"Can you help me think this through?" How pediatric hospitalists learn from informal peer consultation.

Informal peer consultation (IPC), also called curbside consultation, is a common practice in medicine. Research has shown that physicians use IPC but how this learning occurs during the process has not been studied. This basic qualitative study describes how pediatric hospitalists learn during IPC, framed by Kolb's (2015) Experiential Learning Theory of Growth and Development. Eleven pediatric hospitalists were interviewed. Deidentified transcripts were coded for key themes using inductive methods. The main prompt for informal peer consultation was the perception of uncertainty. Three themes describe the learning process: "Eliciting Perspectives," "Thinking Aloud Together," and "Experiencing Validation. A fourth theme, "Acknowledging Value," described the importance of IPC for modeling how to manage uncertainty with patients' caregivers and medical trainees. By describing the learning process, the results have implications for physicians who engage in IPC and may inform faculty-level professional development initiatives to improve the IPC process.

Investigating the dissipation of heat and quantum information from DNA-scaffolded chromophore networks.

Scaffolded molecular networks are important building blocks in biological pigment-protein complexes, and DNA nanotechnology allows analogous systems to be designed and synthesized. System-environment interactions in these systems are responsible for important processes, such as the dissipation of heat and quantum information. This study investigates the role of nanoscale molecular parameters in tuning these vibronic system-environment dynamics. Here, genetic algorithm methods are used to obtain nanoscale parameters for a DNA-scaffolded chromophore network based on comparisons between its calculated and measured optical spectra. These parameters include the positions, orientations, and energy level characteristics within the network. This information is then used to compute the dynamics, including the vibronic population dynamics and system-environment heat currents, using the hierarchical equations of motion. The dissipation of quantum information is identified by the system's transient change in entropy, which is proportional to the heat currents according to the second law of thermodynamics. These results indicate that the dissipation of quantum information is highly dependent on the particular nanoscale characteristics of the molecular network, which is a necessary first step before gleaning the systematic optimization rules. Subsequently, the I-concurrence dynamics are calculated to understand the evolution of the vibronic system's quantum entanglement, which are found to be long-lived compared to these system-bath dissipation processes.

The Medical Student Experience With Prework.

OBJECTIVES: The objective of this study is to better understand the medical student experience with prework to determine what factors influence their motivation to complete prework. INTRODUCTION: Medical education has been shifting to more active learning-type sessions such as flipped classrooms but these activities are unsuccessful when students do not complete the associated prework. The literature is lacking on why students do not complete prework and what would motivate them to do so. This qualitative study aims to answer those questions through the view of expectancy-value motivation theory. METHODS: Thirteen preclinical medical students participated in a semistructured basic interview study investigating their experience with prework. Interview transcripts were coded, and codes were clustered and analyzed for themes. RESULTS: Students develop particular routines they find successful for their studies. They explain how time in their schedules and the amount of time prework takes to complete plays a role in their study environment which must be favorable in order to complete prework. Students view video prework more favorably compared to reading assignments. Students note how the opinions of their peers influence their decision to complete prework. Each of these factors influences student motivation to complete prework. CONCLUSION: This study finds that motivation to complete prework is influenced by the environment, format, and use of prework, student interest and prior knowledge, and peer influence. The combination of these factors determines whether a student believes they are capable of completing prework and if they find it valuable. In order to increase motivation to complete prework, faculty should consider how to address these factors in a way that students are able to fit prework into their study routines. This study provides the first step in understanding the medical student experience with prework and suggests directions for future studies to maximize student motivation to complete prework.

Enzyme assembly on nanoparticle scaffolds enhances cofactor recycling and improves coupled reaction kinetics.

Enzyme activity can be many times enhanced in configurations where they are displayed on a nanoparticle (NP) and this same format sometimes even provides access to channeling phenomena within multienzyme cascades. Here, we demonstrate that such enhancement phenomena can be expanded to enzymatic cofactor recycling along with the coupled enzymatic processes that they are associated with. We begin by showing that the efficiency of glucose driven reduction of nicotinamide adenine dinucleotide (NAD+ → NADH) by glucose dehydrogenase (GDH) is enhanced ca. 5-fold when the enzyme is displayed on nanocrystalline semiconductor quantum dots (QDs) which are utilized as prototypical NP materials in our experimental assays. Coupling this enzymatic step with NADH-dependent lactate dehydrogenase (LDH) conversion of lactate to pyruvate also increases the latter's rate by a similar amount when both enzymes were jointly incorporated into self-assembled QD-based nanoclusters. Detailed agarose gel mobility assays and transmission electron microscopy imaging studies confirm that both tetrameric enzymes assemble to and crosslink the QDs into structured nanoclusters via their multiple-pendant terminal (His)6 sequences. Unexpectedly, control experiments utilizing blocking peptides to prevent enzyme-crosslinking of QDs resulted in even further enhancement of individual enzyme on-QD kinetic activity. This activity was also probed revealing that 200-fold excess peptide/QD addition enhanced individual GDH and LDH on-QD kcat a further 2- and 1.5×, respectively, above that seen just by QD display to a maximum of ∼10-fold GDH enhancement. The potential implications for how these enzyme kinetics-enhancing phenomena can be applied to single and multi-enzyme cascaded reactions in the context of cofactor recycling and cell-free synthetic biology are discussed.

Assessing trainee critical thinking skills using a novel interactive online learning tool.

BACKGROUND: Critical thinking is essential for the accurate diagnosis and management of patients. It is correlated with academic success. OBJECTIVE: Our objective was to design a novel tool for interactive online learning to improve knowledge and to assess trainees' critical thinking skills using the framework of the American Philosophical Association (APA). METHODS: Residents, fellows and students participated in an online, self-directed case-based vignette activity to learn malaria diagnosis and management. Pre and post-tests with multiple choice and open-ended case-based questions assessed knowledge and critical thinking. Comparison between pre and post-test scores across subgroups were performed using paired t-tests or one-way ANOVA. RESULTS: Between 4 April 2017 to 14 July 2019, 62 of 75 (82%) eligible subjects completed both the pre and the post-test. Improved post-test scores occurred in 90% of medical students, p=0.001, 77% of residents, p<0.001, 60% of fellows, p=0.72 and 75% of trainees overall, p=<0.001. Fellows had higher pre-test scores than students or residents but there was no difference by level of training on the post-test. CONCLUSIONS: This interactive online learning activity effectively imparted medical knowledge and improved trainee responses to questions requiring critical thinking. To our knowledge, this is the first time the APA's critical thinking framework has been incorporated into interactive online learning and assessment of critical thinking skills in medical trainees. We applied this innovation specifically in global health education, but there is obvious potential to expand it to a wide variety of areas of clinical training.

Integrating Single Domain Antibodies into Field-Deployable Rapid Assays.

Single domain antibodies (sdAb) are the recombinant variable heavy domains derived from camelid heavy-chain antibodies. While they have binding affinities equivalent to conventional antibodies, sdAb are only one-tenth the size and possess numerous advantages such as excellent thermal stability with the ability to refold following denaturation, and inexpensive production in Escherichia coli or yeast. However, their small size does have drawbacks, one being that they can lose activity upon attachment or adsorption to surfaces, or may fail to adsorb efficiently, as they are highly soluble. This can make the transition from using conventional antibodies to sdAb nontrivial for assay development. Specifically, it is often necessary to re-optimize the protocols and tailor the recombinant sdAb through protein engineering to function efficiently in handheld assays, which currently are utilized for point of care testing and field applications. This work focuses on optimizing the integration of sdAb into rapid vertical flow assays. To achieve this goal, we engineered sdAb-based constructs and developed general protocols for the attachment of the sdAb to both gold nanoparticles and a support membrane. We achieved a limit of detection of 0.11 µg/mL for toxins staphylococcal enterotoxin B and ricin, both potential biothreat agents. Additionally, we demonstrated the ability to detect the nucleocapsid protein of SARS-CoV-2, a common target of antigen tests for COVID-19.

Bivalent single domain antibody constructs for effective neutralization of Venezuelan equine encephalitis.

Venezuelan equine encephalitis virus (VEEV) is a mosquito borne alphavirus which leads to high viremia in equines followed by lethal encephalitis and lateral spread to humans. In addition to naturally occurring outbreaks, VEEV is a potential biothreat agent with no approved human vaccine or therapeutic currently available. Single domain antibodies (sdAb), also known as nanobodies, have the potential to be effective therapeutic agents. Using an immune phage display library derived from a llama immunized with an equine vaccine that included inactivated VEEV, five sdAb sequence families were identified that showed varying ability to neutralize VEEV. One of the sequence families had been identified previously in selections against chikungunya virus, a related alphavirus of public health concern. A key advantage of sdAb is the ability to optimize properties such as neutralization capacity through protein engineering. Neutralization of VEEV was improved by two orders of magnitude by genetically linking sdAb. One of the bivalent constructs showed effective neutralization of both VEEV and chikungunya virus. Several of the bivalent constructs neutralized VEEV in cell-based assays with reductions in the number of plaques by 50% at protein concentrations of 1 ng/mL or lower, making future evaluation of their therapeutic potential compelling.

Simulation for Neonatal Endotracheal Intubation Training: How Different Is It From Clinical Practice?

INTRODUCTION: Neonatal endotracheal intubation is a critical skill that is difficult for learners to acquire even with simulation-based training (SBT). Trainees prefer clinical experiences over SBT. The objective of the study was to explore the differences between SBT and clinical practice in acquiring neonatal intubation skills to inform mannequin design and to improve fidelity. METHODS: A basic qualitative study using semistructured interviews was conducted to determine the experience of newly competent trainees (second- and third-year neonatal-perinatal medicine fellows) and their instructors in developing intubation skills. Participants were asked to compare learning through SBT with clinical practice in terms of context, equipment, and environment. Their responses were analyzed using an inductive approach. RESULTS: Thirty-two participants (20 fellows and 12 faculty) indicated that SBT does not equal the real experience. Specifically, the look, feel, and function of the simulators differ enough from the real patient and the clinical environmental that they do not elicit the desired learning responses. The clinical environment prompted heightened emotions and had a chaotic atmosphere that was not fully captured by SBT. Participants suggested that programs use SBT in the initial phases of training only to gain basic skills and they provided several solutions for mannequin and SBT session design. CONCLUSIONS: Simulation-based training does not fully prepare neonatal-perinatal medicine fellows for neonatal intubation. Mannequins with unique active features, such as multiple airway configurations, slipperiness, secretions, and softer textures should be developed. Realistic environments that replicate the interprofessional nature and stressors of the clinical environment might better prepare learners for the complexity of clinical practice.

Understanding Förster Resonance Energy Transfer in the Sheet Regime with DNA Brick-Based Dye Networks.

Controlling excitonic energy transfer at the molecular level is a key requirement for transitioning nanophotonics research to viable devices with the main inspiration coming from biological light-harvesting antennas that collect and direct light energy with near-unity efficiency using Förster resonance energy transfer (FRET). Among putative FRET processes, point-to-plane FRET between donors and acceptors arrayed in two-dimensional sheets is predicted to be particularly efficient with a theoretical 1/r4 energy transfer distance (r) dependency versus the 1/r6 dependency seen for a single donor-acceptor interaction. However, quantitative validation has been confounded by a lack of robust experimental approaches that can rigidly place dyes in the required nanoscale arrangements. To create such assemblies, we utilize a DNA brick scaffold, referred to as a DNA block, which incorporates up to five two-dimensional planes with each displaying from 1 to 12 copies of five different donor, acceptor, or intermediary relay dyes. Nanostructure characterization along with steady-state and time-resolved spectroscopic data were combined with molecular dynamics modeling and detailed numerical simulations to compare the energy transfer efficiencies observed in the experimental DNA block assemblies to theoretical expectations. Overall, we demonstrate clear signatures of sheet regime FRET, and from this we provide a better understanding of what is needed to realize the benefits of such energy transfer in artificial dye networks along with FRET-based sensing and imaging.

Faculty leadership development: A case study of a synergistic approach.

INTRODUCTION: Ongoing leadership development is essential for academic health center faculty members to respond to increasing environmental complexity. At the George Washington University School of Medicine and Health Sciences, an 8-month program, based on Conger's leadership development approach emphasizing conceptual understanding, skill building, feedback and personal growth was offered to mid-level faculty charged with developing educational programs, clinical services, and/or research initiatives. We studied how specific learning methods catering to different learning approaches contributed to improving leadership competencies. METHODS: Session and program evaluations, participant interviews, mentor surveys, and supervisor interviews were used for data collection. Themes were identified through open coding with use of constant comparative methods to help find patterns in the data. RESULTS: Readings and classroom modules provided a broadened, holistic understanding of leadership; role plays and action plans helped participants apply and practice leadership skills; self-assessments and feedback from peers and mentors provided specifics for focusing development efforts; and personal growth exercises provided opportunities to reflect and consider fresh perspectives. Anchoring learning methods around a real-time project led to improved leadership competencies and personal confidence as reported by participants, supervisors and mentors. CONCLUSION: A faculty leadership development program that integrates understanding, skill building, feedback and personal growth and connects multiple learning methods can provide the synergy to facilitate behavior change and organizational growth.

Single-Domain Antibodies for the Detection of SARS-CoV-2 Nucleocapsid Protein.

The goal of this work was to develop recombinantly expressed variable domains derived from camelid heavy-chain antibodies known as single-domain antibodies (sdAbs) directed against the SARS-CoV-2 nucleocapsid protein for incorporation into detection assays. To achieve this, a llama was immunized using a recombinant SARS-CoV-2 nucleocapsid protein and an immune phage-display library of variable domains was developed. The sdAbs selected from this library segregated into five distinct sequence families. Three of these families bind to unique epitopes with high affinity, low nM to sub-nM KD, as determined by surface plasmon resonance. To further enhance the utility of these sdAbs for the detection of nucleocapsid protein, homobivalent and heterobivalent genetic fusion constructs of the three high-affinity sdAbs were prepared. The effectiveness of the sdAbs for the detection of nucleocapsid protein was evaluated using MagPlex fluid array assays, a multiplexed immunoassay on color-coded magnetic microspheres. Using the optimal bivalent pair, one immobilized on the microsphere and the other serving as the biotinylated recognition reagent, a detection limit as low as 50 pg/mL of recombinant nucleocapsid and of killed virus down to 1.28 × 103 pfu/mL was achieved. The sdAbs described here represent immune reagents that can be tailored to be optimized for a number of detection platforms and may one day aid in the detection of SARS-CoV-2 to assist in controlling the current pandemic.

Stabilization of a Broadly Neutralizing Anti-Chikungunya Virus Single Domain Antibody.

A single domain antibody (clone CC3) previously found to neutralize a vaccine strain of the chikungunya virus (PRNT50 = 2. 5 ng/mL) was found to be broadly neutralizing. Clone CC3 is not only able to neutralize a wild-type (WT) strain of chikungunya virus (CHIKV), but also neutralizes WT strains of Mayaro virus (MAYV) and Ross River virus (RRV); both arthralgic, Old World alphaviruses. Interestingly, CC3 also demonstrated a degree of neutralizing activity against the New World alphavirus, Venezuelan equine encephalitis virus (VEEV); albeit both the vaccine strain, TC-83, and the parental, WT Trinidad donkey strain had PRNT50 values ~1,000-fold higher than that of CHIKV. However, no neutralization activity was observed with Western equine encephalitis virus (WEEV). Ten CC3 variants designed to possess a range of isoelectric points, both higher and lower, were constructed. This approach successfully identified several lower pI mutants which possessed improved thermal stabilities by as much as 10°C over the original CC3 (Tm = 62°C), and excellent refolding abilities while maintaining their capacity to bind and neutralize CHIKV.

Using Trauma-Informed Care in Practice: Evaluation of Internal Medicine Resident Training and Factors Affecting Clinical Use.

INTRODUCTION: Trauma-informed care (TIC) acknowledges that childhood traumas can profoundly affect health outcomes and aims to creates a safe, nurturing medical environment. TIC curricula in graduate medical education are limited and lacking in assessment of the application of TIC in practice. METHODS: This mixed-methods study followed implementation of a 4-hour required training session for 91 internal medicine residents at George Washington University Hospital. Pre- and posttests were administered to determine change in knowledge, attitude, and confidence. Six weeks after the training, individual participant interviews were conducted to ascertain how TIC is practiced in the clinical setting. RESULTS: For the 47 participants with matched pre- and posttest data available, there was a statistically significant score improvement after the session on 13 of 16 items (excluding only 3 attitude items). Six themes emerged: 1) patient characteristics trigger recognition to utilize TIC; 2) time plays a critical role; 3) the acute medical setting is a barrier; 4) the patient-doctor relationship impacts TIC application; 5) concern about next steps influences TIC use; and 6) incorporating TIC requires repetition, practice, and supervisor support. CONCLUSION: This study demonstrated that a 4-hour TIC session can promote growth in resident knowledge and confidence related to TIC. The findings highlight that TIC education needs to account for contextual factors that can impact its clinical application. A focus on work environment factors such as time, resources, and supervisory support can help to maximize TIC learning, retention, and application in practice.

Pediatric Hospitalists' Performance and Perceptions of Script Concordance Testing for Self-Assessment.

OBJECTIVES: The cognitive expertise of Pediatric Hospitalists (PH) lies not in standard knowledge but in making decisions under conditions of uncertainty. To maintain expertise, PH should engage in deliberate practice via self-assessments that promote higher-level cognitive processes necessary to address problems with missing or ambiguous information. Higher levels of cognition are purported with Script Concordance Test (SCT) questions compared to Multiple Choice Questions (MCQ). To determine if PH use higher levels of cognition when answering SCT versus MCQ questions and to analyze participants' perceptions of the utility of using SCT self-assessment for deliberate practice in addressing clinical problems encountered in daily practice. METHODS: This is a mixed methods study comparing the cognitive level expressed according to Bloom's Taxonomy by PH answering MCQ versus SCT questions using a "think aloud" (TA) exercise, followed by qualitative analysis of interviews conducted afterward. RESULTS: A significantly greater percentage of comments were coded as higher cognitive processes (apply, analyze, evaluate, and create) for SCT versus MCQ (74% vs 19%) compared with lower order (remember, understand); chi-square P < .00001. Analysis of interviews revealed 6 themes. CONCLUSION: SCT questions elicited higher level cognition essential to clinical reasoning compared to MCQ questions. PH-indicated MCQ questions measure standard knowledge, while SCT questions better measure decision-making under conditions of uncertainty. PH-perceived SCT could be useful for deliberate practice in Pediatric Hospital Medicine decision-making if they could compare their rationale in answering questions with that of experts.

High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme.

There are currently few approved effective treatments for SARS-CoV-2, the virus responsible for the COVID-19 pandemic. Nanobodies are 12-15 kDa single-domain antibody fragments that can be delivered by inhalation and are amenable to relatively inexpensive large scale production compared to other biologicals. We have isolated nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain and block spike protein interaction with the angiotensin converting enzyme 2 (ACE2) with 1-5 nM affinity. The lead nanobody candidate, NIH-CoVnb-112, blocks SARS-CoV-2 spike pseudotyped lentivirus infection of HEK293 cells expressing human ACE2 with an EC50 of 0.3 µg/mL. NIH-CoVnb-112 retains structural integrity and potency after nebulization. Furthermore, NIH-CoVnb-112 blocks interaction between ACE2 and several high affinity variant forms of the spike protein. These nanobodies and their derivatives have therapeutic, preventative, and diagnostic potential.

Selection and Characterization of Single-Domain Antibodies for Detection of Lassa Nucleoprotein.

Lassa virus is the etiologic agent of Lassa fever, an acute and often fatal illness endemic to West Africa. It is important to develop new reagents applicable either for the specific diagnosis or as improved therapeutics for the treatment of Lassa fever. Here, we describe the development and initial testing of llama-derived single-domain antibodies that are specific for the Lassa virus nucleoprotein. Four sequence families based on complementarity-determining region (CDR) homology were identified by phage-based enzyme-linked immunosorbent assays, however, the highest affinity clones all belonged to the same sequence family which possess a second disulfide bond between Framework 2 and CDR3. The affinity and thermal stability were evaluated for each clone. A MagPlex-based homogeneous sandwich immunoassay for Lassa virus-like particles was also demonstrated to show their potential for further development as diagnostic reagents.

Multi-Enzyme Assembly on T4 Phage Scaffold.

Over the past two decades, various scaffolds have been designed and synthesized to organize enzyme cascades spatially for enhanced enzyme activity based on the concepts of substrate channeling and enhanced stability. The most bio-compatible synthetic scaffolds known for enzyme immobilization are protein and DNA nanostructures. Herein, we examined the utility of the T4 phage capsid to serve as a naturally occurring protein scaffold for the immobilization of a three-enzyme cascade: Amylase, Maltase, and Glucokinase. Covalent constructs between each of the enzymes and the outer capsid protein Hoc were prepared through SpyTag-SpyCatcher pairing and assembled onto phage capsids in vitro with an estimated average of 90 copies per capsid. The capsid-immobilized Maltase has a fourfold higher initial rate relative to Maltase free in solution. Kinetic analysis also revealed that the immobilized three-enzyme cascade has an 18-fold higher converted number of NAD+ to NADH relative to the mixtures in solution. Our results demonstrate that the T4 phage capsid can act as a naturally occurring scaffold with substantial potential to enhance enzyme activity by spatially organizing enzymes on the capsid Hoc.

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays.

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

The process of curricular integration and its effects on anatomical knowledge retention.

INTRODUCTION: Integration has been recognized as an important aspect of medical education. After transitioning from a discipline-specific to a systems-based preclinical curriculum, we examined faculty perceptions of the integrated approach and also whether it would lead to better anatomy knowledge retention. METHODS: To understand faculty perspectives, we reviewed curricular materials, interviewed block directors, and observed educational sessions. We analyzed knowledge retention through a 27-question anatomy test, comparing scores from the last class of the discipline-based curriculum and the first two classes of the integrated curriculum. RESULTS: Planning integrated content involves purposeful ordering, is challenging for faculty, and requires additional resources. Evaluation of the integrated approach for anatomy content demonstrated a significant increase in knowledge retention (p = .012; 56.28% vs. 63.98% for old vs. new curriculum). CONCLUSIONS: This study helps the understanding of what is required for curricular integration. Our anatomy evaluation results corroborated the view that contextually embedded information is easier to learn and retain.

Optimizing the Use of an Online Self-assessment Exam to Promote Self-directed Learning Behaviors in Medical Students.

Online self-assessment exams (SAEs) can be used to encourage self-directed learning (SDL) in medical education, but their effectiveness depends on how they are incorporated into the curriculum. In this pilot, we applied adult learning principles to the curricular design for implementing an online SAE in the neurology clerkship. We examined student perceptions of the efficacy in promoting SDL behaviors by analyzing survey responses from 76 medical students in the clerkship. Factors in the design and implementation of the SAE that promoted SDL behaviors included fostering autonomy, promoting paced study, broadening content exposure, encouraging self-reflection, and providing content with perceived value.