Direct Expression of Fluorinated Proteins in Human Cells for 19F In-Cell NMR Spectroscopy

: In-cell NMR spectroscopy is a powerful approach to study protein structure and function in the native cellular environment. It provides precious insights into the folding, maturation, interactions, and ligand binding of important pharmacological targets directly in human cells. However, its widespread application is hampered by the fact that soluble globular proteins often interact with large cellular components, causing severe line broadening in conventional heteronuclear NMR experiments. 19F NMR can overcome this issue, as fluorine atoms incorporated in proteins can be detected by simple background-free 1D NMR spectra. Here, we show that fluorinated amino acids can be easily incorporated in proteins expressed in human cells by employing a medium switch strategy. This straightforward approach allows the incorporation of different fluorinated amino acids in the protein of interest, reaching fluorination efficiencies up to 60%, as confirmed by mass spectrometry and X-ray crystallography. The versatility of the approach is shown by performing 19F in-cell NMR on several proteins, including those that would otherwise be invisible by 1H-15N in-cell NMR. We apply the approach to observe the interaction between an intracellular target, carbonic anhydrase 2, and its inhibitors, and to investigate how the formation of a complex between superoxide dismutase 1 and its chaperone CCS modulates the interaction of the chaperone subunit with the cellular environment

New Synthetic Analogues of Natural Polyphenols as Sirtuin 1-Activating Compounds

NAD+-dependent deacetylase SIRT1 regulates many different biological processes, thus being involved in pathogenic conditions such as metabolic diseases, neurogenerative disorders and cancer. Notably, experimental evidence underlined that the activation of SIRT1 had promising cardioprotective effects. Consequently, many efforts have been so far devoted to finding new SIRT1 activators, both derived from natural sources or prepared by synthetic procedures. Herein, we discovered new SIRT1-activating derivatives, characterized by phenolic rings spaced by sulfur, nitrogen or oxygen-based central linkers. The newly synthesized derivatives were analyzed in enzymatic assays to determine their ability to activate SIRT1, as compared with that of resveratrol. Among the tested molecules, bisarylaniline compound 10 proved to be the most efficient SIRT1 activator. An evaluation of the effects caused by focused structural variations revealed that its para-hydroxy-substituted diphenyl moiety of 10 was the fundamental structural requirement for achieving good SIRT1 activation. Compound 10 was further investigated in ex vivo studies in isolated and perfused rat hearts submitted to ischemia/reperfusion (I/R), where it showed significant protection of the myocardium against I/R injury. Molecular modeling studies suggest the binding mode of 10 within SIRT1 in the presence of the p53-AMC peptide. Our findings reveal that this chemical scaffold may be used as the starting point to develop a new class of more potent SIRT1 activators as cardioprotective agents

The Rewiring of Ubiquitination Targets in a Pathogenic Yeast Promotes Metabolic Flexibility, Host Colonization and Virulence

Funding: This work was funded by the European Research Council [http://erc.europa.eu/], AJPB (STRIFE Advanced Grant; C-2009-AdG-249793). The work was also supported by: the Wellcome Trust [www.wellcome.ac.uk], AJPB (080088, 097377); the UK Biotechnology and Biological Research Council [www.bbsrc.ac.uk], AJPB (BB/F00513X/1, BB/K017365/1); the CNPq-Brazil [http://cnpq.br], GMA (Science without Borders fellowship 202976/2014-9); and the National Centre for the Replacement, Refinement and Reduction of Animals in Research [www.nc3rs.org.uk], DMM (NC/K000306/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgments We thank Dr. Elizabeth Johnson (Mycology Reference Laboratory, Bristol) for providing strains, and the Aberdeen Proteomics facility for the biotyping of S. cerevisiae clinical isolates, and to Euroscarf for providing S. cerevisiae strains and plasmids. We are grateful to our Microscopy Facility in the Institute of Medical Sciences for their expert help with the electron microscopy, and to our friends in the Aberdeen Fungal Group for insightful discussions.Peer reviewedPublisher PD

Cinnamides Target Leishmania amazonensis Arginase Selectively

Caffeic acid and related natural compounds were previously described as Leishmania amazonensis arginase (L-ARG) inhibitors, and against the whole parasite in vitro. In this study, we tested cinnamides that were previously synthesized to target human arginase. The compound caffeic acid phenethyl amide (CAPA), a weak inhibitor of human arginase (IC50 = 60.3 ± 7.8 μM) was found to have 9-fold more potency against L-ARG (IC50 = 6.9 ± 0.7 μM). The other compounds that did not inhibit human arginase were characterized as L-ARG, showing an IC50 between 1.3-17.8 μM, and where the most active was compound 15 (IC50 = 1.3 ± 0.1 μM). All compounds were also tested against L. amazonensis promastigotes, and only the compound CAPA showed an inhibitory activity (IC50 = 80 μM). In addition, in an attempt to gain an insight into the mechanism of competitive L-ARG inhibitors, and their selectivity over mammalian enzymes, we performed an extensive computational investigation, to provide the basis for the selective inhibition of L-ARG for this series of compounds. In conclusion, our results indicated that the compounds based on cinnamoyl or 3,4-hydroxy cinnamoyl moiety could be a promising starting point for the design of potential antileishmanial drugs based on selective L-ARG inhibitors

The information for catching fly balls: judging and intercepting virtual balls in a CAVE

Visually guided action implies the existence of information as well as a control law relating that information to movement. For ball catching, the Chapman Strategy - keeping constant the rate of change of the tangent of the elevation angle (d(tan(α))/dt) - leads a catcher to the right location at the right time to intercept a fly ball. Previous studies showed the ability to detect the information and the consistency of running patterns with the use of the strategy. However, only direct manipulation of information can show its use. Participants were asked to intercept virtual balls in a Cave Automated Virtual Environment (CAVE) or to judge whether balls would pass behind or in front of them. Catchers in the CAVE successfully intercepted virtual balls with their forehead. Furthermore, the timing of judgments was related to the patterns of changing d(tan(α))/dt. The advantages and disadvantages of a CAVE as a tool for studying interceptive action are discussed

Multicentre randomised placebo-controlled trial of oral anticoagulation with apixaban in systemic sclerosis-related pulmonary arterial hypertension: the SPHInX study protocol

Introduction: Systemic sclerosis (SSc) is a severe and costly multiorgan autoimmune connective tissue disease characterised by vasculopathy and fibrosis. One of the major causes of SSc-related death is pulmonary arterial hypertension (PAH), which develops in 12–15% of patients with SSc and accounts for 30– 40% of deaths. In situ thrombosis in the small calibre peripheral pulmonary vessels resulting from endothelial dysfunction and an imbalance of anticoagulant and prothrombotic mediators has been implicated in the complex pathophysiology of SSc-related PAH (SSc- PAH), with international clinical guidelines recommending the use of anticoagulants for some types of PAH, such as idiopathic PAH. However, anticoagulation has not become part of standard clinical care for patients with SSc-PAH as only observational evidence exists to support its use. Therefore, we present the rationale and methodology of a phase III randomised controlled trial (RCT) to evaluate the efficacy, safety and cost-effectiveness of anticoagulation in SSc-PAH. Methods and analysis: This Australian multicentre RCT will compare 2.5 mg apixaban with placebo, in parallel treatment groups randomised in a 1:1 ratio, both administered twice daily for 3 years as adjunct therapy to stable oral PAH therapy. The composite primary outcome measure will be the time to death or clinical worsening of PAH. Secondary outcomes will include functional capacity, health-related quality of life measures and adverse events. A cost-effectiveness analysis of anticoagulation versus placebo will also be undertaken. Ethics and dissemination: Ethical approval for this RCT has been granted by the Human Research Ethics Committees of all participating centres. An independent data safety monitoring board will review safety and tolerability data for the duration of the trial. The findings of this RCT are to be published in open access journals.Alicia Calderone, Wendy Stevens, David Prior, Harshal Nandurkar, Eli Gabbay, Susanna M Proudman, Trevor Williams, David Celermajer, Joanne Sahhar, Peter K K Wong, Vivek Thakkar, Nathan Dwyer, Jeremy Wrobel, Weng Chin, Danny Liew, Margaret Staples, Rachelle Buchbinder, Mandana Nikpou

A Variable-Density Absorption Event in NGC 3227 mapped with Suzaku and Swift

The morphology of the circumnuclear gas accreting onto supermassive black holes in Seyfert galaxies remains a topic of much debate. As the innermost regions of Active Galactic Nuclei (AGN) are spatially unresolved, X-ray spectroscopy, and in particular line-of-sight absorption variability, is a key diagnostic to map out the distribution of gas. Observations of variable X-ray absorption in multiple Seyferts and over a wide range of timescales indicate the presence of clumps/clouds of gas within the circumnuclear material. Eclipse events by clumps transiting the line of sight allow us to explore the properties of the clumps over a wide range of radial distances from the optical/UV Broad Line Region (BLR) to beyond the dust sublimation radius. Time-resolved absorption events have been extremely rare so far, but suggest a range of density profiles across Seyferts. We resolve a weeks-long absorption event in the Seyfert NGC 3227. We examine six Suzaku and twelve Swift observations from a 2008 campaign spanning 5 weeks. We use a model accounting for the complex spectral interplay of three differently-ionized absorbers. We perform time-resolved spectroscopy to discern the absorption variability behavior. We also examine the IR-to-X-ray spectral energy distribution (SED) to test for reddening by dust. The 2008 absorption event is due to moderately-ionized ($\log \xi\sim 1.2-1.4$) gas covering 90% of the line of sight. We resolve the density profile to be highly irregular, in contrast to a previous symmetric and centrally-peaked event mapped with RXTE in the same object. The UV data do not show significant reddening, suggesting that the cloud is dust-free. The 2008 campaign has revealed a transit by a filamentary, moderately-ionized cloud of variable density that is likely located in the BLR, and possibly part of a disk wind.Comment: Accepted for publication by A&

One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

A four-helix bundle stores copper for methane oxidation

Methane-oxidising bacteria (methanotrophs) require large quantities of copper for the membrane-bound (particulate) methane monooxygenase (pMMO). Certain methanotrophs are also able to switch to using the iron-containing soluble MMO (sMMO) to catalyse methane oxidation, with this switchover regulated by copper. MMOs are Nature’s primary biological mechanism for suppressing atmospheric levels of methane, a potent greenhouse gas. Furthermore, methanotrophs and MMOs have enormous potential in bioremediation and for biotransformations producing bulk and fine chemicals, and in bioenergy, particularly considering increased methane availability from renewable sources and hydraulic fracturing of shale rock. We have discovered and characterised a novel copper storage protein (Csp1) from the methanotroph Methylosinus trichosporium OB3b that is exported from the cytosol, and stores copper for pMMO. Csp1 is a tetramer of 4-helix bundles with each monomer binding up to 13 Cu(I) ions in a previously unseen manner via mainly Cys residues that point into the core of the bundle. Csp1 is the first example of a protein that stores a metal within an established protein-folding motif. This work provides a detailed insight into how methanotrophs accumulate copper for the oxidation of methane. Understanding this process is essential if the wide-ranging biotechnological applications of methanotrophs are to be realised. Cytosolic homologues of Csp1 are present in diverse bacteria thus challenging the dogma that such organisms do not use copper in this location

Spatial effects, sampling errors, and task specialization in the honey bee

Task allocation patterns should depend on the spatial distribution of work within the nest, variation in task demand, and the movement patterns of workers, however, relatively little research has focused on these topics. This study uses a spatially explicit agent based model to determine whether such factors alone can generate biases in task performance at the individual level in the honey bees, Apis mellifera. Specialization (bias in task performance) is shown to result from strong sampling error due to localized task demand, relatively slow moving workers relative to nest size, and strong spatial variation in task demand. To date, specialization has been primarily interpreted with the response threshold concept, which is focused on intrinsic (typically genotypic) differences between workers. Response threshold variation and sampling error due to spatial effects are not mutually exclusive, however, and this study suggests that both contribute to patterns of task bias at the individual level. While spatial effects are strong enough to explain some documented cases of specialization; they are relatively short term and not explanatory for long term cases of specialization. In general, this study suggests that the spatial layout of tasks and fluctuations in their demand must be explicitly controlled for in studies focused on identifying genotypic specialists