toothbrushing, sugar snacking and childhood caries. Attitudes were significantly different in families from deprived and non-deprived backgrounds and in families of children with and without caries. Parents perception of their ability to control their children's toothbrushing and sugar snacking habits were the most significant predictor of whether or not favourable habits were reported. Some differences were found by site and ethnic group.

Conclusions: This study supports the hypothesis that parental attitudes significantly impact on the establishment of habits favourable to oral health. An appreciation of the impact of cultural and ethnic diversity is important in understanding how parental attitudes to oral health vary. Further research should examine in a prospective intervention whether enhancing parenting skills is an effective route to preventing childhood caries.
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March 2004

A multi-country comparison of caries-associated microflora in demographically diverse children.

Community Dent Health 2004 Mar;21(1 Suppl):96-101

Guy's, King's and St Thomas' Dental Institute, London, England, UK.

Objective: The aim of this formative international collaborative research on childhood dental caries was to undertake an initial investigation comparing the dental plaque of young children from diverse ethnic and socioeconomic backgrounds with and without dental caries.

Basic Research Design: The following four null hypotheses were investigated. There were no differences in numbers of individual taxa when comparing plaque samples from: 1) caries-free children from deprived and non-deprived backgrounds; 2) children from deprived and non-deprived backgrounds with at least 3 decayed teeth; 3) children from non-deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth; and, 4) children from deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth.

Participants: 277 children aged 3-4 years from 5 countries.

Main Outcome Measures: A sample of interproximal plaque from anterior teeth was collected using sterile dental floss, and cultured according to accepted international standards.

Results: Analysis of the data found that the first null hypothesis was accepted and that the fourth was rejected. Unexpectedly, the second null hypothesis was rejected as the children with caries from deprived and non-deprived backgrounds had a different caries-associated flora. In particular, children living in deprivation harbored more caries-associated bacteria [mutans streptococci and lactobacilli]. This greater microbial challenge was associated with a higher level of cavitated carious lesions and with more frequent consumption of confectionery. Conclusions Children from deprived backgrounds with caries may be further disadvantaged by having higher levels of caries-associated microflora.
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March 2004