Publications by authors named "John Parkinson"

318 Publications

Zoantharian Endosymbiont Community Dynamics During a Stress Event.

Front Microbiol 2021 28;12:674026. Epub 2021 May 28.

Molecular Invertebrate Systematics and Ecology Laboratory, Department of Chemistry, Biology, and Marine Science, Faculty of Science, University of the Ryukyus, Nishihara, Japan.

Coral reefs are complex ecosystems composed of many interacting species. One ecologically important group consists of zoantharians, which are closely related to reef-building corals. Like corals, zoantharians form mutualistic symbioses with dinoflagellate micro-algae (family Symbiodiniaceae), but their associations remain underexplored. To examine the degree to which zoantharians exhibit altered symbiont dynamics under changing environmental conditions, we reciprocally transplanted colonies of between intertidal (2 m) and subtidal (26 m) depths within a reef in Okinawa, Japan. At this location, can associate with three Symbiodiniaceae species from two genera distributed along a light and depth gradient. We developed species-specific molecular assays and sampled colonies pre- and post-transplantation to analyze symbiont community diversity. Despite large environmental differences across depths, we detected few symbiont compositional changes resulting from transplantation stress. Colonies sourced from the intertidal zone associated with mixtures of a "shallow" sp. and a "shallow" sp. independent of whether they were transplanted to shallow or deep waters. Colonies sourced from the subtidal zone were dominated by a "deep" sp. regardless of transplant depth. Subtidal colonies brought to shallow depths did not transition to the presumably high-light adapted shallow symbionts present in the new environment, but rather bleached and died. These patterns mirror observations of highly stable coral-algal associations subjected to depth transplantation. Our results indicate that -Symbiodiniaceae symbioses remain stable despite stress, suggesting these important reef community members have relatively low capacity to shuffle to more stress-tolerant micro-algae in response to ongoing climate change.
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http://dx.doi.org/10.3389/fmicb.2021.674026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8193574PMC
May 2021

"Just not knowing" can make life sweeter (and saltier): Reward uncertainty alters the sensory experience and consumption of palatable food and drinks.

J Exp Psychol Gen 2021 Mar 18. Epub 2021 Mar 18.

School of Psychology.

Reward uncertainty can prompt exploration and learning, strengthening approach and consummatory behaviors. For humans, these phenomena are exploited in marketing promotions and gambling products, sometimes spurring hedonic consumption. Here, in four experiments, we sought to identify whether reward uncertainty-as a state of "not knowing" that exists between an action and a positively valanced outcome-enhances the in-the-moment consumption and experience of other palatable food and drink rewards. In Experiment 1, we demonstrate that reward uncertainty can increase consumption of commercial alcoholic drinks and energy-dense savory snacks. In Experiment 2, we show that reward uncertainty is unlikely to promote consumption through gross increases in impulsivity (expressed as higher discounting rates) or risk tolerance (expressed as lower probability discounting rates). In Experiment 3, we find that reward uncertainty intensifies the taste of, and hedonic responses to, sucrose solutions in a concentration-dependent manner among individuals with heightened preferences for sweet tastes. Finally, in Experiment 4, we replicate and extend these findings by showing that reward uncertainty intensifies the taste of palatable foods and drinks in ways that are independent of individuals' discounting rates, motor control, reflection impulsivity, and momentary happiness but are strongly moderated by recent depressive symptoms. These data suggest a working hypothesis that (incidental) reward uncertainty, as a state of not knowing, operates as a mood-dependent "taste intensifier" of palatable food and drink rewards, possibly sustaining reward seeking and consumption. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
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http://dx.doi.org/10.1037/xge0001029DOI Listing
March 2021

Modeling the metabolic interplay between a parasitic worm and its bacterial endosymbiont allows the identification of novel drug targets.

Elife 2020 08 11;9. Epub 2020 Aug 11.

Program in Molecular Medicine, Hospital for Sick Children, Toronto, Canada.

The filarial nematode represents a leading cause of disability in the developing world, causing lymphatic filariasis in nearly 40 million people. Currently available drugs are not well-suited to mass drug administration efforts, so new treatments are urgently required. One potential vulnerability is the endosymbiotic bacteria -present in many filariae-which is vital to the worm. Genome scale metabolic networks have been used to study prokaryotes and protists and have proven valuable in identifying therapeutic targets, but have only been applied to multicellular eukaryotic organisms more recently. Here, we present DC625, the first compartmentalized metabolic model of a parasitic worm. We used this model to show how metabolic pathway usage allows the worm to adapt to different environments, and predict a set of 102 reactions essential to the survival of . We validated three of those reactions with drug tests and demonstrated novel antifilarial properties for all three compounds.
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http://dx.doi.org/10.7554/eLife.51850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419141PMC
August 2020

Author Correction: A transcriptomic analysis of the phylum Nematoda.

Nat Genet 2020 Jul;52(7):750

School of Biological Sciences, University of Edinburgh, Edinburgh, UK.

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/s41588-020-0658-6DOI Listing
July 2020

BraInMap Elucidates the Macromolecular Connectivity Landscape of Mammalian Brain.

Cell Syst 2020 04;10(4):333-350.e14

Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA; Broad Institute of Massachusetts Institute of Technology and Harvard University, Boston, MA, USA.

Connectivity webs mediate the unique biology of the mammalian brain. Yet, while cell circuit maps are increasingly available, knowledge of their underlying molecular networks remains limited. Here, we applied multi-dimensional biochemical fractionation with mass spectrometry and machine learning to survey endogenous macromolecules across the adult mouse brain. We defined a global "interactome" comprising over one thousand multi-protein complexes. These include hundreds of brain-selective assemblies that have distinct physical and functional attributes, show regional and cell-type specificity, and have links to core neurological processes and disorders. Using reciprocal pull-downs and a transgenic model, we validated a putative 28-member RNA-binding protein complex associated with amyotrophic lateral sclerosis, suggesting a coordinated function in alternative splicing in disease progression. This brain interaction map (BraInMap) resource facilitates mechanistic exploration of the unique molecular machinery driving core cellular processes of the central nervous system. It is publicly available and can be explored here https://www.bu.edu/dbin/cnsb/mousebrain/.
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http://dx.doi.org/10.1016/j.cels.2020.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938770PMC
April 2020

Discovery and characterization of a Gram-positive Pel polysaccharide biosynthetic gene cluster.

PLoS Pathog 2020 04 1;16(4):e1008281. Epub 2020 Apr 1.

Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada.

Our understanding of the biofilm matrix components utilized by Gram-positive bacteria, and the signalling pathways that regulate their production are largely unknown. In a companion study, we developed a computational pipeline for the unbiased identification of homologous bacterial operons and applied this algorithm to the analysis of synthase-dependent exopolysaccharide biosynthetic systems. Here, we explore the finding that many species of Gram-positive bacteria have operons with similarity to the Pseudomonas aeruginosa pel locus. Our characterization of the pelDEADAFG operon from Bacillus cereus ATCC 10987, presented herein, demonstrates that this locus is required for biofilm formation and produces a polysaccharide structurally similar to Pel. We show that the degenerate GGDEF domain of the B. cereus PelD ortholog binds cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP), and that this binding is required for biofilm formation. Finally, we identify a diguanylate cyclase, CdgF, and a c-di-GMP phosphodiesterase, CdgE, that reciprocally regulate the production of Pel. The discovery of this novel c-di-GMP regulatory circuit significantly contributes to our limited understanding of c-di-GMP signalling in Gram-positive organisms. Furthermore, conservation of the core pelDEADAFG locus amongst many species of bacilli, clostridia, streptococci, and actinobacteria suggests that Pel may be a common biofilm matrix component in many Gram-positive bacteria.
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http://dx.doi.org/10.1371/journal.ppat.1008281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112168PMC
April 2020

A systematic pipeline for classifying bacterial operons reveals the evolutionary landscape of biofilm machineries.

PLoS Comput Biol 2020 04 1;16(4):e1007721. Epub 2020 Apr 1.

Program in Molecular Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada.

In bacteria functionally related genes comprising metabolic pathways and protein complexes are frequently encoded in operons and are widely conserved across phylogenetically diverse species. The evolution of these operon-encoded processes is affected by diverse mechanisms such as gene duplication, loss, rearrangement, and horizontal transfer. These mechanisms can result in functional diversification, increasing the potential evolution of novel biological pathways, and enabling pre-existing pathways to adapt to the requirements of particular environments. Despite the fundamental importance that these mechanisms play in bacterial environmental adaptation, a systematic approach for studying the evolution of operon organization is lacking. Herein, we present a novel method to study the evolution of operons based on phylogenetic clustering of operon-encoded protein families and genomic-proximity network visualizations of operon architectures. We applied this approach to study the evolution of the synthase dependent exopolysaccharide (EPS) biosynthetic systems: cellulose, acetylated cellulose, poly-β-1,6-N-acetyl-D-glucosamine (PNAG), Pel, and alginate. These polymers have important roles in biofilm formation, antibiotic tolerance, and as virulence factors in opportunistic pathogens. Our approach revealed the complex evolutionary landscape of EPS machineries, and enabled operons to be classified into evolutionarily distinct lineages. Cellulose operons show phyla-specific operon lineages resulting from gene loss, rearrangement, and the acquisition of accessory loci, and the occurrence of whole-operon duplications arising through horizonal gene transfer. Our evolution-based classification also distinguishes between PNAG production from Gram-negative and Gram-positive bacteria on the basis of structural and functional evolution of the acetylation modification domains shared by PgaB and IcaB loci, respectively. We also predict several pel-like operon lineages in Gram-positive bacteria and demonstrate in our companion paper (Whitfield et al PLoS Pathogens, in press) that Bacillus cereus produces a Pel-dependent biofilm that is regulated by cyclic-3',5'-dimeric guanosine monophosphate (c-di-GMP).
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http://dx.doi.org/10.1371/journal.pcbi.1007721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7112194PMC
April 2020

A regioselectively 1,1',3,3'-tetrazincated ferrocene complex displaying core and peripheral reactivity.

Chem Sci 2020 Mar 27;11(25):6510-6520. Epub 2020 Mar 27.

WestCHEM, Department of Pure & Applied Chemistry, University of Strathclyde Glasgow G1 1XL UK

Regioselective 1,1',3,3'-tetrazincation [C-H to C-Zn(Bu)] of ferrocene has been achieved by reaction of a fourfold excess of di--butylzinc (BuZn) with sodium 2,2,6,6-tetramethylpiperidide (NaTMP) in hexane solution manifested in the trimetallic iron-sodium-zinc complex [Na(TMP)Zn(Bu){(CH)Fe}], . X-ray crystallographic studies supported by DFT modelling reveal the structure to be an open inverse crown in which two [Na(TMP)Zn(Bu)Na(TMP)Zn(Bu)] cationic units surround a {(CH)Fe} tetraanion. Detailed CD NMR studies have assigned the plethora of H and C chemical shifts of this complex. It exists in a major form in which capping and bridging TMP groups interchange, as well as a minor form that appears to be an intermediate in this complicated exchange phenomenon. Investigation of has uncovered two distinct reactivities. Two of its peripheral -butyl carbanions formally deprotonate toluene at the lateral methyl group to generate benzyl ligands that replace these carbanions in [Na(TMP)Zn(Bu)(CHPh){(CH)Fe}], , which retains its tetrazincated ferrocenyl core. Benzyl-Na π-arene interactions are a notable feature of . In contrast, reaction with pyridine affords the crystalline product {[Na·4py][Zn(py*)(Bu)·py]}, , where py is neutral pyridine (CHN) and py* is the anion (4-CHN), a rare example of pyridine deprotonated/metallated at the 4-position. This ferrocene-free complex appears to be a product of core reactivity in that the core-positioned ferrocenyl anions of , in company with TMP anions, have formally deprotonated the heterocycle.
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http://dx.doi.org/10.1039/d0sc01612hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152701PMC
March 2020

Host and Symbiont Cell Cycle Coordination Is Mediated by Symbiotic State, Nutrition, and Partner Identity in a Model Cnidarian-Dinoflagellate Symbiosis.

mBio 2020 03 10;11(2). Epub 2020 Mar 10.

Department of Integrative Biology, Oregon State University, Corvallis, Oregon, USA.

The cell cycle is a critical component of cellular proliferation, differentiation, and response to stress, yet its role in the regulation of intracellular symbioses is not well understood. To explore host-symbiont cell cycle coordination in a marine symbiosis, we employed a model for coral-dinoflagellate associations: the tropical sea anemone Aiptasia () and its native microalgal photosymbionts ( and ). Using fluorescent labeling and spatial point-pattern image analyses to characterize cell population distributions in both partners, we developed protocols that are tailored to the three-dimensional cellular landscape of a symbiotic sea anemone tentacle. Introducing cultured symbiont cells to symbiont-free adult hosts increased overall host cell proliferation rates. The acceleration occurred predominantly in the symbiont-containing gastrodermis near clusters of symbionts but was also observed in symbiont-free epidermal tissue layers, indicating that the presence of symbionts contributes to elevated proliferation rates in the entire host during colonization. Symbiont cell cycle progression differed between cultured algae and those residing within hosts; the endosymbiotic state resulted in increased S-phase but decreased G/M-phase symbiont populations. These phenotypes and the deceleration of cell cycle progression varied with symbiont identity and host nutritional status. These results demonstrate that host and symbiont cells have substantial and species-specific effects on the proliferation rates of their mutualistic partners. This is the first empirical evidence to support species-specific regulation of the symbiont cell cycle within a single cnidarian-dinoflagellate association; similar regulatory mechanisms likely govern interpartner coordination in other coral-algal symbioses and shape their ecophysiological responses to a changing climate. Biomass regulation is critical to the overall health of cnidarian-dinoflagellate symbioses. Despite the central role of the cell cycle in the growth and proliferation of cnidarian host cells and dinoflagellate symbionts, there are few studies that have examined the potential for host-symbiont coregulation. This study provides evidence for the acceleration of host cell proliferation when in local proximity to clusters of symbionts within cnidarian tentacles. The findings suggest that symbionts augment the cell cycle of not only their enveloping host cells but also neighboring cells in the epidermis and gastrodermis. This provides a possible mechanism for rapid colonization of cnidarian tissues. In addition, the cell cycles of symbionts differed depending on nutritional regime, symbiotic state, and species identity. The responses of cell cycle profiles to these different factors implicate a role for species-specific regulation of symbiont cell cycles within host cnidarian tissues.
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http://dx.doi.org/10.1128/mBio.02626-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064764PMC
March 2020

New reductive rearrangement of -arylindoles triggered by the Grubbs-Stoltz reagent EtSiH/KO Bu.

Chem Sci 2020 Mar 11;11(14):3719-3726. Epub 2020 Mar 11.

Department of Pure and Applied Chemistry, University of Strathclyde 295 Cathedral Street Glasgow G1 1XL UK

-Arylindoles are transformed into dihydroacridines in a new type of rearrangement, through heating with triethylsilane and potassium butoxide. Studies indicate that the pathway involves (i) the formation of indole radical anions followed by fragmentation of the indole C2-N bond, and (ii) a ring-closing reaction that follows a potassium-ion dependent hydrogen atom transfer step. Unexpected behaviors of 'radical-trap' substrates prove very helpful in framing the proposed mechanism.
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http://dx.doi.org/10.1039/d0sc00361aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8152433PMC
March 2020

Complete structure of the chemosensory array core signalling unit in an E. coli minicell strain.

Nat Commun 2020 02 6;11(1):743. Epub 2020 Feb 6.

Institut de Biologie Structurale, Université Grenoble Alpes, CEA, CNRS, IBS, 71 Avenue des martyrs, F-38044, Grenoble, France.

Motile bacteria sense chemical gradients with transmembrane receptors organised in supramolecular signalling arrays. Understanding stimulus detection and transmission at the molecular level requires precise structural characterisation of the array building block known as a core signalling unit. Here we introduce an Escherichia coli strain that forms small minicells possessing extended and highly ordered chemosensory arrays. We use cryo-electron tomography and subtomogram averaging to provide a three-dimensional map of a complete core signalling unit, with visible densities corresponding to the HAMP and periplasmic domains. This map, combined with previously determined high resolution structures and molecular dynamics simulations, yields a molecular model of the transmembrane core signalling unit and enables spatial localisation of its individual domains. Our work thus offers a solid structural basis for the interpretation of a wide range of existing data and the design of further experiments to elucidate signalling mechanisms within the core signalling unit and larger array.
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http://dx.doi.org/10.1038/s41467-020-14350-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005262PMC
February 2020

N-Linked Surface Glycan Biosynthesis, Composition, Inhibition, and Function in Cnidarian-Dinoflagellate Symbiosis.

Microb Ecol 2020 Jul 25;80(1):223-236. Epub 2020 Jan 25.

Department of Chemistry, Oregon State University, Corvallis, OR, USA.

The success of symbioses between cnidarian hosts (e.g., corals and sea anemones) and micro-algal symbionts hinges on the molecular interactions that govern the establishment and maintenance of intracellular mutualisms. As a fundamental component of innate immunity, glycan-lectin interactions impact the onset of marine endosymbioses, but our understanding of the effects of cell surface glycome composition on symbiosis establishment remains limited. In this study, we examined the canonical N-glycan biosynthesis pathway in the genome of the dinoflagellate symbiont Breviolum minutum (family Symbiodiniaceae) and found it to be conserved with the exception of the transferase GlcNAc-TII (MGAT2). Using coupled liquid chromatography-mass spectrometry (LC-MS/MS), we characterized the cell surface N-glycan content of B. minutum, providing the first insight into the molecular composition of surface glycans in dinoflagellates. We then used the biosynthesis inhibitors kifunensine and swainsonine to alter the glycan composition of B. minutum. Successful high-mannose enrichment via kifunensine treatment resulted in a significant decrease in colonization of the model sea anemone Aiptasia (Exaiptasia pallida) by B. minutum. Hybrid glycan enrichment via swainsonine treatment, however, could not be confirmed and did not impact colonization. We conclude that functional Golgi processing of N-glycans is critical for maintaining appropriate cell surface glycan composition and for ensuring colonization success by B. minutum.
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http://dx.doi.org/10.1007/s00248-020-01487-9DOI Listing
July 2020

Accumulation of genetic variants associated with immunity in the selective breeding of broilers.

BMC Genet 2020 01 17;21(1). Epub 2020 Jan 17.

Department of Biochemistry, University of Toronto, Toronto, M5S 1A8, ON, Canada.

Background: To satisfy an increasing demand for dietary protein, the poultry industry has employed genetic selection to increase the growth rate of broilers by over 400% in the past 50 years. Although modern broilers reach a marketable weight of ~ 2 kg in a short span of 35 days, a speed twice as fast as a broiler 50 years ago, the expedited growth has been associated with several negative detrimental consequences. Aside from heart and musculoskeletal problems, which are direct consequences of additional weight, the immune response is also thought to be altered in modern broilers.

Results: Given that identifying the underlying genetic basis responsible for a less sensitive innate immune response would be economically beneficial for poultry breeding, we decided to compare the genomes of two unselected meat control strains that are representative of broilers from 1957 and 1978, and a current commercial broiler line. Through analysis of genetic variants, we developed a custom prioritization strategy to identify genes and pathways that have accumulated genetic changes and are biologically relevant to immune response and growth performance. Our results highlight two genes, TLR3 and PLIN3, with genetic variants that are predicted to enhance growth performance at the expense of immune function.

Conclusions: Placing these new genomes in the context of other chicken lines, reveal genetic changes that have specifically arisen in selective breeding programs that were implemented in the last 50 years.
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http://dx.doi.org/10.1186/s12863-020-0807-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6969402PMC
January 2020

Bis(phosphine)hydridorhodacarborane Derivatives of 1,1'-Bis(-carborane) and Their Catalysis of Alkene Isomerization and the Hydrosilylation of Acetophenone.

Inorg Chem 2020 Feb 16;59(3):2011-2023. Epub 2020 Jan 16.

Institute of Chemical Sciences , Heriot-Watt University , Edinburgh EH14 4AS , United Kingdom.

Deprotonation of [7-(1'--1',2'-CBH)--7,8-CBH] and reaction with [Rh(PPh)Cl] results in isomerization of the metalated cage and the formation of [8-(1'--1',2'-CBH)-2-H-2,2-(PPh)--2,1,8-RhCBH] (). Similarly, deprotonation/metalation of [8'-(7--7,8-CBH)-2'-(-cymene)--2',1',8'-RuCBH] and [8'-(7--7,8-CBH)-2'-Cp*--2',1',8'-CoCBH] affords [8-{8'-2'-(-cymene)--2',1',8'-RuCBH}-2-H-2,2-(PPh)--2,1,8-RhCBH] () and [8-(8'-2'-Cp*--2',1',8'-CoCBH)-2-H-2,2-(PPh)--2,1,8-RhCBH] (), respectively, as diastereoisomeric mixtures. The performances of compounds - as catalysts in the isomerization of 1-hexene and in the hydrosilylation of acetophenone are compared with those of the known single-cage species [3-H-3,3-(PPh)--3,1,2-RhCBH] () and [2-H-2,2-(PPh)--2,1,12-RhCBH] (), the last two compounds also being the subjects of Rh NMR spectroscopic studies, the first such investigations of rhodacarboranes. In alkene isomerization all the 2,1,8- or 2,1,12-RhCB species (-, ) outperform the 3,1,2-RhCB compound , while for hydrosilylation the single-cage compounds and are better catalysts than the double-cage species -.
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http://dx.doi.org/10.1021/acs.inorgchem.9b03351DOI Listing
February 2020

Structure and dynamics of the E. coli chemotaxis core signaling complex by cryo-electron tomography and molecular simulations.

Commun Biol 2020 01 10;3(1):24. Epub 2020 Jan 10.

Department of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15260, USA.

To enable the processing of chemical gradients, chemotactic bacteria possess large arrays of transmembrane chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW, organized as coupled core-signaling units (CSU). Despite decades of study, important questions surrounding the molecular mechanisms of sensory signal transduction remain unresolved, owing especially to the lack of a high-resolution CSU structure. Here, we use cryo-electron tomography and sub-tomogram averaging to determine a structure of the Escherichia coli CSU at sub-nanometer resolution. Based on our experimental data, we use molecular simulations to construct an atomistic model of the CSU, enabling a detailed characterization of CheA conformational dynamics in its native structural context. We identify multiple, distinct conformations of the critical P4 domain as well as asymmetries in the localization of the P3 bundle, offering several novel insights into the CheA signaling mechanism.
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http://dx.doi.org/10.1038/s42003-019-0748-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954272PMC
January 2020

Investigation of the Factors That Dictate the Preferred Orientation of Lexitropsins in the Minor Groove of DNA.

J Med Chem 2019 11 8;62(22):10423-10440. Epub 2019 Nov 8.

WestCHEM, Department of Pure and Applied Chemistry , University of Strathclyde , 295 Cathedral Street , Glasgow G1 1XL , United Kingdom.

Lexitropsins are small molecules that bind to the minor groove of DNA as antiparallel dimers in a specific orientation. These molecules have shown therapeutic potential in the treatment of several diseases; however, the development of these molecules to target particular genes requires revealing the factors that dictate their preferred orientation in the minor grooves, which to date have not been investigated. In this study, a distinct structure (thzC) was carefully designed as an analog of a well-characterized lexitropsin (thzA) to reveal the factors that dictate the preferred binding orientation. Comparative evaluations of the biophysical and molecular modeling results of both compounds showed that the position of the dimethylaminopropyl group and the orientation of the amide links of the ligand with respect to the 5'-3'-ends; dictate the preferred orientation of lexitropsins in the minor grooves. These findings could be useful in the design of novel lexitropsins to selectively target specific genes.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01534DOI Listing
November 2019

Under Elevated c-di-GMP in Escherichia coli, YcgR Alters Flagellar Motor Bias and Speed Sequentially, with Additional Negative Control of the Flagellar Regulon via the Adaptor Protein RssB.

J Bacteriol 2019 12 6;202(1). Epub 2019 Dec 6.

Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas, USA

In and , the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of restores wild-type motor behavior in , indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In , c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in Instead, we found through reversion analysis that deletion of , which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σ for degradation, restored wild-type motility in the mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression. Flagellum-driven motility has been studied in and for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in , we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σ regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated.
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http://dx.doi.org/10.1128/JB.00578-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6932234PMC
December 2019

Metabolomic Profiling of the Immune Stimulatory Effect of Eicosenoids on PMA-Differentiated THP-1 Cells.

Vaccines (Basel) 2019 Oct 9;7(4). Epub 2019 Oct 9.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK.

Honey bee venom has been established to have significant effect in immunotherapy. In the present study, (Z)-11-eicosenol-a major constituent of bee venom, along with its derivations methyl cis-11-eicosenoate and cis-11-eicosenoic acid, were synthesised to investigate their immune stimulatory effect and possible use as vaccine adjuvants. Stimuli that prime and activate the immune system have exerted profound effects on immune cells, particularly macrophages; however, the effectiveness of bee venom constituents as immune stimulants has not yet been established. Here, the abilities of these compounds to act as pro-inflammatory stimuli were assessed, either alone or in combination with lipopolysaccharide (LPS), by examining the secretion of tumour necrosis factor-α (TNF-α) and the cytokines interleukin-1β (IL-1β), IL-6 and IL-10 by THP-1 macrophages. The compounds clearly increased the levels of IL-1β and decreased IL-10, whereas a decrease in IL-6 levels suggested a complex mechanism of action. A more in-depth profile of macrophage behaviour was therefore obtained by comprehensive untargeted metabolic profiling of the cells using liquid chromatography mass spectrometry (LC-MS) to confirm the ability of the eicosanoids to trigger the immune system. The level of 358 polar and 315 non-polar metabolites were changed significantly ( < 0.05) by all treatments. The LPS-stimulated production of most of the inflammatory metabolite biomarkers in glycolysis, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, purine, pyrimidine and fatty acids metabolism were significantly enhanced by all three compounds, and particularly by methyl cis-11-eicosenoate and cis-11-eicosenoic acid. These findings support the proposed actions of (Z)-11-eicosenol, methyl cis-11-eicosenoate and cis-11-eicosenoic acid as immune system stimulators.
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http://dx.doi.org/10.3390/vaccines7040142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963534PMC
October 2019

Commensal gut microbiota can modulate adaptive immune responses in chickens vaccinated with whole inactivated avian influenza virus subtype H9N2.

Vaccine 2019 10 18;37(44):6640-6647. Epub 2019 Sep 18.

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Electronic address:

Variations in the composition of commensal gut microbiota have been reported to be major contributors to differences in responses to vaccination among individuals. In chickens, there is limited information on the role of gut microbiota in responses to vaccination. The current study studied the role of gut microbiota in cell- and antibody-mediated immune responses to vaccination with a whole inactivated avian influenza virus, subtype H9N2. A total of 166 one-day-old specific pathogen free layer chickens (SPF) were randomly assigned to treatments, where a combination of antibiotic depletion, and probiotics (a combination of five Lactobacillus species) or fecal microbial transplant (FMT) reconstitution were used to study the dynamics of cell- and antibody-mediated immune responses to primary and secondary vaccinations at days 15 and 29 of age, respectively. Overall, at days 7 and 14 post primary vaccination (p.p.v.), administration of probiotics to non-depleted chickens resulted in significantly higher mean hemagglutination (HI) titre compared to antibiotic treated chickens. Furthermore, at day 21 p.p.v., chickens treated with probiotics or FMT post-antibiotic treatment showed a significantly higher mean HI titre compared to non-depleted chickens treated with probiotics. At day 7 p.p.v., a significantly higher virus specific IgM and IgG titres were observed in non-depleted chickens administered with probiotics compared to antibiotic depleted chickens, and a significantly higher IgG titre was observed in chickens treated with FMT following antibiotic treatment compared to only antibiotic treatment. Analysis of interferon gamma expression in splenocytes to assess cell-mediated immune responses showed a significantly lower expression in antibiotic-treated chickens compared to non-depleted chickens and FMT reconstituted chickens. Taken together, the current study suggests that shifts in the composition of gut microbiota of chickens may result in changes in cell- and antibody-mediated immune responses to vaccination against influenza viruses. Further studies will be needed to highlight the mechanisms involved in this modulation.
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http://dx.doi.org/10.1016/j.vaccine.2019.09.046DOI Listing
October 2019

Identification of a Kinase-Active CheA Conformation in Escherichia coli Chemoreceptor Signaling Complexes.

J Bacteriol 2019 12 5;201(23). Epub 2019 Nov 5.

School of Biological Sciences, University of Utah, Salt Lake City, Utah, USA

chemotaxis relies on control of the autophosphorylation activity of the histidine kinase CheA by transmembrane chemoreceptors. Core signaling units contain two receptor trimers of dimers, one CheA homodimer, and two monomeric CheW proteins that couple CheA activity to receptor control. Core signaling units appear to operate as two-state devices, with distinct kinase-on and kinase-off CheA output states whose structural nature is poorly understood. A recent all-atom molecular dynamic simulation of a receptor core unit revealed two alternative conformations, "dipped" and "undipped," for the ATP-binding CheA.P4 domain that could be related to kinase activity states. To explore possible signaling roles for the dipped CheA.P4 conformation, we created CheA mutants with amino acid replacements at residues (R265, E368, and D372) implicated in promoting the dipped conformation and examined their signaling consequences with Förster resonance energy transfer (FRET)-based kinase assays. We used cysteine-directed cross-linking reporters for the dipped and undipped conformations to assess mutant proteins for these distinct CheA.P4 domain configurations. Phenotypic suppression analyses revealed functional interactions among the conformation-controlling residues. We found that structural interactions between R265, located at the N terminus of the CheA.P3 dimerization domain, and E368/D372 in the CheA.P4 domain played a critical role in stabilizing the dipped conformation and in producing kinase-on output. Charge reversal replacements at any of these residues abrogated the dipped cross-linking signal, CheA kinase activity, and chemotactic ability. We conclude that the dipped conformation of the CheA.P4 domain is critical to the kinase-active state in core signaling units. Regulation of CheA kinase in chemoreceptor arrays is critical for chemotaxis. However, to date, little is known about the CheA conformations that lead to the kinase-on or kinase-off states. Here, we explore the signaling roles of a distinct conformation of the ATP-binding CheA.P4 domain identified by all-atom molecular dynamics simulation. Amino acid replacements at residues predicted to stabilize the so-called "dipped" CheA.P4 conformation abolished the kinase activity of CheA and its ability to support chemotaxis. Our findings indicate that the dipped conformation of the CheA.P4 domain is critical for reaching the kinase-active state in chemoreceptor signaling arrays.
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http://dx.doi.org/10.1128/JB.00543-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6832072PMC
December 2019

Understanding how and why de-implementation works in health and care: research protocol for a realist synthesis of evidence.

Syst Rev 2019 08 5;8(1):194. Epub 2019 Aug 5.

Nottingham University Business School, Nottingham University, Nottingham, UK.

Background: Strategies to improve the effectiveness and quality of health and care have predominantly emphasised the implementation of new research and evidence into service organisation and delivery. A parallel, but less understood issue is how clinicians and service leaders stop existing practices and interventions that are no longer evidence based, where new evidence supersedes old evidence, or interventions are replaced with those that are more cost effective. The aim of this evidence synthesis is to produce meaningful programme theory and practical guidance for policy makers, managers and clinicians to understand how and why de-implementation processes and procedures can work.

Methods And Analysis: The synthesis will examine the attributes or characteristics that constitute the concept of de-implementation. The research team will then draw on the principles of realist inquiry to provide an explanatory account of how, in what context and for whom to explain the successful processes and impacts of de-implementation. The review will be conducted in four phases over 18 months. Phase 1: develop a framework to map the preliminary programme theories through an initial scoping of the literature and consultation with key stakeholders. Phase 2: systematic searches of the evidence to develop the theories identified in phase 1. Phase 3: validation and refinement of programme theories through stakeholder interviews. Phase 4: formulating actionable recommendations for managers, commissioners and service leaders about what works through different approaches to de-implementation.

Discussion: This evidence synthesis will address gaps in knowledge about de-implementation across health and care services and ensure that guidance about strategies and approaches accounts for contextual factors, which may be operating at different organisational and decision-making levels. Through the development of the programme theory, which explains what works, how and under which circumstances, findings from the evidence synthesis will support managers and service leaders to make measured decisions about de-implementation.

Systematic Review Registration: PROSPERO CRD42017081030.
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http://dx.doi.org/10.1186/s13643-019-1111-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683493PMC
August 2019

Considerations for maximizing the adaptive potential of restored coral populations in the western Atlantic.

Ecol Appl 2019 12 19;29(8):e01978. Epub 2019 Aug 19.

Department of Biology, Pennsylvania State University, University Park, Pennsylvania, 16803, USA.

Active coral restoration typically involves two interventions: crossing gametes to facilitate sexual larval propagation; and fragmenting, growing, and outplanting adult colonies to enhance asexual propagation. From an evolutionary perspective, the goal of these efforts is to establish self-sustaining, sexually reproducing coral populations that have sufficient genetic and phenotypic variation to adapt to changing environments. Here, we provide concrete guidelines to help restoration practitioners meet this goal for most Caribbean species of interest. To enable the persistence of coral populations exposed to severe selection pressure from many stressors, a mixed provenance strategy is suggested: genetically unique colonies (genets) should be sourced both locally as well as from more distant, environmentally distinct sites. Sourcing three to four genets per reef along environmental gradients should be sufficient to capture a majority of intraspecies genetic diversity. It is best for practitioners to propagate genets with one or more phenotypic traits that are predicted to be valuable in the future, such as low partial mortality, high wound healing rate, high skeletal growth rate, bleaching resilience, infectious disease resilience, and high sexual reproductive output. Some effort should also be reserved for underperforming genets because colonies that grow poorly in nurseries sometimes thrive once returned to the reef and may harbor genetic variants with as yet unrecognized value. Outplants should be clustered in groups of four to six genets to enable successful fertilization upon maturation. Current evidence indicates that translocating genets among distant reefs is unlikely to be problematic from a population genetic perspective but will likely provide substantial adaptive benefits. Similarly, inbreeding depression is not a concern given that current practices only raise first-generation offspring. Thus, proceeding with the proposed management strategies even in the absence of a detailed population genetic analysis of the focal species at sites targeted for restoration is the best course of action. These basic guidelines should help maximize the adaptive potential of reef-building corals facing a rapidly changing environment.
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http://dx.doi.org/10.1002/eap.1978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916196PMC
December 2019

Conformational shifts in a chemoreceptor helical hairpin control kinase signaling in .

Proc Natl Acad Sci U S A 2019 07 17;116(31):15651-15660. Epub 2019 Jul 17.

School of Biological Sciences, University of Utah, Salt Lake City, UT 84112

Motile cells use chemoreceptor signaling arrays to track chemical gradients with exquisite precision. Highly conserved residues in the cytoplasmic hairpin tip of chemoreceptor molecules promote assembly of trimer-based signaling complexes and modulate the activity of their CheA kinase partners. To explore hairpin tip output states in the serine receptor Tsr, we characterized the signaling consequences of amino acid replacements at the salt-bridge residue pair E385-R388. All mutant receptors assembled trimers and signaling complexes, but most failed to support serine chemotaxis in soft agar assays. Small side-chain replacements at either residue produced OFF- or ON-shifted outputs that responded to serine stimuli in wild-type fashion, suggesting that these receptors, like the wild-type, operate as two-state signaling devices. Larger aliphatic or aromatic side chains caused slow or partial kinase control responses that proved dependent on the connections between core signaling units that promote array cooperativity. In a mutant lacking one of two key adapter-kinase contacts (interface 2), those mutant receptors exhibited more wild-type behaviors. Lastly, mutant receptors with charged amino acid replacements assembled signaling complexes that were locked in kinase-ON (E385K|R) or kinase-OFF (R388D|E) output. The hairpin tips of mutant receptors with these more aberrant signaling properties probably have nonnative structures or dynamic behaviors. Our results suggest that chemoeffector stimuli and adaptational modifications influence the cooperative connections between core signaling units. This array remodeling process may involve activity-dependent changes in the relative strengths of interface 1 and 2 interactions between the CheW and CheA.P5 components of receptor core signaling complexes.
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http://dx.doi.org/10.1073/pnas.1902521116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681711PMC
July 2019

Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States.

mBio 2019 07 2;10(4). Epub 2019 Jul 2.

Institute of Biology, Leiden University, Leiden, The Netherlands

Tsr, the serine chemoreceptor in , transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture. In , membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.
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http://dx.doi.org/10.1128/mBio.00973-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6606802PMC
July 2019

Inter-partner specificity limits the acquisition of thermotolerant symbionts in a model cnidarian-dinoflagellate symbiosis.

ISME J 2019 10 11;13(10):2489-2499. Epub 2019 Jun 11.

School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, 6140, New Zealand.

The ability of corals and other cnidarians to survive climate change depends partly on the composition of their endosymbiont communities. The dinoflagellate family Symbiodiniaceae is genetically and physiologically diverse, and one proposed mechanism for cnidarians to acclimate to rising temperatures is to acquire more thermally tolerant symbionts. However, cnidarian-dinoflagellate associations vary in their degree of specificity, which may limit their capacity to alter symbiont communities. Here, we inoculated symbiont-free polyps of the sea anemone Exaiptasia pallida (commonly referred to as 'Aiptasia'), a model system for the cnidarian-dinoflagellate symbiosis, with simultaneous or sequential mixtures of thermally tolerant and thermally sensitive species of Symbiodiniaceae. We then monitored symbiont success (relative proportional abundance) at normal and elevated temperatures across two to four weeks. All anemones showed signs of bleaching at high temperature. During simultaneous inoculations, the native, thermally sensitive Breviolum minutum colonized polyps most successfully regardless of temperature when paired against the non-native but more thermally tolerant Symbiodinium microadriaticum or Durusdinium trenchii. Furthermore, anemones initially colonized with B. minutum and subsequently exposed to S. microadriaticum failed to acquire the new symbiont. These results highlight how partner specificity may place strong limitations on the ability of certain cnidarians to acquire more thermally tolerant symbionts, and hence their adaptive potential under climate change.
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http://dx.doi.org/10.1038/s41396-019-0429-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776018PMC
October 2019

Sequence-Selective Minor Groove Recognition of a DNA Duplex Containing Synthetic Genetic Components.

J Am Chem Soc 2019 06 4;141(24):9555-9563. Epub 2019 Jun 4.

Department of Pure and Applied Chemistry , University of Strathclyde , Thomas Graham Building, 295 Cathedral Street , Glasgow G1 1XL , United Kingdom.

The structural basis of minor groove recognition of a DNA duplex containing synthetic genetic information by hairpin pyrrole-imidazole polyamides is described. Hairpin polyamides induce a higher melting stabilization of a DNA duplex containing the unnatural P·Z base-pair when an imidazole unit is aligned with a P nucleotide. An NMR structural study showed that the incorporation of two isolated P·Z pairs enlarges the minor groove and slightly narrows the major groove at the site of this synthetic genetic information, relative to a DNA duplex consisting entirely of Watson-Crick base-pairs. Pyrrole-imidazole polyamides bind to a P·Z-containing DNA duplex to form a stable complex, effectively mimicking a G·C pair. A structural hallmark of minor groove recognition of a P·Z pair by a polyamide is the reduced level of allosteric distortion induced by binding of a polyamide to a DNA duplex. Understanding the molecular determinants that influence minor groove recognition of DNA containing synthetic genetic components provides the basis to further develop unnatural base-pairs for synthetic biology applications.
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http://dx.doi.org/10.1021/jacs.8b12444DOI Listing
June 2019
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