Metal selectivity and translocation mechanism characterization in proteoliposomes of the transmembrane NiCoT transporter NixA from Helicobacter pylori

Essential trace metals play key roles in the survival, replication, and virulence of bacterial pathogens. Helicobacter pylori (H. pylori), the main bacterial cause of gastric ulcers, requires Ni(ii) to colonize and persist in the acidic environment inside the stomach, exploiting the nickel-containing enzyme urease to catalyze the hydrolysis of urea to ammonia and bicarbonate and create a pH-buffered microenvironment. Urease utilizes Ni(ii) as a catalytic cofactor for its activity. In ureolytic bacteria, unique transmembrane (TM) transporters evolved to guarantee the selective uptake and efflux of Ni(ii) across cellular membranes to meet the cellular requirements. NixA is an essential Ni(ii) transporter expressed by H. pylori when the extracellular environment experiences a drop in pH. This Class I nickel-cobalt transporter of the NiCoT family catalyzes the uptake of Ni(ii) across the inner membrane from the periplasm. In this study, we characterized NixA using a platform whereby, for the first time on a NiCoT transporter, recombinantly expressed and purified NixA and key mutants in the translocation pathway have been reconstituted in artificial lipid bilayer vesicles (proteoliposomes). Fluorescent sensors responsive to Ni(ii) transport (Fluozin-3-Zn(ii)), luminal pH changes (pyranine), and membrane potential (oxonol VI) were encapsulated in the proteoliposomes lumen to monitor, in real-time, NixA transport properties and translocation mechanism. Kinetic transport analysis revealed that NixA is highly selective for Ni(ii) with no substrate promiscuity towards Co(ii), the other putative metal substrate of the NiCoT family, nor Zn(ii). NixA-mediated Ni(ii) transport exhibited a Michaelis-Menten-type saturable substrate concentration dependence, with an experimental KM, Ni(ii) = 31.0 ± 1.2 μM. Ni(ii) transport by NixA was demonstrated to be electrogenic, and metal translocation did not require a proton motive force, resulting in the generation of a positive-inside transmembrane potential in the proteoliposome lumen. Mutation analysis characterized key transmembrane residues for substrate recognition, binding, and/or transport, suggesting the presence of a three-step transmembrane translocation conduit. Taken together, these investigations reveal that NixA is a Ni(ii)-selective Class I NiCoT electrogenic uniporter. The work also provides an in vitro approach to characterize the transport properties of metal transporters responsible for Ni(ii) acquisition and extrusion in prokaryotes

Do All Lives Have the Same Value? Support for International Military Interventions as a Function of Political System and Public Opinion of the Target States

This research examined the support for international military interventions as a function of the political system and the public opinion of the target country. In two experiments, we informed participants about a possible military intervention by the international community towards a sovereign country whose government planned to use military force against a secessionist region. They were then asked whether they would support this intervention whilst being reminded that it would cause civilian deaths. The democratic or nondemocratic political system of the target country was experimentally manipulated, and the population support for its belligerent government policy was either assessed (Experiment 1) or manipulated (Experiment 2). Results showed greater support for the intervention when the target country was nondemocratic, as compared to the democratic and the control conditions, but only when its population supported the belligerent government policy. Support for the external intervention was low when the target country was democratic, irrespective of national public opinion. These findings provide support for the democracy-as-value hypothesis applied to international military interventions, and suggest that civilian deaths (collateral damage) are more acceptable when nondemocratic populations support their government's belligerent policy

Detection and elimination of cellular bottlenecks in protein-producing yeasts

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented

Autoinjectors Preferred for Intramuscular Epinephrine in Anaphylaxis and Allergic Reactions

Introduction: Epinephrine is the treatment of choice for anaphylaxis. We surveyed emergency department (ED) health care providers regarding 2 methods of intramuscular (IM) epinephrine administration (autoinjector and manual injection) for the management of anaphylaxis and allergic reactions and identified provider perceptions and preferred method of medication delivery.Methods: This observational study adhered to survey reporting guidelines. It was performed through a Web-based survey completed by health care providers at an academic ED. The participants consisted of all ED providers, including staff physicians, resident physicians, pharmacists, advanced practice providers, and nurses. The primary outcomes were assessment of provider perceptions and identification of the preferred IM epinephrine administration method by ED health care providers.Results: Of 217 ED health care providers invited to participate, 172 (79%) completed the survey. Overall, 82% of respondents preferred the autoinjector method of epinephrine administration. Providers rated the autoinjector method more favorably with regard to time required for training, ease of use, convenience, satisfaction with weight-based dosing, risk of dosing errors, and speed of administration (P<.001 for all comparisons). However, manual injection use was rated more favorably with regard to risk of provider self-injury and patient cost (P<.001 for both comparisons). Three participants (2%) reported a finger stick injury from an epinephrine autoinjector.Conclusion: ED health care providers preferred the autoinjector method of IM epinephrine administration for the management of anaphylaxis or allergic reactions. Epinephrine autoinjector use may reduce barriers to epinephrine administration for the management of anaphylaxis in the ED