Development of a Real-Time Pectic Oligosaccharide-Detecting Biosensor Using the Rapid and Flexible Computational Identification of Non-Disruptive Conjugation Sites (CINC) Biosensor Design Platform.

Fluorescently labeled, solute-binding proteins that change their fluorescent output in response to ligand binding are frequently used as biosensors for a wide range of applications. We have previously developed a "Computational Identification of Non-disruptive Conjugation sites" (CINC) approach, an in silico pipeline utilizing molecular dynamics simulations for the rapid design and construction of novel protein-fluorophore conjugate-type biosensors. Here, we report an improved in silico scoring algorithm for use in CINC and its use in the construction of an oligogalacturonide-detecting biosensor set. Using both 4,5-unsaturated and saturated oligogalacturonides, we demonstrate that signal transmission from the ligand-binding pocket of the starting protein scaffold to the CINC-selected reporter positions is effective for multiple different ligands. The utility of an oligogalacturonide-detecting biosensor is shown in Carbohydrate Active Enzyme (CAZyme) activity assays, where the biosensor is used to follow product release upon polygalacturonic acid (PGA) depolymerization in real time. The oligogalacturonide-detecting biosensor set represents a novel enabling tool integral to our rapidly expanding platform for biosensor-based carbohydrate detection, and moving forward, the CINC pipeline will continue to enable the rational design of biomolecular tools to detect additional chemically distinct oligosaccharides and other solutes.

Construction of a highly selective and sensitive carbohydrate-detecting biosensor utilizing Computational Identification of Non-disruptive Conjugation sites (CINC) for flexible and streamlined biosensor design.

Fluorescently-labeled solute-binding proteins that alter their fluorescence output in response to ligand binding have been utilized as biosensors for a variety of applications. Coupling protein ligand binding to altered fluorescence output often requires trial and error-based testing of both multiple labeling positions and fluorophores to produce a functional biosensor with the desired properties. This approach is laborious and can lead to reduced ligand binding affinity or altered ligand specificity. Here we report the Computational Identification of Non-disruptive Conjugation sites (CINC) for streamlined identification of fluorophore conjugation sites. By exploiting the structural dynamics properties of proteins, CINC identifies positions where conjugation of a fluorophore results in a fluorescence change upon ligand binding without disrupting protein function. We show that a CINC-developed maltooligosaccharide (MOS)-detecting biosensor is capable of rapid (kon = 20 µM-1s-1), sensitive (sub-µM KD) and selective MOS detection. The MOS-detecting biosensor is modular with respect to the spectroscopic properties and demonstrates portability to detecting MOS released via α-amylase-catalyzed depolymerization of starch using both a stopped-flow and a microplate reader assay. Our MOS-detecting biosensor represents a first-in-class probe whose design was guided by changes in localized dynamics of individual amino acid positions, supporting expansion of the CINC pipeline as an indispensable tool for a wide range of protein engineering applications.

A Mixed Methods Study of Senior Medical Student Help Calling in an Individual, Acute Care Simulation Experience.

INTRODUCTION: The ability to appropriately seek help is a key skill for medical students transitioning to residency. We designed a simulation activity for senior medical students and became interested in studying help calling behaviors and attitudes. METHODS: We collected quantitative data regarding help calling for 2 simulation cases and qualitative data using a survey. We undertook a structured thematic analysis based on grounded theory methodology. We identified and compared groups who did and did not call for help. RESULTS: One hundred thirty-four students participated and 122 (91%) completed an evaluation. More students called for help without prompting in the second case (34/134, 25% in first case; 110/134, 82% in second case, P < 0.001). Most students did not call for help in the first case but called in the second case (81, 60%). Our qualitative analysis identified 5 themes: (1) students seek to avoid shame and burdening their team, (2) prior institutional simulation and testing experience may imprint help calling behaviors, (3) students view help calling primarily through an individual lens, (4) students overestimate the complexity of the help calling process, and (5) the simulation environment and intentional experimentation make it difficult to observe natural behavior. When compared with help callers, more non-help callers had phrases coded as "perception of expectations" (37% vs. 10%, P = 0.03). CONCLUSIONS: Learners participating in simulation exercises designed to promote help-calling behaviors face training-based barriers related to shame and the desire for autonomy and simulation-based challenges related to assessment.

Emergency Department Thoracotomy: A Cost-Effective Model for Simulation Training.

BACKGROUND: Simulation provides a safe learning environment where high-stakes, low-frequency procedures can be practiced without the fear of being unsuccessful or causing harm. Emergency department thoracotomy (EDT) is one such procedure. Realistic thoracotomy models are expensive and not readily available. OBJECTIVE: Our objective is to describe a cost-effective, realistic, reproducible, and reusable thoracotomy model for simulation training. METHODS: We modified a commercially available clothes mannequin torso to expose the chest and abdominal cavity. A plastic skeleton composed of a spinal cord and ribs was placed inside the torso. Tubing was used to simulate the aorta and esophagus; both tubes were secured to the distal spine with zip ties. Commercially available lungs and heart were placed inside the chest cavity. A small rubber ball simulated the left lung to be able to maneuver the lung. The heart was covered with plastic wrap to simulate the pericardium. Thick tape was used to simulate the pleural cavity. Yoga mats were used to simulate the intercostal muscles, subcutaneous tissue, and skin. RESULTS: This model was tested with Emergency Medicine (EM) residents during a simulation session. A voluntary survey was available for residents to provide feedback. Survey results confirmed that the model provided valuable education, with overall positive feedback. CONCLUSION: This EDT model provides a valuable teaching opportunity to EM residents who otherwise might not have the opportunity to perform this procedure. Residents agreed that the model improved their confidence and is an effective method in providing the opportunity to practice this low-frequency, high-stakes procedure.

Streamlined purification of fluorescently labeled Escherichia coli phosphate-binding protein (PhoS) suitable for rapid-kinetics applications.

Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (Pi) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of Pi binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni2+-affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the Pi release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a Pi detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.

Does emergency physician empathy reduce thoughts of litigation? A randomised trial.

BACKGROUND: We hypothesised the addition of brief empathetic statements to physician-patient interaction might decrease thoughts regarding litigation. METHODS: We enrolled a convenience sample of adults in our emergency department (ED) waiting room into a randomised, double-blind controlled trial. Subjects watched videos of simulated discharge conversations between physicians and patient actors; half of the videos differed only by the inclusion of two brief empathetic statements: verbalisations that (1) the physician recognises that the patient is concerned about their symptoms and (2) the patient knows their typical state of health better than a physician seeing them for the first time and did the right thing by seeking evaluation. After watching the video subjects were asked to score a five-point Likert scale their thoughts regarding suing this physician in the event of a missed outcome leading to lost work (primary outcome), and four measures of satisfaction with the physician encounter (secondary outcomes). RESULTS: We enrolled and randomised 437 subjects. 213 in the empathy group and 208 in the non-empathy group completed the trial. Sixteen subjects did not complete the trial due to computer malfunction or incomplete data sheets. Empathy group subjects reported statistically significant less thoughts of litigation than the non-empathy group (mean Likert scale 2.66 vs 2.95, difference -0.29, 95% CI -0.04 to -0.54, p=0.0176). All four secondary measures of satisfaction with the physician encounter were better in the empathy group. CONCLUSIONS: In this study, the addition of brief empathetic statements to ED discharge scenarios was associated with a statistically significant reduction in thoughts regarding litigation. CLINICAL TRIAL REGISTRATION: NCT01837706.

Introducing a class of standardized and interchangeable parts utilizing programmed ribosomal frameshifts for synthetic biology applications.

Synthetic biology and the rational design of biological devices depend on the availability of standardized and interchangeable biological parts with diverse range of functions. Reliable access to different reading frames during translation has largely been overlooked as functionality for bioengineering applications. Here we report the construction and initial characterization of the first member of such a class of biological parts that conforms to the BioBrick Standard (RFC25), allowing its interchangeable use in biological devices. Using our standardized frameshifting signal consisting of a UUUAAAG slippery sequence, a 6 nt spacer and an engineered pseudoknot based on the infectious bronchitis virus pseudoknot PK401 embedded in a dual reporter construct, we confirm that the frameshifting activity is comparable to the previously published frequency despite the introduced sequence changes. The frameshifting activity is demonstrated using SDS-PAGE and fluorescence spectroscopy. Standardized programmable ribosomal frameshift parts with specific frameshifting frequencies will be of utility for applications such as double coding DNA sequences by expanding the codable space into the -1 frame. Programmed shifting into the -1 frame to bypass a stop codon allows labeling of a protein pool with a fixed stoichiometry of fusion protein, as well as the construction of multi-enzyme expression constructs with specific expression ratios. A detailed understanding of the structural basis of programmed frameshifting will provide the opportunities to rationally design frameshifting elements with a wide range of applications in synthetic biology, including signals that are regulated by small ligands.

Measuring cardiac index with a focused cardiac ultrasound examination in the ED.

OBJECTIVES: Noninvasive technology may assist the emergency department (ED) physician in determining the hemodynamic status in critically ill patients. The objective of our study was to show that ED physicians can accurately measure cardiac index (CI) by performing a bedside focused cardiac ultrasound examination. METHODS: A convenience sample of adult subjects were prospectively enrolled. Cardiac index, left ventricular outflow tract (LVOT) diameter, velocity time integral (VTI), stroke volume index, and heart rate were obtained by trained ED physicians and a certified cardiac sonographer. The primary outcome was percent of optimal LVOT diameter and VTI measurements as verified by an expert cardiologist. RESULTS: One hundred patients were enrolled, with obtainable CI measurements in 97 patients. Cardiac index, LVOT diameter, VTI, stroke volume index, and heart rate measurements by ED physician were 2.42 ± 0.70 L min(-1) m(-2), 2.07 ± 0.22 cm, 18.30 ± 3.71 cm, 32.34 ± 7.92 mL beat(-1) m(-2), and 75.32 ± 13.45 beats/min, respectively. Measurements of LVOT diameter by ED physicians and sonographer were optimal in 90.0% (95% confidence interval, 82.6%-94.5) and 91.3% (73.2%-97.6%) of patients, respectively. Optimal VTI measurements were obtained in 78.4% (69.2%-85.4%) and 78.3% (58.1%-90.3%) of patients, respectively. In 23 patients, the correlation (r) for CI between ED physician and sonographer was 0.82 (0.60-0.92), with bias and limits of agreement of -0.11 (-1.06 to 0.83) L min(-1) m(-2) and percent difference of 12.4% ± 10.1%. CONCLUSIONS: Emergency department ED physicians can accurately measure CI using standard bedside ultrasound. A focused ultrasound cardiac examination to derive CI has potential use in the management of critical ill patients in the ED.

Thoracic aortic dissection in an 18-year-old woman with no risk factors.

Spontaneous aortic dissection is a rare, life-threatening cause of chest pain, and has a higher prevalence when traditional risks such as age, hypertension, dyslipidemia, or connective tissue disorders are present. However, even in the absence of risk factors, non-traumatic rupture of an aortic dissection may occur. Most are found in patients over 40 years of age. Younger victims of this disease often also suffer from other conditions such as cystic medial necrosis, connective tissue disorders such as Marfan's syndrome, or vasculitis. We present the case of an 18-year-old, previously healthy woman who was country line dancing when she began to complain of severe, cramping chest and back pain. She was hemodynamically stable on initial presentation but experienced two seizures while in the emergency department and was intubated. Subsequently, her blood pressure dropped and she developed cardiac arrest, and despite vigorous resuscitation that included blood products and emergency department thoracotomy, she was refractory to all attempts. At autopsy she was found to have a spontaneous, non-traumatic rupture of an aortic dissection. This patient had no discernable risk factors for aortic dissection or discoverable cause on necropsy. We present this case to raise awareness among physicians and review other reported cases in the literature of aortic dissection in patients under age 40 years.