Travis Whitfill

Molecular basis for a protein-mediated DNA-bridging mechanism that functions in condensation of the E. coli chromosome.

The E. coli chromosome is condensed into insulated regions termed macrodomains (MDs), which are essential for genomic packaging. How chromosomal MDs are specifically organized and compacted is unknown. Here, we report studies revealing the molecular basis for Terminus-containing (Ter) chromosome condensation by the Ter-specific factor MatP. MatP contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix and C-terminal coiled-coil. Strikingly, MatP-matS structures show that the MatP coiled-coils form bridged tetramers that flexibly link distant matS sites. Atomic force microscopy and electron microscopy studies demonstrate that MatP alone loops DNA. Mutation of key coiled-coil residues destroys looping and causes a loss of Ter condensation in vivo. Thus, these data reveal the molecular basis for a protein-mediated DNA-bridging mechanism that mediates condensation of a large chromosomal domain in enterobacteria.

SlmA forms a higher-order structure on DNA that inhibits cytokinetic Z-ring formation over the nucleoid

The spatial and temporal control of Filamenting temperature sensitive mutant Z (FtsZ) Z-ring formation is crucial for proper cell division in bacteria. In Escherichia coli, the synthetic lethal with a defective Min system (SlmA) protein helps mediate nucleoid occlusion, which prevents chromosome fragmentation by binding FtsZ and inhibiting Z-ring formation over the nucleoid. However, to perform its function, SlmA must be bound to the nucleoid. To deduce the basis for this chromosomal requirement, we performed biochemical, cellular, and structural studies. Strikingly, structures show that SlmA dramatically distorts DNA, allowing it to bind as an orientated dimer-of-dimers. Biochemical data indicate that SlmA dimer-of-dimers can spread along the DNA. Combined structural and biochemical data suggest that this DNA-activated SlmA oligomerization would prevent FtsZ protofilament propagation and bundling. Bioinformatic analyses localize SlmA DNA sites near membrane-tethered chromosomal regions, and cellular studies show that SlmA inhibits FtsZ reservoirs from forming membrane-tethered Z rings. Thus, our combined data indicate that SlmA DNA helps block Z-ring formation over chromosomal DNA by forming higher-order protein-nucleic acid complexes that disable FtsZ filaments from coalescing into proper structures needed for Z-ring creation.

Impact of Just-in-Time and Just-in-Place Simulation on Intern Success With Infant Lumbar Puncture.

BACKGROUND AND OBJECTIVE: Simulation-based skill trainings are common; however, optimal instructional designs that improve outcomes are not well specified. We explored the impact of just-in-time and just-in-place training (JIPT) on interns’ infant lumbar puncture (LP) success. METHODS: This prospective study enrolled pediatric and emergency medicine interns from 2009 to 2012 at 34 centers. Two distinct instructional design strategies were compared. Cohort A (2009–2010) completed simulation-based training at commencement of internship, receiving individually coached practice on the LP simulator until achieving a predefined mastery performance standard. Cohort B (2010–2012) had the same training plus JIPT sessions immediately before their first clinical LP. Main outcome was LP success, defined as obtaining fluid with first needle insertion and <1000 red blood cells per high-power field. Process measures included use of analgesia, early stylet removal, and overall attempts. RESULTS: A total of 436 first infant LPs were analyzed. The LP success rate in cohort A was 35% (13/37), compared with 38% (152/399) in cohort B (95% confidence interval for difference [CI diff], −15% to +18%). Cohort B exhibited greater analgesia use (68% vs 19%; 95% CI diff, 33% to 59%), early stylet removal (69% vs 54%; 95% CI diff, 0% to 32%), and lower mean number of attempts (1.4 ± 0.6 vs 2.1 ± 1.6, P < .01) compared with cohort A. CONCLUSIONS: Across multiple institutions, intern success rates with infant LP are poor. Despite improving process measures, adding JIPT to training bundles did not improve success rate. More research is needed on optimal instructional design strategies for infant LP.

Do you see what I see? Insights from using google glass for disaster telemedicine triage.

INTRODUCTION Disasters are high-stakes, low-frequency events. Telemedicine may offer a useful adjunct for paramedics performing disaster triage. The objective of this study was to determine the feasibility of telemedicine in disaster triage, and to determine whether telemedicine has an effect on the accuracy of triage or the time needed to perform triage. METHODS This is a feasibility study in which an intervention team of two paramedics used the mobile device Google Glass (Google Inc; Mountain View, California USA) to communicate with an off-site physician disaster expert. The paramedic team triaged simulated disaster victims at the triennial drill of a commercial airport. The simulated victims had preassigned expected triage levels. The physician had an audio-video interface with the paramedic team and was able to observe the victims remotely. A control team of two paramedics performed disaster triage in the usual fashion. Both teams used the SMART Triage System (TSG Associates LLP; Halifax, England), which assigns patients into Red, Yellow, Green, and Black triage categories. The paramedics were video recorded, and their time required to triage was logged. It was determined whether the intervention team and the control team varied regarding accuracy of triage. Finally, the amount of time the intervention team needed to triage patients when telemedicine was used was compared to when that team did not use telemedicine. RESULTS The two teams triaged the same 20 patients. There was no significant difference between the two groups in overall triage accuracy (85.7% for the intervention group vs 75.9% for the control group; P = .39). Two patients were triaged with telemedicine. For the intervention group, there was a significant difference in time to triage patients with telemedicine versus those without telemedicine (35.5 seconds; 95% CI, 72.5-143.5 vs 18.5 seconds; 95% CI, 13.4-23.6; P = .041). CONCLUSION There was no increase in triage accuracy when paramedics evaluating disaster victims used telemedicine, and telemedicine required more time than conventional triage. There are a number of obstacles to available technology that, if overcome, might improve the utility of telemedicine in disaster response.

Differences in the Quality of Pediatric Resuscitative Care Across a Spectrum of Emergency Departments

Importance The quality of pediatric resuscitative care delivered across the spectrum of emergency departments (EDs) in the United States is poorly described. In a recent study, more than 4000 EDs completed the Pediatric Readiness Survey (PRS); however, the correlation of PRS scores with the quality of simulated or real patient care has not been described. Objective To measure and compare the quality of resuscitative care delivered to simulated pediatric patients across a spectrum of EDs and to examine the correlation of PRS scores with quality measures. Design, Setting, and Participants This prospective multicenter cohort study evaluated 58 interprofessional teams in their native pediatric or general ED resuscitation bays caring for a series of 3 simulated critically ill patients (sepsis, seizure, and cardiac arrest). Main Outcomes and Measures A composite quality score (CQS) was measured as the sum of 4 domains: (1) adherence to sepsis guidelines, (2) adherence to cardiac arrest guidelines, (3) performance on seizure resuscitation, and (4) teamwork. Pediatric Readiness Survey scores and health care professional demographics were collected as independent data. Correlations were explored between CQS and individual domain scores with PRS. Results Overall, 58 teams from 30 hospitals participated (8 pediatric EDs [PEDs], 22 general EDs [GEDs]). The mean CQS was 71 (95% CI, 68-75); PEDs had a higher mean CQS (82; 95% CI, 79-85) vs GEDs (66; 95% CI, 63-69) and outperformed GEDs in all domains. However, when using generalized estimating equations to estimate CQS controlling for clustering of the data, PED status did not explain a higher CQS (β = 4.28; 95% CI, -4.58 to 13.13) while the log of pediatric patient volume did explain a higher CQS (β = 9.57; 95% CI, 2.64-16.49). The correlation of CQS to PRS was moderate (r = 0.51; P < .001). The correlation was weak for cardiac arrest (r = 0.24; P = .07), weak for sepsis (ρ = 0.45; P < .001) and seizure (ρ = 0.43; P = .001), and strong for teamwork (ρ = 0.71; P < .001). Conclusions and Relevance This multicenter study noted significant differences in the quality of simulated pediatric resuscitative care across a spectrum of EDs. The CQS was higher in PEDs compared with GEDs. However, when controlling for pediatric patient volume and other variables in a multivariable model, PED status does not explain a higher CQS while pediatric patient volume does. The correlation of the PRS was moderate for simulation-based measures of quality.

Structures of the Bacillus subtilis Glutamine Synthetase Dodecamer Reveal Large Intersubunit Catalytic Conformational Changes Linked to a Unique Feedback Inhibition Mechanism.

Background: B. subtilis GS catalyzes the production of glutamine, a key metabolite in nitrogen assimilation. Results: B. subtilis GS forms a dodecamer that undergoes large intersubunit conformational changes during catalysis. Conclusion: B. subtilis GS structures reveal a heretofore unseen active site restructuring that is linked to a novel feedback regulatory mechanism. Significance: The GS specific regulatory/catalytic mechanism may be used to target Gram-positive pathogens. Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in nitrogen metabolism. There are two main bacterial GS isoenzymes, GSI-α and GSI-β. GSI-α enzymes, which have not been structurally characterized, are uniquely feedback-inhibited by Gln. To gain insight into GSI-α function, we performed biochemical and cellular studies and obtained structures for all GSI-α catalytic and regulatory states. GSI-α forms a massive 600-kDa dodecameric machine. Unlike other characterized GS, the Bacillus subtilis enzyme undergoes dramatic intersubunit conformational alterations during formation of the transition state. Remarkably, these changes are required for active site construction. Feedback inhibition arises from a hydrogen bond network between Gln, the catalytic glutamate, and the GSI-α-specific residue, Arg62, from an adjacent subunit. Notably, Arg62 must be ejected for proper active site reorganization. Consistent with these findings, an R62A mutation abrogates Gln feedback inhibition but does not affect catalysis. Thus, these data reveal a heretofore unseen restructuring of an enzyme active site that is coupled with an isoenzyme-specific regulatory mechanism. This GSI-α-specific regulatory network could be exploited for inhibitor design against Gram-positive pathogens.

Disparities in Adherence to Pediatric Sepsis Guidelines across a Spectrum of Emergency Departments: A Multicenter, Cross-Sectional Observational In Situ Simulation Study

BACKGROUND Each year in the United States, 72,000 pediatric patients develop septic shock, at a cost of

Pediatric Disaster Triage: Multiple Simulation Curriculum Improves Prehospital Care Providers' Assessment Skills

4.8 billion. Adherence to practice guidelines can significantly reduce mortality; however, few methods to compare performance across a spectrum of emergency departments (EDs) have been described. OBJECTIVES We employed standardized, in situ simulations to measure and compare adherence to pediatric sepsis guidelines across a spectrum of EDs. We hypothesized that pediatric EDs (PEDs) would have greater adherence to the guidelines than general EDs (GEDs). We also explored factors associated with improved performance. METHODS This multi-center observational study examined in situ teams caring for a simulated infant in septic shock. The primary outcome was overall adherence to the pediatric sepsis guideline as measured by six subcomponent metrics. Characteristics of teams were compared using multivariable logistic regression to describe factors associated with improved performance. RESULTS We enrolled 47 interprofessional teams from 24 EDs. Overall, 21/47 teams adhered to all six sepsis metrics (45%). PEDs adhered to all six metrics more than GEDs (93% vs. 22%; difference 71%, 95% confidence interval [CI] 43-84). Adherent teams had significantly higher Emergency Medical Services for Children readiness scores, MD composition of physicians to total team members, teamwork scores, provider perceptions of pediatric preparedness, and provider perceptions of sepsis preparedness. In a multivariable regression model, only greater composite team experience had greater adjusted odds of achieving an adherent sepsis score (adjusted odds ratio 1.38, 95% CI 1.01-1.88). CONCLUSIONS Using standardized in situ scenarios, we revealed high variability in adherence to the pediatric sepsis guideline across a spectrum of EDs. PEDs demonstrated greater adherence to the guideline than GEDs; however, in adjusted analysis, only composite team experience level of the providers was associated with improved guideline adherence.

The Correlation of Workplace Simulation-Based Assessments With Interns' Infant Lumbar Puncture Success: A Prospective, Multicenter, Observational Study.

ABSTRACT Objective: Paramedics and emergency medical technicians (EMTs) triage pediatric disaster victims infrequently. The objective of this study was to measure the effect of a multiple-patient, multiple-simulation curriculum on accuracy of pediatric disaster triage (PDT). Methods: Paramedics, paramedic students, and EMTs from three sites were enrolled. Triage accuracy was measured three times (Time 0, Time 1 [two weeks later], and Time 2 [6 months later]) during a disaster simulation, in which high and low fidelity manikins and actors portrayed 10 victims. Accuracy was determined by participant triage decision concordance with predetermined expected triage level (RED [Immediate], YELLOW [Delayed], GREEN [Ambulatory], BLACK [Deceased]) for each victim. Between Time 0 and Time 1, participants completed an interactive online module, and after each simulation there was an individual debriefing. Associations between participant level of training, years of experience, and enrollment site were determined, as were instances of the most dangerous mistriage, when RED and YELLOW victims were triaged BLACK. Results: The study enrolled 331 participants, and the analysis included 261 (78.9%) participants who completed the study, 123 from the Connecticut site, 83 from Rhode Island, and 55 from Massachusetts. Triage accuracy improved significantly from Time 0 to Time 1, after the educational interventions (first simulation with debriefing, and an interactive online module), with a median 10% overall improvement (p < 0.001). Subgroup analyses showed between Time 0 and Time 1, paramedics and paramedic students improved more than EMTs (p = 0.002). Analysis of triage accuracy showed greatest improvement in overall accuracy for YELLOW triage patients (Time 0 50% accurate, Time1 100%), followed by RED patients (Time 0 80%, Time 1 100%). There was no significant difference in accuracy between Time 1 and Time 2 (p = 0.073). Conclusion: This study shows that the multiple-victim, multiple-simulation curriculum yields a durable 10% improvement in simulated triage accuracy. Future iterations of the curriculum can target greater improvements in EMT triage accuracy.

Do you see what I see? A randomised pilot study to evaluate the effectiveness and efficiency of simulation-based training with videolaryngoscopy for neonatal intubation

Introduction Little data are available to guide supervisors’ decisions regarding when trainees are prepared to safely perform their first procedure on a patient. We aimed to describe the correlation of simulation-based assessments, in the workplace, with interns’ first clinical infant lumbar puncture (ILP) success. Methods This is a prospective, observational subcomponent of a larger study of incoming interns at 33 academic medical centers (July 2010 to June 2012) assessing the impact of just-in-time training. When an intern’s patient required an ILP, a just-in-time simulation-based skills refresher was conducted with his or her supervisor. At the end of the refresher, supervisors assessed interns’ ILP skills on a simulator in the workplace before clinical performance using a four point anchored scale. The primary outcome was the correlation of supervisors’ assessment and interns’ procedural success. The number needed to assess for this instrument (1 / absolute risk reduction) was calculated. Results A total of 1600 interns were eligible to participate, and 1215 were enrolled. A total of 297 completed an assessment and a subsequent clinical ILP. Success rates for each scale rating were 29% (18/63) for novice, 39% (51/130) for beginner, 55% (46/83) for competent, and 43% (9/21) for proficient. The correlation coefficient was 0.161 (95% confidence interval, 0.057–0.265), indicating a weak correlation between supervisor rating and success. Success rate was 53% for the ratings of competent or proficient compared with 35% for the ratings of novice or beginner. Using the global rating scale for the summative assessment to determine procedural readiness could lead to 1 fewer patient experiencing a failed ILP for every 6 interns tested (6.2; 95% confidence interval, 4.0–8.5). Conclusions A simulation-based assessment of interns conducted in the workplace before their first ILP has some value in predicting clinical ILP success.