A Dynamic Core in Human NQO1 Controls the Functional and Stability Effects of Ligand Binding and Their Communication across the Enzyme Dimer

Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson’s and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale

Guidance for family about comfort care in dementia: a comparison of an educational booklet adopted in six jurisdictions over a 15 year timespan

BackgroundTo support family caregivers of people with dementia in end-of-life decision making, a family booklet on comfort care has been adapted and adopted by several European jurisdictions since the original publication in Canada in 2005.MethodsWe analyzed and compared the adaptations to the family booklets used in Canada, the Czech Republic, Italy, the Netherlands, the UK and Ireland that were made up to 2021. Qualitative content analysis was used to create a typology of changes to the original booklet. Interviews with the teams that adapted the booklets contributed to methodological triangulation. Further, using an established framework, we assessed whether the contents of the booklets addressed all domains relevant to optimal palliative dementia care.ResultsThe booklets differed in the types of treatment addressed, in particular tube feeding, euthanasia, and spiritual care. There was also variability in the extent to which medical details were provided, an emphasis on previously expressed wishes in medical decision making, addressing of treatment dilemmas at the end of life, the tone of the messages (indirect or explicit) and the discussion of prognosis (as more or less positive), and the involvement of various healthcare professionals and family caregivers in care. All booklets addressed all domains of palliative dementia care.ConclusionsWe identified core elements in providing information on end-of-life care to family caregivers of people with dementia as related to optimal palliative care in dementia. Additionally, local adaptations and updates are required to account for socio-cultural, clinical, and legal differences which may also change over time. These results may inform development of educational and advance care planning materials for different contexts.</div

Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L\u2192G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity ( 4820 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance

A Very Intense Neutrino Super Beam Experiment for Leptonic CP Violation Discovery based on the European Spallation Source Linac: A Snowmass 2013 White Paper

Very intense neutrino beams and large neutrino detectors will be needed in order to enable the discovery of CP violation in the leptonic sector. We propose to use the proton linac of the European Spallation Source currently under construction in Lund, Sweden to deliver, in parallel with the spallation neutron production, a very intense, cost effective and high performance neutrino beam. The baseline program for the European Spallation Source linac is that it will be fully operational at 5 MW average power by 2022, producing 2 GeV 2.86 ms long proton pulses at a rate of 14 Hz. Our proposal is to upgrade the linac to 10 MW average power and 28 Hz, producing 14 pulses/s for neutron production and 14 pulses/s for neutrino production. Furthermore, because of the high current required in the pulsed neutrino horn, the length of the pulses used for neutrino production needs to be compressed to a few $\mu$s with the aid of an accumulator ring. A long baseline experiment using this Super Beam and a megaton underground Water Cherenkov detector located in existing mines 300-600 km from Lund will make it possible to discover leptonic CP violation at 5 $\sigma$ significance level in up to 50% of the leptonic Dirac CP-violating phase range. This experiment could also determine the neutrino mass hierarchy at a significance level of more than 3 $\sigma$ if this issue will not already have been settled by other experiments by then. The mass hierarchy performance could be increased by combining the neutrino beam results with those obtained from atmospheric neutrinos detected by the same large volume detector. This detector will also be used to measure the proton lifetime, detect cosmological neutrinos and neutrinos from supernova explosions. Results on the sensitivity to leptonic CP violation and the neutrino mass hierarchy are presented.Comment: 28 page

Synchronization of the Distributed Readout Frontend Electronics of the Baby MIND Detector

Baby MIND is a new downstream muon range detector for the WGASCI experiment. This article discusses the distributed readout system and its timing requirements. The paper presents the design of the synchronization subsystem and the results of its test

Baby MIND: A magnetised spectrometer for the WAGASCI experiment

The WAGASCI experiment being built at the J-PARC neutrino beam line will measure the difference in cross sections from neutrinos interacting with a water and scintillator targets, in order to constrain neutrino cross sections, essential for the T2K neutrino oscillation measurements. A prototype Magnetised Iron Neutrino Detector (MIND), called Baby MIND, is being constructed at CERN to act as a magnetic spectrometer behind the main WAGASCI target to be able to measure the charge and momentum of the outgoing muon from neutrino charged current interactions.Comment: Poster presented at NuPhys2016 (London, 12-14 December 2016). Title + 4 pages, LaTeX, 6 figure

Baby MIND Experiment Construction Status

Baby MIND is a magnetized iron neutrino detector, with novel design features, and is planned to serve as a downstream magnetized muon spectrometer for the WAGASCI experiment on the T2K neutrino beam line in Japan. One of the main goals of this experiment is to reduce systematic uncertainties relevant to CP-violation searches, by measuring the neutrino contamination in the anti-neutrino beam mode of T2K. Baby MIND is currently being constructed at CERN, and is planned to be operational in Japan in October 2017.Comment: Poster presented at NuPhys2016 (London, 12-14 December 2016). 4 pages, LaTeX, 7 figure

Baby MIND: A magnetized segmented neutrino detector for the WAGASCI experiment

T2K (Tokai-to-Kamioka) is a long-baseline neutrino experiment in Japan designed to study various parameters of neutrino oscillations. A near detector complex (ND280) is located 280~m downstream of the production target and measures neutrino beam parameters before any oscillations occur. ND280's measurements are used to predict the number and spectra of neutrinos in the Super-Kamiokande detector at the distance of 295~km. The difference in the target material between the far (water) and near (scintillator, hydrocarbon) detectors leads to the main non-cancelling systematic uncertainty for the oscillation analysis. In order to reduce this uncertainty a new WAter-Grid-And-SCintillator detector (WAGASCI) has been developed. A magnetized iron neutrino detector (Baby MIND) will be used to measure momentum and charge identification of the outgoing muons from charged current interactions. The Baby MIND modules are composed of magnetized iron plates and long plastic scintillator bars read out at the both ends with wavelength shifting fibers and silicon photomultipliers. The front-end electronics board has been developed to perform the readout and digitization of the signals from the scintillator bars. Detector elements were tested with cosmic rays and in the PS beam at CERN. The obtained results are presented in this paper.Comment: In new version: modified both plots of Fig.1 and added one sentence in the introduction part explaining Baby MIND role in WAGASCI experiment, added information for the affiliation

The proposal of a GSSP for the Berriasian Stage (Cretaceous System): Part 1.

Here in the first part of this publication we discuss the possibilities for the selection of a GSSP for the Berriasian Stage of the Cretaceous System, based on the established methods for correlation in the Tithonian/Berriasian interval. This will be followed, in the second part, by an account of the stratigraphic evidence that justifies the locality of Tré Maroua (Hautes-Alpes, SE France) as the proposed GSSP. Here we discuss the possibilities for correlation in the historical J/K boundary interval, and the evolution of thinking on the positioning of the boundary over recent generations, and in relation to research in the last ten years. The Tithonian/Berriasian boundary level is accepted as occurring within magnetosubzone M19n.2n. The detailed distribution of calpionellids has been recorded at numerous sites, tied to magnetostratigraphy, and the base of the calpionellid Alpina Zone is taken to define the base of the Berriasian Stage. This is at a level just below the distinctive reversed magnetic subzone M19n.1r (the so-called Brodno reversal). We discuss a wide range of magnetostratigraphic and biostratigraphic data from key localities globally, in the type Berriasian areas of France and wider regions (Le Chouet, Saint Bertrand, Puerto Escaño, Rio Argos, Bosso, Brodno, Kurovice, Theodosia etc.). The characteristic datums that typify the J/K boundary interval in Tethys and its extensions are detailed, and the correlative viability of various fossil groups is discussed. The boundary level is correlated to well-known J/K sections globally, and a series of secondary markers and proxies are indicated which assist wider correlation. Particularly significant are the primary basal Berriasian marker, the base of the Alpina Subzone (marked by dominance of small Calpionella alpina, Crassicollaria parvula and Tintinopsella carpathica) and secondary markers bracketing the base of the Calpionella Zone, notably the FOs of the calcareous nannofossil species Nannoconus wintereri (just below the boundary) and the FO of Nannoconus steinmannii minor (just above). Notable proxies for the boundary are: 1) the base of the Arctoteuthis tehamaensis Zone in boreal and subboreal regions, 2) the dated base of the Alpina Subzone at 140.22 ± 0.14 Ma, which also gives a precise age estimate for the system boundary; and 3) the base of radiolarian “unitary zone” 14, which is situated just above the base of the Alpina Subzone