11 research outputs found
Assessing Interactions Between a Polytopic Membrane Protein and Lipid Bilayers Using Differential Scanning Calorimetry and Solid-State NMR
It is known that the lipid composition
within a cellular membrane
can influence membrane protein structure and function. In this Article,
we investigated how structural changes to a membrane protein upon
substrate binding can impact the lipid bilayer. To carry out this
study, we reconstituted the secondary active drug transporter EmrE
into a variety of phospholipid bilayers varying in headgroup and chain
length and carried out differential scanning calorimetry (DSC) and
solid-state NMR experiments. The DSC results revealed a difference
in cooperativity of the lipid phase transition for drug-free EmrE
protonated at glutamic acid 14 (i.e., proton-loaded form) and the
tetraphenylphosphonium (TPP<sup>+</sup>) bound form of the protein
(i.e., drug-loaded form). To complement these findings, we acquired
magic-angle-spinning (MAS) spectra in the presence and absence of
TPP<sup>+</sup> by directly probing the phospholipid headgroup using <sup>31</sup>P NMR. These spectra showed a reduction in lipid line widths
around the main phase transition for samples where EmrE was bound
to TPP<sup>+</sup> compared to the drug free form. Finally, we collected
oriented solid-state NMR spectra on isotopically enriched EmrE that
displayed chemical shift perturbations to both transmembrane and loop
residues upon TPP<sup>+</sup> binding. All of these results prompt
us to propose a mechanism whereby substrate-induced changes to the
structural dynamics of EmrE alters the surrounding lipids within the
bilayer
Intrinsic Conformational Plasticity of Native EmrE Provides a Pathway for Multidrug Resistance
EmrE is a multidrug resistance efflux
pump with specificity to
a wide range of antibiotics and antiseptics. To obtain atomic-scale
insight into the attributes of the native state that encodes the broad
specificity, we used a hybrid of solution and solid-state NMR methods
in lipid bilayers and bicelles. Our results indicate that the native
EmrE dimer oscillates between inward and outward facing structural
conformations at an exchange rate (<i>k</i><sub>ex</sub>) of ∼300 s<sup>–1</sup> at 37 °C (millisecond
motions), which is ∼50-fold faster relative to the tetraÂphenylÂphosphoÂnium
(TPP<sup>+</sup>) substrate-bound form of the protein. These observables
provide quantitative evidence that the rate-limiting step in the TPP<sup>+</sup> transport cycle is not the outward–inward conformational
change in the absence of drug. In addition, using differential scanning
calorimetry, we found that the width of the gel-to-liquid crystalline
phase transition was 2 °C broader in the absence of the TPP<sup>+</sup> substrate versus its presence, which suggested that changes
in transporter dynamics can impact the phase properties of the membrane.
Interestingly, experiments with cross-linked EmrE showed that the
millisecond inward-open to outward-open dynamics was not the culprit
of the broadening. Instead, the calorimetry and NMR data supported
the conclusion that faster time scale structural dynamics (nanosecond–microsecond)
were the source and therefore impart the conformationally plastic
character of native EmrE capable of binding structurally diverse substrates.
These findings provide a clear example how differences in membrane
protein transporter structural dynamics between drug-free and bound
states can have a direct impact on the physical properties of the
lipid bilayer in an allosteric fashion
Development of glandular models from human nasal progenitor cells.
Hyperplasia/hypertrophy of submucosal glands contributes to mucus overproduction in chronic diseases of the upper and lower respiratory tracts, especially in adult and pediatric chronic rhinosinusitis. Mechanisms that lead to glandular hyperplasia/hypertrophy are markedly understudied, reflecting a lack of in vitro model systems wherein airway epithelial progenitor cells differentiate into glandular cells. In this study, we developed and compared several in vitro three-dimensional systems using human nasal epithelial basal cells (HNEBCs) cultured by different methods on two types of extracellular matrices. We demonstrate that HNEBCs cultured on Matrigel (Corning, Tewksbury, MA) form glandular acini-like structures, whereas HNEBCs embedded in a collagen type I matrix form a network of tubules. Fibroblast-conditioned medium increases tubule formation in collagen type I. In contrast, HNEBCs cocultured with fibroblasts self-aggregate into organotypic structures with tubules and acini. These observations provide morphological evidence that HNEBCs are pluripotent and retain the capacity to differentiate into structures resembling specific structural components of submucosal glands depending on the extracellular matrices and culture conditions. The resultant models should prove useful in targeting cross-talk between epithelial cells and fibroblasts to decipher molecular mechanisms and specific signals responsible for the development of glandular hyperplasia/hypertrophy, which in turn may lead to new therapeutic strategies for chronic rhinosinusitis and other inflammatory respiratory diseases characterized by glandular hyperplasia/hypertrophy
Evaluation of Communication and Safety Behaviors During Hospital-Wide Code Response Simulation
INTRODUCTION: To understand the baseline quality of team communication behaviors at our organization, we implemented institution-wide simulation training and measured the performance of safety behaviors of ad hoc teams in emergent situations. METHODS: Clinicians participated in 2 interprofessional video-recorded simulation scenarios, each followed by debriefing. Using a standardized evaluation instrument, 2 reviewers independently evaluated the presence or absence of desired team safety behaviors, including escalating care, sharing a mental model, establishing leadership, thinking out loud, and identifying roles and responsibilities. We also scored the quality of sharing the mental model, closed-loop communication, and overall team performance on a 7-point scale. Discordant reviews were resolved with scoring by an additional reviewer. RESULTS: A total of 1404 clinicians participated in 398 simulation scenarios, resulting in 257 usable videos. Overall, teams exhibited desired behaviors at the following frequencies: escalating care, 85%; sharing mental models, 66%; verbally establishing leadership, 6%; thinking out loud, 87%; and identifying roles and responsibilities, 27%. Across all reviews, the quality of the graded behaviors (of 7 points) was 2.8 for shared mental models, 3.3 for closed-loop communication, and 3.2 for overall team performance. CONCLUSIONS: In a simulation setting with ad hoc teams, there was variable performance on completing safety behaviors and only a fair quality of graded communication behaviors. These results establish a baseline assessment of communication and teamwork behaviors and will guide future quality improvement interventions
Evaluation of Communication and Safety Behaviors During Hospital-Wide Code Response Simulation
INTRODUCTION: To understand the baseline quality of team communication behaviors at our organization, we implemented institution-wide simulation training and measured the performance of safety behaviors of ad hoc teams in emergent situations. METHODS: Clinicians participated in 2 interprofessional video-recorded simulation scenarios, each followed by debriefing. Using a standardized evaluation instrument, 2 reviewers independently evaluated the presence or absence of desired team safety behaviors, including escalating care, sharing a mental model, establishing leadership, thinking out loud, and identifying roles and responsibilities. We also scored the quality of sharing the mental model, closed-loop communication, and overall team performance on a 7-point scale. Discordant reviews were resolved with scoring by an additional reviewer. RESULTS: A total of 1404 clinicians participated in 398 simulation scenarios, resulting in 257 usable videos. Overall, teams exhibited desired behaviors at the following frequencies: escalating care, 85%; sharing mental models, 66%; verbally establishing leadership, 6%; thinking out loud, 87%; and identifying roles and responsibilities, 27%. Across all reviews, the quality of the graded behaviors (of 7 points) was 2.8 for shared mental models, 3.3 for closed-loop communication, and 3.2 for overall team performance. CONCLUSIONS: In a simulation setting with ad hoc teams, there was variable performance on completing safety behaviors and only a fair quality of graded communication behaviors. These results establish a baseline assessment of communication and teamwork behaviors and will guide future quality improvement interventions