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Crystallophores are lanthanide complexes that have demonstrated outstanding induction of crystallization for various proteins. This article explores the effect of tailored modifications of the crystallophore first generation and their impact on the nucleating properties and protein crystal structures. Through high-throughput crystallization experiments and dataset analysis, we evaluated the effectiveness of these variants, in comparison to the first crystallophore generation G1. In particular, the V1 variant, featuring a propanol pendant arm, demonstrated the ability to produce new crystallization conditions for the proteins tested (hen-egg white lysozyme, proteinase K and thaumatin). Structural analysis performed in the case of hen egg-white lysozyme along with Molecular Dynamics simulations, highlights V1's unique behavior, taking advantage of the flexibility of its propanol arm to explore different protein surfaces and form versatile supramolecular interactions.
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Simulación de Dinámica Molecular , Muramidasa , Muramidasa/química , Muramidasa/metabolismo , Endopeptidasa K/química , Endopeptidasa K/metabolismo , Elementos de la Serie de los Lantanoides/química , Cristalización , Animales , Cristalografía por Rayos X , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Pollos , Proteínas/química , Proteínas/metabolismo , Complejos de Coordinación/químicaRESUMEN
In this work, we experimentally investigate the potency of high pressure to drive a protein toward an excited state where an inhibitor targeted for this state can bind. Ras proteins are small GTPases cycling between active GTP-bound and inactive GDP-bound states. Various states of GTP-bound Ras in active conformation coexist in solution, amongst them, state 2 which binds to effectors, and state 1, weakly populated at ambient conditions, which has a low affinity for effectors. Zn2+-cyclen is an allosteric inhibitor of Ras protein, designed to bind specifically to the state 1. In H-Ras(wt).Mg2+.GppNHp crystals soaked with Zn2+-cyclen, no binding could be observed, as expected in the state 2 conformation which is the dominant state at ambient pressure. Interestingly, Zn2+-cyclen binding is observed at 500â MPa pressure, close to the nucleotide, in Ras protein that is driven by pressure to a state 1 conformer. The unknown binding mode of Zn2+-cyclen to H-Ras can thus be fully characterized in atomic details. As a more general conjunction from our study, high pressure x-ray crystallography turns out to be a powerful method to induce transitions allowing drug binding in proteins that are in low-populated conformations at ambient conditions, enabling the design of specific inhibitors.
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Ciclamas , Zinc , Zinc/química , Zinc/metabolismo , Cristalografía por Rayos X , Ciclamas/química , Ciclamas/farmacología , Regulación Alostérica , Presión , Unión Proteica , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Humanos , Sitios de UniónRESUMEN
Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.
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Evidence of stable liquid water oceans beneath the ice crust of moons within the Solar System is of great interest for astrobiology. In particular, subglacial oceans may present hydrothermal processes in their abysses, similarly to terrestrial hydrothermal vents. Therefore, terrestrial extremophilic deep life can be considered a model for putative icy moon extraterrestrial life. However, the comparison between putative extraterrestrial abysses and their terrestrial counterparts suffers from a potentially determinant difference. Indeed, some icy moons oceans may be so deep that the hydrostatic pressure would exceed the maximal pressure at which hydrothermal vent organisms have been isolated. While terrestrial microorganisms that are able to survive in such conditions are known, the effect of high pressure on fundamental biochemical processes is still unclear. In this study, the effects of high hydrostatic pressure on DNA synthesis catalyzed by DNA polymerases are investigated for the first time. The effect on both strand displacement and primer extension activities is measured, and pressure tolerance is compared between enzymes of various thermophilic organisms isolated at different depths.
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Luna , Agua , Polimerizacion , Agua/química , Exobiología , ADNRESUMEN
Lactate dehydrogenase (LDH, EC.1.1.127) is an important enzyme engaged in the anaerobic metabolism of cells, catalyzing the conversion of pyruvate to lactate and NADH to NAD+. LDH is a relevant enzyme to investigate structure-function relationships. The present work provides the missing link in our understanding of the evolution of LDHs. This allows to explain (i) the various evolutionary origins of LDHs in eukaryotic cells and their further diversification and (ii) subtle phenotypic modifications with respect to their regulation capacity. We identified a group of cyanobacterial LDHs displaying eukaryotic-like LDH sequence features. The biochemical and structural characterization of Cyanobacterium aponinum LDH, taken as representative, unexpectedly revealed that it displays homotropic and heterotropic activation, typical of an allosteric enzyme, whereas it harbors a long N-terminal extension, a structural feature considered responsible for the lack of allosteric capacity in eukaryotic LDHs. Its crystallographic structure was solved in 2 different configurations typical of the R-active and T-inactive states encountered in allosteric LDHs. Structural comparisons coupled with our evolutionary analyses helped to identify 2 amino acid positions that could have had a major role in the attenuation and extinction of the allosteric activation in eukaryotic LDHs rather than the presence of the N-terminal extension. We tested this hypothesis by site-directed mutagenesis. The resulting C. aponinum LDH mutants displayed reduced allosteric capacity mimicking those encountered in plants and human LDHs. This study provides a new evolutionary scenario of LDHs that unifies descriptions of regulatory properties with structural and mutational patterns of these important enzymes.
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L-Lactato Deshidrogenasa , Lactato Deshidrogenasas , Humanos , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismoRESUMEN
High pressure is a convenient thermodynamic parameter to probe the dynamics of proteins as it is intimately related to volume which is essential for protein function. To be biologically active, a protein fluctuates between different substates. Pressure perturbation can promote some hidden substates by modifying the population between them. High pressure macromolecular crystallography (HPMX) is a perfect tool to capture and to characterize such substates at a molecular level providing new insights on protein dynamics. The present chapter describes the use of the diamond anvil cell to perform HPMX experiments. It also provides tips on sample preparation and optimal data collection as well as on efficient analysis of the resulting high-pressure structures.
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Manejo de Especímenes , Cristalografía , Sustancias Macromoleculares , TermodinámicaRESUMEN
Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl2 , CaCl2 and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl2 for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments.
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Haloferax volcanii , Marte , Proteoma , Sales (Química) , Medio Ambiente Extraterrestre , Haloferax volcanii/genética , BiomarcadoresRESUMEN
Small-angle X-ray scattering (SAXS) has become an indispensable tool in structural biology, complementing atomic-resolution techniques. It is sensitive to the electron-density difference between solubilized biomacromolecules and the buffer, and provides information on molecular masses, particle dimensions and interactions, low-resolution conformations and pair distance-distribution functions. When SAXS data are recorded at multiple contrasts, i.e. at different solvent electron densities, it is possible to probe, in addition to their overall shape, the internal electron-density profile of biomacromolecular assemblies. Unfortunately, contrast-variation SAXS has been limited by the range of solvent electron densities attainable using conventional co-solutes (for example sugars, glycerol and salt) and by the fact that some biological systems are destabilized in their presence. Here, SAXS contrast data from an oligomeric protein and a protein-RNA complex are presented in the presence of iohexol and Gd-HPDO3A, two electron-rich molecules that are used in biomedical imaging and that belong to the families of iodinated and lanthanide-based complexes, respectively. Moderate concentrations of both molecules allowed solvent electron densities matching those of proteins to be attained. While iohexol yielded higher solvent electron densities (per mole), it interacted specifically with the oligomeric protein and precipitated the protein-RNA complex. Gd-HPDO3A, while less efficient (per mole), did not disrupt the structural integrity of either system, and atomic models could be compared with the SAXS data. Due to their elevated solubility and electron density, their chemical inertness, as well as the possibility of altering their physico-chemical properties, lanthanide-based complexes represent a class of molecules with promising potential for contrast-variation SAXS experiments on diverse biomacromolecular systems.
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Medios de Contraste , Elementos de la Serie de los Lantanoides , Yohexol , Proteínas/química , ARN/química , Dispersión del Ángulo Pequeño , Solventes , Difracción de Rayos XRESUMEN
Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.
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Bacillus thuringiensis , Nanopartículas , Animales , Proteínas Bacterianas/toxicidad , Endotoxinas , Proteínas Hemolisinas/toxicidad , Larva , Control de MosquitosRESUMEN
The proteasome system allows the elimination of functional or structurally impaired proteins. This includes the degradation of nascent peptides. In Archaea, how the proteasome complex interacts with the translational machinery remains to be described. Here, we characterized a small orphan protein, Q9UZY3 (UniProt ID), conserved in Thermococcales. The protein was identified in native pull-down experiments using the proteasome regulatory complex (proteasome-activating nucleotidase [PAN]) as bait. X-ray crystallography and small-angle X-ray scattering experiments revealed that the protein is monomeric and adopts a ß-barrel core structure with an oligonucleotide/oligosaccharide-binding (OB)-fold, typically found in translation elongation factors. Mobility shift experiment showed that Q9UZY3 displays transfer ribonucleic acid (tRNA)-binding properties. Pull-downs, co-immunoprecipitation and isothermal titration calorimetry (ITC) studies revealed that Q9UZY3 interacts in vitro with PAN. Native pull-downs and proteomic analysis using different versions of Q9UZY3 showed that the protein interacts with the assembled PAN-20S proteasome machinery in Pyrococcus abyssi (Pa) cellular extracts. The protein was therefore named Pbp11, for Proteasome-Binding Protein of 11 kDa. Interestingly, the interaction network of Pbp11 also includes ribosomal proteins, tRNA-processing enzymes and exosome subunits dependent on Pbp11's N-terminal domain that was found to be essential for tRNA binding. Together these data suggest that Pbp11 participates in an interface between the proteasome and the translational machinery.
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Proteínas Arqueales , Complejo de la Endopetidasa Proteasomal , Archaea/metabolismo , Proteínas Arqueales/metabolismo , Proteínas Portadoras , Cristalografía por Rayos X , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica , ARN de TransferenciaRESUMEN
In this work, we experimentally investigate the allosteric transitions between conformational states on the Ras oncogene protein using high pressure crystallography. Ras protein is a small GTPase involved in central regulatory processes occurring in multiple conformational states. Ras acts as a molecular switch between active GTP-bound, and inactive GDP-bound states, controlling essential signal transduction pathways. An allosteric network of interactions between the effector binding regions and the membrane interacting regions is involved in Ras cycling. The conformational states which coexist simultaneously in solution possess higher Gibbs free energy than the ground state. Equilibria between these states can be shifted by applying pressure favouring conformations with lower partial molar volume, and has been previously analyzed by high-pressure NMR spectroscopy. High-pressure macromolecular crystallography (HPMX) is a powerful tool perfectly complementary to high-pressure NMR, allowing characterization at the molecular level with a high resolution the different allosteric states involved in the Ras cycling. We observe a transition above 300 MPa in the crystal leading to more stable conformers. Thus, we compare the crystallographic structures of Ras(wt)·Mg2+·GppNHp and Ras(D33K)·Mg2+·GppNHp at various high hydrostatic pressures. This gives insight into per-residue descriptions of the structural plasticity involved in allosteric equilibria between conformers. We have mapped out at atomic resolution the different segments of Ras protein which remain in the ground-state conformation or undergo structural changes, adopting excited-energy conformations corresponding to transient intermediate states. Such in crystallo phase transitions induced by pressure open the possibility to finely explore the structural determinants related to switching between Ras allosteric sub-states without any mutations nor exogenous partners.
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The recent discovery of extrasolar Earth-like planets that orbit in their habitable zone of their system, and the latest clues of the presence of liquid water in the subsurface of Mars and in the subglacial ocean of Jupiter's and Saturn's moons, has reopened debates about habitability and limits of life. Although liquid water, widely accepted as an absolute requirement for terrestrial life, may be present in other bodies of the solar system or elsewhere, physical and chemical conditions, such as temperature, pressure, and salinity, may limit this habitability. However, extremophilic microorganisms found in various extreme terrestrial environments are adapted to thrive in permanently extreme ranges of physicochemical conditions. This review first describes promising environments for life in the Solar System and the microorganisms that inhabit similar environments on the Earth. The effects of extreme temperatures, salt, and hydrostatic pressure conditions on biomolecules will be explained in some detail, and recent advances in understanding biophysical and structural adaptation strategies allowing microorganisms to cope with extreme physicochemical conditions are reviewed to discuss promising environments for life in the Solar System in terms of habitability.
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Exobiología , Extremófilos , Planeta Tierra , Medio Ambiente Extraterrestre , PlanetasRESUMEN
The stability of the tetrameric enzyme urate oxidase in complex with excess of 8-azaxanthine was investigated either under high hydrostatic pressure per se or under a high pressure of argon. The active site is located at the interface of two subunits, and the catalytic activity is directly related to the integrity of the tetramer. This study demonstrates that applying pressure to a protein-ligand complex drives the thermodynamic equilibrium towards ligand saturation of the complex, revealing a new binding site. A transient dimeric intermediate that occurs during the pressure-induced dissociation process was characterized under argon pressure and excited substates of the enzyme that occur during the catalytic cycle can be trapped by pressure. Comparison of the different structures under pressure infers an allosteric role of the internal hydrophobic cavity in which argon is bound, since this cavity provides the necessary flexibility for the active site to function.
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Urato Oxidasa , Argón , Sitios de Unión , Presión Hidrostática , Ligandos , Urato Oxidasa/química , Urato Oxidasa/metabolismoRESUMEN
Adaption to environmental conditions is reflected by protein adaptation. In particular, proteins of extremophiles display distinctive traits ensuring functional, structural and dynamical properties under permanently extreme physical and chemical conditions. While it has mostly been studied with approaches focusing on specific proteins, biophysical approaches have also confirmed this link between environmental and protein adaptation at the more complex and diverse scale of the proteome. However, studies of this type remain challenging and often require large amounts of biological material. We report here the use of nanoDSF as a tool to study proteome stability and solubility in cell lysates of the model halophilic archaeon Haloarcula marismortui. Notably, our results show that, as with single halophilic protein studies, proteome stability was correlated to the concentration of NaCl or KCl under which the cells were lysed and hence the proteome exposed. This work highlights that adaptation to environmental conditions can be experimentally observed at the scale of the proteome. Still, we show that the biochemical properties of single halophilic proteins can only be partially extrapolated to the whole proteome.
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Halobacteriales , Proteoma , Adaptación Fisiológica , Haloarcula marismortui , Cloruro de SodioRESUMEN
The use of lanthanide complexes as powerful auxiliaries for biocrystallography prompted us to systematically analyze the influence of the commercial crystallization kit composition on the efficiency of two lanthanide additives: [Eu(DPA)3]3- and Tb-Xo4. This study reveals that the tris(dipicolinate) complex presents a lower chemical stability and a strong tendency toward false positives, which are detrimental for its use in a high-throughput robotized crystallization platform. In particular, the crystal structures of (Mg(H2O)6)3[Eu(DPA)3]2·7H2O (1), {(Ca(H2O)4)3[Eu(DPA)3]2}n·10nH2O (2), and {Cu(DPA)(H2O)2}n (3), resulting from spontaneous crystallization in the presence of a divalent alkaline-earth cation and transmetalation, are reported. On the other hand, Tb-Xo4 is perfectly soluble in the crystallization media, stable in the presence of alkaline-earth dications, and slowly decomposes (within days) by transmetalation with transition metals. The original structure of [Tb4L4(H2O)4]Cl4·15H2O (4) is also described, where L represents a bis(pinacolato)triazacyclononane ligand. This paper also highlights a potential synergy of interactions between Tb-Xo4 and components of the crystallization mixtures, leading to the formation of complex adducts like {AdkA/Tb-Xo4/Mg2+/glycerol} in the protein binding sites. The observation of such multicomponent adducts illustrated the complexity and versatility of the supramolecular chemistry occurring at the surface of the proteins.
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Cationes Bivalentes/química , Complejos de Coordinación/química , Elementos de la Serie de los Lantanoides/química , Cristalografía por Rayos X , Modelos Moleculares , Estructura Molecular , Tamaño de la PartículaRESUMEN
In this work, we combined biochemical and structural investigations with molecular dynamics (MD) simulations to analyze the very different thermal-dependent allosteric behavior of two lactate dehydrogenases (LDH) from thermophilic bacteria. We found that the enzyme from Petrotoga mobilis (P. mob) necessitates an absolute requirement of the allosteric effector (fructose 1, 6-bisphosphate) to ensure functionality. In contrast, even without allosteric effector, the LDH from Thermus thermophilus (T. the) is functional when the temperature is raised. We report the crystal structure of P. mob LDH in the Apo state solved at 1.9 Å resolution. We used this structure and the one from T. the, obtained previously, as a starting point for MD simulations at various temperatures. We found clear differences between the thermal dynamics, which accounts for the behavior of the two enzymes. Our work demonstrates that, within an allosteric enzyme, some areas act as local gatekeepers of signal transmission, allowing the enzyme to populate either the T-inactive or the R-active states with different degrees of stringency.
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Extremófilos , Lactato Deshidrogenasas , Regulación Alostérica , Extremófilos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Thermus thermophilusRESUMEN
Extreme halophilic Archaea thrive in high salt, where, through proteomic adaptation, they cope with the strong osmolarity and extreme ionic conditions of their environment. In spite of wide fundamental interest, however, studies providing insights into this adaptation are scarce, because of practical difficulties inherent to the purification and characterization of halophilic enzymes. In this work, we describe the evolutionary history of malate dehydrogenases (MalDH) within Halobacteria (a class of the Euryarchaeota phylum). We resurrected nine ancestors along the inferred halobacterial MalDH phylogeny, including the Last Common Ancestral MalDH of Halobacteria (LCAHa) and compared their biochemical properties with those of five modern halobacterial MalDHs. We monitored the stability of these various MalDHs, their oligomeric states and enzymatic properties, as a function of concentration for different salts in the solvent. We found that a variety of evolutionary processes, such as amino acid replacement, gene duplication, loss of MalDH gene and replacement owing to horizontal transfer resulted in significant differences in solubility, stability and catalytic properties between these enzymes in the three Halobacteriales, Haloferacales, and Natrialbales orders since the LCAHa MalDH. We also showed how a stability trade-off might favor the emergence of new properties during adaptation to diverse environmental conditions. Altogether, our results suggest a new view of halophilic protein adaptation in Archaea.
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Euryarchaeota , Halobacterium , Malatos , Filogenia , ProteómicaRESUMEN
BACKGROUND: Since December 2016, the basic military training (BMT) facility for the Canadian Armed Forces (CAF) has experienced repeated outbreaks of Group A Streptococcus (GAS). In 2018, a voluntary mass antibiotic prophylaxis (MAP) program was implemented to interrupt GAS transmission among recruits. The objective of this study was to describe the epidemiology of three GAS outbreaks and a period of increased pharyngitis infections at the CAF BMT facility in Québec over a two-year span, and to detail the prevention and control measures implemented to mitigate the risk to recruit health. METHODS: Descriptive data were collected on invasive and severe GAS cases along with laboratory data including genotyping of throat swabs from recruits presenting with pharyngitis. A laboratory-based acute respiratory infection surveillance system was used to aid in monitoring and decision-making. Close contacts of recruits were assessed for asymptomatic GAS carriage and MAP adverse events surveillance was conducted. RESULTS: Three distinct GAS outbreaks occurred at the Canadian Forces Leadership and Recruit School totaling eight invasive (iGAS) and 13 severe (sGAS) cases over two years. All iGAS/sGAS cases, apart from one instructor, were among recruits. The predominant strain in all three outbreaks was type emm6.4. A total of 11,293 recruits received MAP (penicillin G benzathine or azithromycin) between March 7, 2018 and November 18, 2019. There were eight reported serious adverse events related to penicillin administration. CONCLUSION: The CAF BMT facility experienced three GAS outbreaks over the course of two years, and despite the use of enhanced hygiene measures, only MAP has been effective in quelling these outbreaks.
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BACKGROUND: Between December 2016 and March 2018, two outbreaks of Group A Streptococcus (GAS) infection occurred at the Canadian Forces Leadership and Recruit School. A voluntary mass antibiotic prophylaxis (MAP) program was implemented in March 2018, to interrupt an ongoing GAS outbreak, and to prevent future outbreaks. METHODS: Instructors and recruits were offered a one-time intramuscular injection of 1.2 million units penicillin G benzathine (PGB). Individuals with a penicillin allergy were offered azithromycin; 500 mg orally once weekly for four consecutive weeks. Instructors and recruits were also asked to complete a voluntary and anonymous survey one week after receipt of MAP, to detect MAP-related adverse events. RESULTS: MAP was offered to 2,749 individuals; 2,707 of whom agreed to receive it (98.5% uptake). The majority of personnel experienced adverse events in the days following MAP; 92.3% of personnel who received PGB reported localized pain at the injection site, and 70.2% of personnel who received azithromycin reported gastrointestinal symptoms. However, only five cases of serious adverse events were reported, and less than 1% of recruits could not complete their basic military training course because of MAP-related adverse events. CONCLUSION: The MAP program implemented in March 2018 was the first of its kind in the Canadian Armed Forces, and the largest single use of PGB in a defined group in Canada. It resulted in very few serious adverse events and with minimal impact on military recruits' successful completion of recruit training.
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F420H2-oxidase (FprA) catalyses the four-electron reduction of O2 to 2H2O using the reduced form of F420 as electron donor. The hydrophobic O2-channel detected by Kr-derivatization and the concerted movement of a gating loop could contribute to prevent unwanted side-reaction between the catalytic intermediates and solvents, therefore preventing reactive oxygen species formation.