Mg-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are major drivers of multidrug resistance among gram-negative bacteria. Cations, such as Mg, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell. Here, we reveal an interplay between Mg and pH in modulating the structural dynamics of the periplasmic adapter protein, AcrA, and its function within the prototypical AcrAB-TolC multidrug pump from Escherichia coli.

Mg-dependent mechanism of environmental versatility in a multidrug efflux pump.

Tripartite resistance nodulation and cell division multidrug efflux pumps span the periplasm and are a major driver of multidrug resistance among Gram-negative bacteria. The periplasm provides a distinct environment between the inner and outer membranes of Gram-negative bacteria. Cations, such as Mg, become concentrated within the periplasm and, in contrast to the cytoplasm, its pH is sensitive to conditions outside the cell.

Systematic evaluation of benzoylation for liquid chromatography-mass spectrometry analysis of different analyte classes.

LC-MS is an indispensable tool for small molecule analysis in many fields; however, many small molecules require chemical derivatization to improve retention on commonly used reversed-phase columns and increase ionization. Benzoyl chloride (BzCl) derivatization is commonly used for derivatization of primary and secondary amines and phenolic alcohols, though evidence exists that with proper reaction conditions (i.e.

3D Printing in a hospital: Centralized clinical implementation and applications for comprehensive care.

This educational article discusses the use of 3D printing or additive manufacturing in hospitals, not just for rapid prototyping but also for creating end-use products, such as clinical, diagnostic, and educational tools. The flexibility of 3D printing is valuable for creating patient-specific medical devices, custom surgical tools, anatomical models, implants, research tools and on-demand parts, among others. The advantages of and requirements for implementing a clinical 3D printing service in a hospital environment are discussed, including centralized 3D printing management, technology, example use cases, and considerations for implementation.

A phase 1, randomized, placebo-controlled, dose-ranging study to evaluate the safety and immunogenicity of an mRNA-based chikungunya virus vaccine in healthy adults.

Background: Chikungunya, a mosquito-borne viral disease caused by the chikungunya virus (CHIKV), causes a significant global health burden, and there is currently no approved vaccine to prevent chikungunya disease. In this study, the safety and immunogenicity of a CHIKV mRNA vaccine candidate (mRNA-1388) were evaluated in healthy participants in a CHIKV-nonendemic region.

Methods: This phase 1, first-in-human, randomized, placebo-controlled, dose-ranging study enrolled healthy adults (ages 18-49 years) between July 2017 and March 2019 in the United States.

Capillary electrophoresis Western blot using inkjet transfer to membrane.

Traditional Western blots are commonly used to separate and assay proteins; however, they have limitations including a long, cumbersome process and large sample requirements. Here, we describe a system for Western blotting where capillary gel electrophoresis is used to separate sodium dodecyl sulfate-protein complexes. The capillary outlet is threaded into a piezoelectric inkjetting head that deposits the separated proteins in a quasi-continuous stream of <100 pL droplets onto a moving membrane.

Methods to study folding of alpha-helical membrane proteins in lipids.

How alpha-helical membrane proteins fold correctly in the highly hydrophobic membrane interior is not well understood. Their folding is known to be highly influenced by the lipids within the surrounding bilayer, but the majority of folding studies have focused on detergent-solubilized protein rather than protein in a lipid environment. There are different ways to study folding in lipid bilayers, and each method has its own advantages and disadvantages.

The Impact on Ambulance Mobilisations of an Increasing Age Profile of Telecare Service Users Receiving Advanced Proactive, Personalised Telecare in Spain-a Longitudinal Study 2014-2018.

Advanced proactive personalised telecare services in Spain have helped service users to live independently in their own homes for longer. Concern was however noted regarding potential impacts on ambulance mobilisations as time in the service, and mean age at cessation, increased. The purpose of this study was to investigate these impacts.

How lipids affect the energetics of co-translational alpha helical membrane protein folding.

Membrane proteins need to fold with precision in order to function correctly, with misfolding potentially leading to disease. The proteins reside within a hydrophobic lipid membrane and must insert into the membrane and fold correctly, generally whilst they are being translated by the ribosome. Favourable and unfavourable free energy contributions are present throughout each stage of insertion and folding.

Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein.

The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome.

Cell-Free Synthesis Strategies to Probe Co-translational Folding of Proteins Within Lipid Membranes.

In order to comprehend the molecular basis of transmembrane protein biogenesis, methods are required that are capable of investigating the co-translational folding of these hydrophobic proteins. Equally, in artificial cell studies, controllable methods are desirable for in situ synthesis of membrane proteins that then direct reactions in the synthetic cell membrane. Here we describe a method that exploits cell-free expression systems and tunable membrane mimetics to facilitate co-translational studies.

Integrating Membrane Transporter Proteins into Droplet Interface Bilayers.

Droplet interface bilayers (DIBs) are an emerging tool within synthetic biology that aims to recreate biological processes in artificial cells. A critical component for the utility of these bilayers is controlled flow between compartments and, notably, uphill transport against a substrate concentration gradient. A versatile method to achieve the desired flow is to exploit the specificity of membrane proteins that regulate the movement of ions and transport of specific metabolic compounds.

The membrane transporter lactose permease increases lipid bilayer bending rigidity.

Cellular life relies on membranes, which provide a resilient and adaptive cell boundary. Many essential processes depend upon the ease with which the membrane is able to deform and bend, features that can be characterized by the bending rigidity. Quantitative investigations of such mechanical properties of biological membranes have primarily been undertaken in solely lipid bilayers and frequently in the absence of buffers.

Activating mechanosensitive channels embedded in droplet interface bilayers using membrane asymmetry.

Droplet microcompartments linked by lipid bilayers show great promise in the construction of synthetic minimal tissues. Central to controlling the flow of information in these systems are membrane proteins, which can gate in response to specific stimuli in order to control the molecular flux between membrane separated compartments. This has been demonstrated with droplet interface bilayers (DIBs) using several different membrane proteins combined with electrical, mechanical, and/or chemical activators.

Membrane protein mediated bilayer communication in networks of droplet interface bilayers.

Droplet interface bilayers (DIBs) are model membranes formed between lipid monolayer-encased water droplets in oil. Compared to conventional methods, one of the most unique properties of DIBs is that they can be connected together to generate multi-layered 'tissue-like' networks, however introducing communication pathways between these compartments typically relies on water-soluble pores that are unable to gate. Here, we show that network connectivity can instead be achieved using a water-insoluble membrane protein by successfully reconstituting a chemically activatable mutant of the mechanosensitive channel MscL into a network of DIBs.

Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer.

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells.

Dehydration in older people: A systematic review of the effects of dehydration on health outcomes, healthcare costs and cognitive performance.

Objective: To systematically examine the effect of dehydration on health outcomes, identify associated financial costs and consider impacts on cognitive performance in older adults.

Design: A systematic review of English-language articles via OVID using MEDLINE, PsychINFO, EMBASE, and others, to March 2018. Included studies examined the relationship between hydration status and health, care costs or cognitive outcome.

Hydrogen-deuterium exchange mass spectrometry captures distinct dynamics upon substrate and inhibitor binding to a transporter.

Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE.

Capturing Membrane Protein Ribosome Nascent Chain Complexes in a Native-like Environment for Co-translational Studies.

Co-translational folding studies of membrane proteins lag behind cytosolic protein investigations largely due to the technical difficulty in maintaining membrane lipid environments for correct protein folding. Stalled ribosome-bound nascent chain complexes (RNCs) can give snapshots of a nascent protein chain as it emerges from the ribosome during biosynthesis. Here, we demonstrate how SecM-facilitated nascent chain stalling and native nanodisc technologies can be exploited to capture -generated membrane protein RNCs within their native lipid compositions.

Cell-free expression tools to study co-translational folding of alpha helical membrane transporters.

Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins.

Delineating the Rules for Structural Adaptation of Membrane-Associated Proteins to Evolutionary Changes in Membrane Lipidome.

Membrane function is fundamental to life. Each species explores membrane lipid diversity within a genetically predefined range of possibilities. How membrane lipid composition in turn defines the functional space available for evolution of membrane-centered processes remains largely unknown.

The use of bacterial polysaccharides in bioprinting.

Additive manufacturing or 3D printing has spearheaded a revolution in the biomedical sector allowing the rapid prototyping of medical devices. The recent advancements in bioprinting technology are enabling the development of potential new therapeutic options with respect to tissue engineering and regenerative medicines. Bacterial polysaccharides have been shown to be a central component of the inks used in a variety of bioprinting processes influencing their key features such as the mechanical and thermal properties, printability, biocompatibility, and biodegradability.