Nanoscale dynamics of the cadherin-catenin complex bound to vinculin revealed by neutron spin echo spectroscopy.

We report a neutron spin echo (NSE) study of the nanoscale dynamics of the cell-cell adhesion cadherin-catenin complex bound to vinculin. Our measurements and theoretical physics analyses of the NSE data reveal that the dynamics of full-length α-catenin, ß-catenin, and vinculin residing in the cadherin-catenin-vinculin complex become activated, involving nanoscale motions in this complex. The cadherin-catenin complex is the central component of the cell-cell adherens junction (AJ) and is fundamental to embryogenesis, tissue wound healing, neuronal plasticity, cancer metastasis, and cardiovascular health and disease. A highly dynamic cadherin-catenin-vinculin complex provides the molecular dynamics basis for the flexibility and elasticity that are necessary for the AJs to function as force transducers. Our theoretical physics analysis provides a way to elucidate these driving nanoscale motions within the complex without requiring large-scale numerical simulations, providing insights not accessible by other techniques. We propose a three-way "motorman" entropic spring model for the dynamic cadherin-catenin-vinculin complex, which allows the complex to function as a flexible and elastic force transducer.

Activated nanoscale actin-binding domain motion in the catenin-cadherin complex revealed by neutron spin echo spectroscopy.

As the core component of the adherens junction in cell-cell adhesion, the cadherin-catenin complex transduces mechanical tension between neighboring cells. Structural studies have shown that the cadherin-catenin complex exists as an ensemble of flexible conformations, with the actin-binding domain (ABD) of α-catenin adopting a variety of configurations. Here, we have determined the nanoscale protein domain dynamics of the cadherin-catenin complex using neutron spin echo spectroscopy (NSE), selective deuteration, and theoretical physics analyses. NSE reveals that, in the cadherin-catenin complex, the motion of the entire ABD becomes activated on nanosecond to submicrosecond timescales. By contrast, in the α-catenin homodimer, only the smaller disordered C-terminal tail of ABD is moving. Molecular dynamics (MD) simulations also show increased mobility of ABD in the cadherin-catenin complex, compared to the α-catenin homodimer. Biased MD simulations further reveal that the applied external forces promote the transition of ABD in the cadherin-catenin complex from an ensemble of diverse conformational states to specific states that resemble the actin-bound structure. The activated motion and an ensemble of flexible configurations of the mechanosensory ABD suggest the formation of an entropic trap in the cadherin-catenin complex, serving as negative allosteric regulation that impedes the complex from binding to actin under zero force. Mechanical tension facilitates the reduction in dynamics and narrows the conformational ensemble of ABD to specific configurations that are well suited to bind F-actin. Our results provide a protein dynamics and entropic explanation for the observed force-sensitive binding behavior of a mechanosensitive protein complex.

An ensemble of cadherin-catenin-vinculin complex employs vinculin as the major F-actin binding mode.

The cell-cell adhesion cadherin-catenin complexes recruit vinculin to the adherens junction (AJ) to modulate the mechanical couplings between neighboring cells. However, it is unclear how vinculin influences the AJ structure and function. Here, we identified two patches of salt bridges that lock vinculin in the head-tail autoinhibited conformation and reconstituted the full-length vinculin activation mimetics bound to the cadherin-catenin complex. The cadherin-catenin-vinculin complex contains multiple disordered linkers and is highly dynamic, which poses a challenge for structural studies. We determined the ensemble conformation of this complex using small-angle x-ray and selective deuteration/contrast variation small-angle neutron scattering. In the complex, both α-catenin and vinculin adopt an ensemble of flexible conformations, but vinculin has fully open conformations with the vinculin head and actin-binding tail domains well separated from each other. F-actin binding experiments show that the cadherin-catenin-vinculin complex binds and bundles F-actin. However, when the vinculin actin-binding domain is removed from the complex, only a minor fraction of the complex binds to F-actin. The results show that the dynamic cadherin-catenin-vinculin complex employs vinculin as the primary F-actin binding mode to strengthen AJ-cytoskeleton interactions.

An ensemble of flexible conformations underlies mechanotransduction by the cadherin-catenin adhesion complex.

The cadherin-catenin adhesion complex is the central component of the cell-cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•ß-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and ß-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of ß-catenin forms a flexible "tongue" that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin-catenin adhesion complex in mechanotransduction.

Implementation of Drive-Through Testing for COVID-19 Using an External Emergency Department Triage.

BACKGROUND: During the coronavirus disease 2019 (COVID-19) pandemic, healthcare systems in many regions of the country were being overwhelmed by large numbers of patients needing care. In this paper, we discuss use of an external emergency department (ED) site by a hospital system based in Charlotte, North Carolina to address concerns of a local surge similar to those seen around the country. OBJECTIVE: Demonstrate how expansion of ED facilities can increase efficiency of care for patients while also improving safety for clinicians, staff, and non-infected patients. METHODS: We describe development and implementation of our external ED drive-through testing sites during the COVID-19 pandemic. We collected data from three external ED sites in the Atrium Health system between March 15th and April 15th, 2020. Patients were included if they were seen at one of the sites and tested for COVID-19. There were no exclusion criteria. We analyzed the data to identify any differences in patient demographics between sites. RESULTS: We saw 580 patients across the three sites, 302 of whom met criteria for COVID-19 testing. The majority of patients tested were Caucasian females. The majority who tested positive, however, were males. Thirteen patients were redirected into the hospital ED for further medical evaluation. CONCLUSIONS: External expansion of the ED is an important strategy that can allow hospitals to accommodate potentially infectious patients while maintaining appropriate isolation and rapid throughput. Proper implementation of the right system to meet hospital-specific needs can prove effective for the healthcare system.

α-Catenin Structure and Nanoscale Dynamics in Solution and in Complex with F-Actin.

As a core component of the adherens junction, α-catenin stabilizes the cadherin/catenin complexes to the actin cytoskeleton for the mechanical coupling of cell-cell adhesion. α-catenin also modulates actin dynamics, cell polarity, and cell-migration functions that are independent of the adherens junction. We have determined the solution structures of the α-catenin monomer and dimer using in-line size-exclusion chromatography small-angle X-ray scattering, as well as the structure of α-catenin dimer in complex to F-actin filament using selective deuteration and contrast-matching small angle neutron scattering. We further present the first observation, to our knowledge, of the nanoscale dynamics of α-catenin by neutron spin-echo spectroscopy, which explicitly reveals the mobile regions of α-catenin that are crucial for binding to F-actin. In solution, the α-catenin monomer is more expanded than either protomer shown in the crystal structure dimer, with the vinculin-binding M fragment and the actin-binding domain being able to adopt different configurations. The α-catenin dimer in solution is also significantly more expanded than the dimer crystal structure, with fewer interdomain and intersubunit contacts than the crystal structure. When in complex to F-actin, the α-catenin dimer has an even more open and extended conformation than in solution, with the actin-binding domain further separated from the main body of the dimer. The α-catenin-assembled F-actin bundle develops into an ordered filament packing arrangement at increasing α-catenin/F-actin molar ratios. Together, the structural and dynamic studies reveal that α-catenin possesses dynamic molecular conformations that prime this protein to function as a mechanosensor protein.

Case Report: Life Saving Application of Commercial Tourniquet in Pediatric Extremity Hemorrhage.

Hemorrhage is the leading preventable cause of death in civilian and military trauma. Recent data from the ongoing conflicts in Iraq and Afghanistan suggest that early and aggressive tourniquet utilization is a safe and effective way to dramatically reduce mortality from extremity hemorrhage. As a result, prehospital tourniquet use is now endorsed by a majority of professional emergency medicine, emergency medical service and trauma professional societies. However, there currently exists scant evidence supporting the efficacy of commercially available tourniquets in controlling extremity hemorrhage in pediatric trauma patients.

Translating Tactical Combat Casualty Care Lessons Learned to the High-Threat Civilian Setting: Tactical Emergency Casualty Care and the Hartford Consensus.

Combat operations necessitate bold thought and afford the opportunity to rapidly evolve and improve trauma care. The development and maturation of Tactical Combat Casualty Care (TCCC) is an important example of a critical process improvement strategy that reduced mortality in high-threat combat-related trauma. The Committee for Tactical Emergency Casualty Care (C-TECC) adapted the lessons of TCCC to the civilian high-threat environment and provided important all-hazards response principles for austere, dynamic, and resource-limited environments. The Hartford Consensus mobilized the resources of the American College of Surgeons to drive public policy regarding a more singular focus: hemorrhage control. The combined efforts of C-TECC and Hartford Consensus have helped redefine the practice of trauma care in high-threat scenarios across the United States.

Phosphatidylinositol 4,5-bisphosphate clusters the cell adhesion molecule CD44 and assembles a specific CD44-Ezrin heterocomplex, as revealed by small angle neutron scattering.

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.

Tragedies Provide Learning Opportunities.

With the increasing frequency of mass-casualty events, the American College of Emergency Physicians has assembled a High Threat Task Force to look for ways to improve the emergency response to these events. The panel intends to focus on training and operations, but the ultimate goal is to find ways to eliminate preventable deaths. The panel intends to standardize and unify improvement efforts that have thus far been disparate. The co-chairman of the panel says one of the biggest gaps is the lack of evidence-based guidelines for how emergency personnel should respond to mass-shooting events. Noting that the incident command structure was created for fighting wildfires, the co-chairman of the task force notes that mass-shooting events require a more dynamic leadership response.

Law enforcement-applied tourniquets: a case series of life-saving interventions.

Although the epidemiology of civilian trauma is distinct from that encountered in combat, in both settings, extremity hemorrhage remains a major preventable cause of potential mortality. The current paper describes the largest case series in the literature in which police officers arriving prior to emergency medical services applied commercially available field tourniquets to civilian victims of violent trauma. Although all 3 patients with vascular injury arrived at the receiving emergency department in extremis, they were successfully resuscitated and survived to discharge without major morbidity. While this outcome is likely multifactorial and highlights the exceptional care delivered by the modern trauma system, tourniquet application appears to have kept critically injured patients alive long enough to reach definitive trauma care. No patient had a tourniquet-related complication. This case series suggests that law enforcement officers can effectively identify indications for tourniquets and rapidly apply such life-saving interventions.

Integration of energy analytics and smart energy microgrid into mobile medicine operations for the 2012 Democratic National Convention.

INTRODUCTION: Disasters create major strain on energy infrastructure in affected communities. Advances in microgrid technology offer the potential to improve "off-grid" mobile disaster medical response capabilities beyond traditional diesel generation. The Carolinas Medical Center's mobile emergency medical unit (MED-1) Green Project (M1G) is a multi-phase project designed to demonstrate the benefits of integrating distributive generation (DG), high-efficiency batteries, and "smart" energy utilization in support of major out-of-hospital medical response operations. METHODS: Carolinas MED-1 is a mobile medical facility composed of a fleet of vehicles and trailers that provides comprehensive medical care capacities to support disaster response and special-event operations. The M1G project partnered with local energy companies to deploy energy analytics and an energy microgrid in support of mobile clinical operations for the 2012 Democratic National Convention (DNC) in Charlotte, North Carolina (USA). Energy use data recorded throughout the DNC were analyzed to create energy utilization models that integrate advanced battery technology, solar photovoltaic (PV), and energy conservation measures (ECM) to improve future disaster response operations. RESULTS: The generators that supply power for MED-1 have a minimum loading ratio (MLR) of 30 kVA. This means that loads below 30 kW lead to diesel fuel consumption at the same rate as a 30 kW load. Data gathered from the two DNC training and support deployments showed the maximum load of MED-1 to be around 20 kW. This discrepancy in MLR versus actual load leads to significant energy waste. The lack of an energy storage system reduces generator efficiency and limits integration of alternative energy generation strategies. A storage system would also allow for alternative generation sources, such as PV, to be incorporated. Modeling with a 450 kWh battery bank and 13.5 kW PV array showed a 2-fold increase in potential deployment times using the same amount of fuel versus the current conventional system. CONCLUSIONS: The M1G Project demonstrated that the incorporation of a microgrid energy management system and a modern battery system maximize the MED-1 generators' output. Using a 450 kWh battery bank and 13.5 kW PV array, deployment operations time could be more than doubled before refueling. This marks a dramatic increase in patient care capabilities and has significant public health implications. The results highlight the value of smart-microgrid technology in developing energy independent mobile medical capabilities and expanding cost-effective, high-quality medical response.

Open conformation of ezrin bound to phosphatidylinositol 4,5-bisphosphate and to F-actin revealed by neutron scattering.

Ezrin is a member of the ezrin-radixin-moesin family (ERM) of adapter proteins that are localized at the interface between the cell membrane and the cortical actin cytoskeleton, and they regulate a variety of cellular functions. The structure representing a dormant and closed conformation of an ERM protein has previously been determined by x-ray crystallography. Here, using contrast variation small angle neutron scattering, we reveal the structural changes of the full-length ezrin upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) and to F-actin. Ezrin binding to F-actin requires the simultaneous binding of ezrin to PIP(2). Once bound to F-actin, the opened ezrin forms more extensive contacts with F-actin than generally depicted, suggesting a possible role of ezrin in regulating the interfacial structure and dynamics between the cell membrane and the underlying actin cytoskeleton. In addition, using gel filtration, we find that the conformational opening of ezrin in response to PIP(2) binding is cooperative, but the cooperativity is disrupted by a phospho-mimic mutation S249D in the 4.1-ezrin/radixin/moesin (FERM) domain of ezrin. Using surface plasmon resonance, we show that the S249D mutation weakens the binding affinity and changes the kinetics of 4.1-ERM to PIP(2) binding. The study provides the first structural view of the activated ezrin bound to PIP(2) and to F-actin.

Nanoscale protein dynamics: a new frontier for neutron spin echo spectroscopy.

Recent studies show that neutron spin echo spectroscopy (NSE) can reveal long-range protein domain motions on nanometer lengthscales and on nanosecond to microsecond timescales. This unique capability of NSE provides new opportunities to understand protein dynamics and functions, such as how binding signals are propagated in a protein to distal sites. Here we review our applications of NSE to the study of nanoscale protein domain motions in a set of cell signaling proteins. We summarize the theoretical framework we have developed, which allows one to interpret the NSE data (Biophys. J. 99, 3473 (2010) and Proc. Natl. Acad. Sci. USA 102, 17646 (2005)). Our theoretical framework uses simple concepts from nonequilibrium statistical mechanics, and does not require elaborate molecular dynamics simulations, complex fits to rotational motion, or elastic network models. It is thus more robust than multiparameter techniques that require untestable assumptions. We also demonstrate our experimental scheme involving deuterium labeling of a protein domain or a subunit in a protein complex. We show that our selective deuteration scheme can highlight and resolve specific domain dynamics from the abundant global translational and rotational motions in a protein. Our approach thus clears significant hurdles to the application of NSE for the study of protein dynamics in solution.

Incidence of fatal airway obstruction in police officers feloniously killed in the line of duty: a 10-year retrospective analysis.

BACKGROUND: According to US military data, airway obstruction is the third leading cause of possibly preventable death in combat. In the absence of law enforcement-specific medical training, military experience has been translated to the law enforcement sector. The purpose of this study was to determine whether airway obstruction represents a significant cause of possibly preventable death in police officers, and whether current military combat lifesaver training programs might have prevented these fatalities. METHODS: De-identified, open-source US Federal Bureau of Investigation (FBI) Uniform Crime Report Law Enforcement Officers Killed and Assaulted (LEOKA) data for the years 1998-2007 were reviewed. Cases were included if officers were on duty at the time of fatal injury and died within one hour from time of wounding from penetrating face or neck trauma. After case identification, letters requesting autopsy reports were sent to the departments of victim officers. Reports were abstracted into a Microsoft Excel database. RESULTS: During the study period, 42 of 533 victim officers met inclusion criteria. Departmental response rate was 85.7%. Autopsy reports were provided for 29 officers; 23 (54.8%) cases remained in the final analysis. All officers died from gunshot wounds. No coroner specifically identified airway obstruction as either a direct cause of death or contributing factor. Based upon autopsy findings, three of 341 officers possibly succumbed to airway trauma (0.9%; 95% CI, 0.0%-1.9%). Endotracheal intubation was the most common advanced airway management technique utilized during attempted resuscitation. CONCLUSION: The limited LEOKA data suggests that acute airway obstruction secondary to penetrating trauma appears to be a rare cause of possibly preventable death in police officers. Based upon the nature of airway trauma, nasopharyngeal airways would not be expected to be an effective lifesaving intervention. This study highlights the requirement for a comprehensive mortality and "near miss" database for law enforcement officers.

Observational study of organisational responses of 17 US hospitals over the first year of the COVID-19 pandemic.

OBJECTIVES: The COVID-19 pandemic has required significant modifications of hospital care. The objective of this study was to examine the operational approaches taken by US hospitals over time in response to the COVID-19 pandemic. DESIGN, SETTING AND PARTICIPANTS: This was a prospective observational study of 17 geographically diverse US hospitals from February 2020 to February 2021. OUTCOMES AND ANALYSIS: We identified 42 potential pandemic-related strategies and obtained week-to-week data about their use. We calculated descriptive statistics for use of each strategy and plotted percent uptake and weeks used. We assessed the relationship between strategy use and hospital type, geographic region and phase of the pandemic using generalised estimating equations (GEEs), adjusting for weekly county case counts. RESULTS: We found heterogeneity in strategy uptake over time, some of which was associated with geographic region and phase of pandemic. We identified a body of strategies that were both commonly used and sustained over time, for example, limiting staff in COVID-19 rooms and increasing telehealth capacity, as well as those that were rarely used and/or not sustained, for example, increasing hospital bed capacity. CONCLUSIONS: Hospital strategies during the COVID-19 pandemic varied in resource intensity, uptake and duration of use. Such information may be valuable to health systems during the ongoing pandemic and future ones.

Dignitary medicine: adapting prehospital, preventive, tactical and travel medicine to new populations.

Dignitary Protection Medicine (DPM) is a new area of medical expertise that incorporates elements of virtually all medical and surgical specialties, drawing heavily from travel, tactical and expedition medicine. The fundamentals of DPM stem from the experiences of White House, State Department and other physicians who have traveled extensively with dignitaries. Furthermore, increased international travel of business executives and political dignitaries has mandated a need for proficiency in this realm. We sought to define the requisite knowledge base and skill sets that form the foundation of this new area of specialization.

Disaster mobile health technology: lessons from Haiti.

INTRODUCTION: Mobile health (mHealth) technology can play a critical role in improving disaster victim tracking, triage, patient care, facility management, and theater-wide decision-making. PROBLEM: To date, no disaster mHealth application provides responders with adequate capabilities to function in an austere environment. METHODS: The Operational Medicine Institute (OMI) conducted a qualitative trial of a modified version of the off-the-shelf application iChart at the Fond Parisien Disaster Rescue Camp during the large-scale response to the January 12, 2010 earthquake in Haiti. RESULTS: The iChart mHealth system created a patient log of 617 unique entries used by on-the-ground medical providers and field hospital administrators to facilitate provider triage, improve provider handoffs, and track vulnerable populations such as unaccompanied minors, pregnant women, traumatic orthopedic injuries and specified infectious diseases. CONCLUSION: The trial demonstrated that even a non-disaster specific application with significant programmatic limitations was an improvement over existing patient tracking and facility management systems. A unified electronic medical record and patient tracking system would add significant value to first responder capabilities in the disaster response setting.

How Should Tactical Clinicians Help Make Use of Force More Just?

Law enforcement (LE) use of force (UOF) is a complex social, medical, and legal issue. In recent years, highly visible and publicized UOF incidents have sparked public outrage about racial inequity and motivated broad institutional reflection on sanctioning violence in the name of protecting communities. A public health approach to violence and to LE-UOF is required to develop 21st-century policing recommendations. Tactical emergency medical services physicians are uniquely positioned to convene and to serve as advisors, educators, and advocates to LE departments, community leaders, and public policy developers in order to improve UOF policies.

El uso de la fuerza (UOF, por sus siglas en inglés) por parte de las fuerzas de orden público (LE, por sus siglas en inglés) es un tema complejo a nivel social, médico y legal. En los últimos años, ha habido incidentes de UOF muy visibles y publicitados que han enfurecido a la sociedad respecto de la inequidad racial y han motivado una reflexión institucional amplia sobre la autorización de la violencia en pos de proteger a las comunidades. Se requiere un enfoque desde la salud pública a la violencia y al UOF de las LE a fin de desarrollar recomendaciones para la policía del siglo XXI. Los médicos de los servicios de emergencia táctica cuentan con una posición única para mediar y dar servicio en calidad de asesores, educadores y defensores de departamentos, líderes de la comunidad y creadores de políticas públicas de las LE a fin de mejorar las políticas del UOF.