NADH oxidation drives respiratory Na+ transport in mitochondria from Yarrowia lipolytica

It is generally assumed that respiratory complexes exclusively use protons to energize the inner mitochondrial membrane. Here we show that oxidation of NADH by submitochondrial particles (SMPs) from the yeast Yarrowia lipolytica is coupled to protonophore-resistant Na+ uptake, indicating that a redox-driven, primary Na+ pump is operative in the inner mitochondrial membrane. By purification and reconstitution into proteoliposomes, a respiratory NADH dehydrogenase was identified which coupled NADH-dependent reduction of ubiquinone (1.4μmolmin−1mg−1) to Na+ translocation (2.0μmolmin−1mg−1). NADH-driven Na+ transport was sensitive towards rotenone, a specific inhibitor of complex I. We conclude that mitochondria from Y. lipolytica contain a NADH-driven Na+ pump and propose that it represents the complex I of the respiratory chain. Our study indicates that energy conversion by mitochondria does not exclusively rely on the proton motive force but may benefit from the electrochemical Na+ gradient established by complex

Transport of Na+ and K+ by an antiporter-related subunit from the Escherichia coli NADH dehydrogenase I produced in Saccharomyces cerevisiae

The NADH dehydrogenase I from Escherichia coli is a bacterial homolog of the mitochondrial complex I which translocates Na+ rather than H+. To elucidate the mechanism of Na+ transport, the C-terminally truncated NuoL subunit (NuoLN) which is related to Na+/H+ antiporters was expressed as a protein A fusion protein (ProtA-NuoLN) in the yeast Saccharomyces cerevisiae which lacks an endogenous complex I. The fusion protein inserted into membranes from the endoplasmatic reticulum (ER), as confirmed by differential centrifugation and Western analysis. Membrane vesicles containing ProtA-NuoLN catalyzed the uptake of Na+ and K+ at rates which were significantly higher than uptake by the control vesicles under identical conditions, demonstrating that ProtA-NuoLN translocated Na+ and K+ independently from other complex I subunits. Na+ transport by ProtA-NuoLN was inhibited by EIPA (5-(N-ethyl-N-isopropyl)-amiloride) which specifically reacts with Na+/H+ antiporters. The cation selectivity and function of the NuoL subunit as a transporter module of the NADH dehydrogenase complex is discusse

Vibrio natriegens as Host for Expression of Multisubunit Membrane Protein Complexes

Escherichia coli is a convenient host for the expression of proteins, but the heterologous production of large membrane protein complexes often is hampered by the lack of specific accessory genes required for membrane insertion or cofactor assembly. In this study we introduce the non-pathogenic and fast-growing Vibrio natriegens as a suitable expression host for membrane-bound proteins from Vibrio cholerae. We achieved production of the primary Na+ pump, the NADH:quinone oxidoreductase (NQR), from V. cholerae in an active state, as indicated by increased overall NADH:quinone oxidoreduction activity of membranes from the transformed V. natriegens, and the sensitivity toward Ag+, a specific inhibitor of the NQR. Complete assembly of V. cholerae NQR expressed in V. natriegens was demonstrated by BN PAGE followed by activity staining. The secondary transport system Mrp from V. cholerae, another membrane-bound multisubunit complex, was also produced in V. natriegens in a functional state, as demonstrated by in vivo Li+ transport. V. natriegens is a promising expression host for the production of membrane protein complexes from Gram-negative pathogens

Proceedings of Abstracts Engineering and Computer Science Research Conference 2019

© 2019 The Author(s). This is an open-access work distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. For further details please see https://creativecommons.org/licenses/by/4.0/. Note: Keynote: Fluorescence visualisation to evaluate effectiveness of personal protective equipment for infection control is © 2019 Crown copyright and so is licensed under the Open Government Licence v3.0. Under this licence users are permitted to copy, publish, distribute and transmit the Information; adapt the Information; exploit the Information commercially and non-commercially for example, by combining it with other Information, or by including it in your own product or application. Where you do any of the above you must acknowledge the source of the Information in your product or application by including or linking to any attribution statement specified by the Information Provider(s) and, where possible, provide a link to this licence: http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/This book is the record of abstracts submitted and accepted for presentation at the Inaugural Engineering and Computer Science Research Conference held 17th April 2019 at the University of Hertfordshire, Hatfield, UK. This conference is a local event aiming at bringing together the research students, staff and eminent external guests to celebrate Engineering and Computer Science Research at the University of Hertfordshire. The ECS Research Conference aims to showcase the broad landscape of research taking place in the School of Engineering and Computer Science. The 2019 conference was articulated around three topical cross-disciplinary themes: Make and Preserve the Future; Connect the People and Cities; and Protect and Care

Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na⁺-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

The Na⁺ translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na⁺-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na⁺-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na⁺-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na⁺ pump(s) can compensate for Na⁺ pumping activity of Na⁺-NQR. Overall, our study provides important insights into the contribution of Na⁺-NQR to V. cholerae physiology

Evaluation of the long-term efficacy and safety of an imidacloprid 10%/flumethrin 4.5% polymer matrix collar (Seresto®) in dogs and cats naturally infested with fleas and/or ticks in multicentre clinical field studies in Europe

<p>Abstract</p> <p>Background</p> <p>The objective of these two GCP multicentre European clinical field studies was to evaluate the long-term efficacy and safety of a new imidacloprid/flumethrin collar (Seresto^®, Bayer AnimalHealth, Investigational Veterinary Product(IVP)) in dogs and cats naturally infested with fleas and/or ticks in comparison to a dimpylat collar ("Ungezieferband fuer Hunde/fuer Katzen", Beaphar, Control Product (CP)).</p> <p>Methods</p> <p>232 (IVP) and 81 (CP) cats and 271(IVP) and 129 (CP) dogs were treated with either product according to label claims and formed the safety population. Flea and tick counts were conducted in monthly intervals for up to 8 months in the efficacy subpopulation consisting of 118 (IVP) + 47 (CP) cats and 197 (IVP) + 94 (CP) dogs. Efficacy was calculated as reduction of infestation rate within the same treatment group and statistically compared between the two treatment groups.</p> <p>Results</p> <p>Preventive efficacy against fleas in cats/dogs varied in the IVP group between 97.4%/94.1% and 100%/100% (overall mean: 98.3%/96.7%) throughout the 8 month period and in the CP group between 57.1%/28.2% and 96.1%/67.8% (overall mean: 79.3%/57.9%). Preventive efficacy against ticks in cats/dogs varied in the IVP group between 94.0%/91.2% and 100%/100% (overall mean: 98.4%/94.7%) throughout the 8 month period and in the CP group between 90.7%/79.9% and 100%/88.0% (overall mean: 96.9%/85.6%). The IVP group was statistically non-inferior to the CP group, and on various assessment days, statistical superiority was proven for flea and tick count reduction in dogs and cats. Both treatments proved to be safe in dogs and cats with mainly minor local observations at the application site. There was moreover, no incidence of any mechanical problem with the collar in dogs and cats during the entire study period.</p> <p>Conclusions</p> <p>The imidacloprid/flumethrin collar proved to reduce tick counts by at least 90% and flea counts by at least 95% for a period of at least 7-8 months in cats and dogs under field conditions. Therefore, it can be used as sustainable long-term preventative, covering the whole flea and tick season.</p

26th Annual Computational Neuroscience Meeting (CNS*2017): Part 3 - Meeting Abstracts - Antwerp, Belgium. 15–20 July 2017

This work was produced as part of the activities of FAPESP Research,\ud Disseminations and Innovation Center for Neuromathematics (grant\ud 2013/07699-0, S. Paulo Research Foundation). NLK is supported by a\ud FAPESP postdoctoral fellowship (grant 2016/03855-5). ACR is partially\ud supported by a CNPq fellowship (grant 306251/2014-0)

25th annual computational neuroscience meeting: CNS-2016

The same neuron may play different functional roles in the neural circuits to which it belongs. For example, neurons in the Tritonia pedal ganglia may participate in variable phases of the swim motor rhythms [1]. While such neuronal functional variability is likely to play a major role the delivery of the functionality of neural systems, it is difficult to study it in most nervous systems. We work on the pyloric rhythm network of the crustacean stomatogastric ganglion (STG) [2]. Typically network models of the STG treat neurons of the same functional type as a single model neuron (e.g. PD neurons), assuming the same conductance parameters for these neurons and implying their synchronous firing [3, 4]. However, simultaneous recording of PD neurons shows differences between the timings of spikes of these neurons. This may indicate functional variability of these neurons. Here we modelled separately the two PD neurons of the STG in a multi-neuron model of the pyloric network. Our neuron models comply with known correlations between conductance parameters of ionic currents. Our results reproduce the experimental finding of increasing spike time distance between spikes originating from the two model PD neurons during their synchronised burst phase. The PD neuron with the larger calcium conductance generates its spikes before the other PD neuron. Larger potassium conductance values in the follower neuron imply longer delays between spikes, see Fig. 17.Neuromodulators change the conductance parameters of neurons and maintain the ratios of these parameters [5]. Our results show that such changes may shift the individual contribution of two PD neurons to the PD-phase of the pyloric rhythm altering their functionality within this rhythm. Our work paves the way towards an accessible experimental and computational framework for the analysis of the mechanisms and impact of functional variability of neurons within the neural circuits to which they belong