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

Multicopy plasmid integration in Komagataella phaffii mediated by a defective auxotrophic marker

Background: A commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele. Results: A K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth. Conclusions: In this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii

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

Developing and Implementing a Multisource Feedback Tool to Assess Competencies of Emergency Medicine Residents in the United States

Objectives: Multisource feedback (MSF) has potential value in learner assessment, but has not been broadly implemented nor studied in emergency medicine (EM). This study aimed to adapt existing MSF instruments for emergency department implementation, measure feasibility, and collect initial validity evidence to support score interpretation for learner assessment. Methods: Residents from eight U.S. EM residency programs completed a self-assessment and were assessed by eight physicians, eight nonphysician colleagues, and 25 patients using unique instruments. Instruments included a five-point rating scale to assess interpersonal and communication skills, professionalism, systems-based practice, practice-based learning and improvement, and patient care. MSF feasibility was measured by percentage of residents who collected the target number of instruments. To develop internal structure validity evidence, Cronbach\u27s alpha was calculated as a measure of internal consistency. Results: A total of 125 residents collected a mean of 7.0 physician assessments (n = 752), 6.7 nonphysician assessments (n = 775), and 17.8 patient assessments (n = 2,100) with respective response rates of 67.2, 75.2, and 77.5%. Cronbach\u27s alpha values for physicians, nonphysicians, patients, and self were 0.97, 0.97, 0.96, and 0.96, respectively. Conclusions: This study demonstrated that MSF implementation is feasible, although challenging. The tool and its scale demonstrated excellent internal consistency. EM educators may find the adaptation process and tools applicable to their learners