Structure of orthoreovirus RNA chaperone σNS, a component of viral replication factories.

The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication.

Some viruses need to phase-separate to replicate.

Replication of rotavirus, an important cause of gastroenteritis in children, proceeds in large, easily discernible cytoplasmic structures, called viroplasms or viral factories, but mechanisms underlying their formation and function in infected cells have remained mysterious. In this issue, Geiger et al (2021) used a combination of in silico, in vitro, and cell-based approaches to define how two essential rotavirus nonstructural proteins, NSP2 and NSP5, form liquid-liquid phase-separated condensates as the structural foundation of rotavirus factories.

Reovirus Nonstructural Protein σNS Recruits Viral RNA to Replication Organelles.

The function of the mammalian orthoreovirus (reovirus) σNS nonstructural protein is enigmatic. σNS is an RNA-binding protein that forms oligomers and enhances the stability of bound RNAs, but the mechanisms by which it contributes to reovirus replication are unknown. To determine the function of σNS-RNA binding in reovirus replication, we engineered σNS mutants deficient in RNA-binding capacity. We found that alanine substitutions of positively charged residues in a predicted RNA-binding domain decrease RNA-dependent oligomerization. To define steps in reovirus replication facilitated by the RNA-binding property of σNS, we established a complementation system in which wild-type or mutant forms of σNS could be tested for the capacity to overcome inhibition of σNS expression. Mutations in σNS that disrupt RNA binding also diminish viral replication and σNS distribution to viral factories. Moreover, viral mRNAs only incorporate into viral factories or factory-like structures (formed following expression of nonstructural protein µNS) when σNS is present and capable of binding RNA. Collectively, these findings indicate that σNS requires positively charged residues in a putative RNA-binding domain to recruit viral mRNAs to sites of viral replication and establish a function for σNS in reovirus replication. IMPORTANCE Viral replication requires the formation of neoorganelles in infected cells to concentrate essential viral and host components. However, for many viruses, it is unclear how these components coalesce into neoorganelles to form factories for viral replication. We discovered that two mammalian reovirus nonstructural proteins act in concert to form functioning viral factories. Reovirus µNS proteins assemble into exclusive factory scaffolds that require reovirus σNS proteins for efficient viral mRNA incorporation. Our results demonstrate a role for σNS in RNA recruitment to reovirus factories and, more broadly, show how a cytoplasmic non-membrane-enclosed factory is formed by an RNA virus. Understanding the mechanisms of viral factory formation will help identify new targets for antiviral therapeutics that disrupt assembly of these structures and inform the use of nonpathogenic viruses for biotechnological applications.

A modified lysosomal organelle mediates nonlytic egress of reovirus.

Mammalian orthoreoviruses (reoviruses) are nonenveloped viruses that replicate in cytoplasmic membranous organelles called viral inclusions (VIs) where progeny virions are assembled. To better understand cellular routes of nonlytic reovirus exit, we imaged sites of virus egress in infected, nonpolarized human brain microvascular endothelial cells (HBMECs) and observed one or two distinct egress zones per cell at the basal surface. Transmission electron microscopy and 3D electron tomography (ET) of the egress zones revealed clusters of virions within membrane-bound structures, which we term membranous carriers (MCs), approaching and fusing with the plasma membrane. These virion-containing MCs emerged from larger, LAMP-1-positive membranous organelles that are morphologically compatible with lysosomes. We call these structures sorting organelles (SOs). Reovirus infection induces an increase in the number and size of lysosomes and modifies the pH of these organelles from ∼4.5-5 to ∼6.1 after recruitment to VIs and before incorporation of virions. ET of VI-SO-MC interfaces demonstrated that these compartments are connected by membrane-fusion points, through which mature virions are transported. Collectively, our results show that reovirus uses a previously undescribed, membrane-engaged, nonlytic egress mechanism and highlights a potential new target for therapeutic intervention.

Direct, operando observation of the bilayer solid electrolyte interphase structure: Electrolyte reduction on a non-intercalating electrode.

The solid electrolyte interphase (SEI) remains a central challenge to lithium-ion battery durability, in part due to poor understanding of the basic chemistry responsible for its formation and evolution. In this study, the SEI on a non-intercalating tungsten anode is measured by operando neutron reflectometry and quartz crystal microbalance. A dual-layer SEI is observed, with a 3.7 nm thick inner layer and a 15.4 nm thick outer layer. Such structures have been proposed in the literature, but have not been definitively observed via neutron reflectometry. The SEI mass per area was 1207.2 ng/cm2, and QCM provides insight into the SEI formation dynamics during a negative-going voltage sweep and its evolution over multiple cycles. Monte Carlo simulations identify SEI chemical compositions consistent with the combined measurements. The results are consistent with a primarily inorganic, dense inner layer and a primarily organic, porous outer layer, directly confirming structures proposed in the literature. Further refinement of techniques presented herein, coupled with additional complementary measurements and simulations, can give quantitative insight into SEI formation and evolution as a function of battery materials and cycling conditions. This, in turn, will enable scientifically-guided design of durable, conductive SEI layers for Li-ion batteries for a range of applications.

Reovirus σNS and µNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles.

Like most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and µNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.IMPORTANCE Viruses modify cellular structures to build replication organelles. These organelles serve as sites of viral genome replication and particle morphogenesis and are essential for viral infection. However, how these organelles are constructed is not well understood. We found that the replication organelles of mammalian reoviruses are formed by collections of membranous tubules and vesicles derived from extensive remodeling of the peripheral endoplasmic reticulum (ER). We also observed that ER tubulation and vesiculation are triggered by the reovirus σNS and µNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles and provide functions for two enigmatic reovirus replication proteins. Most importantly, this research uncovers a new mechanism by which viruses form factories for particle assembly.

EMS activation of the cardiac catheterization laboratory is associated with process improvements in the care of myocardial infarction patients.

INTRODUCTION: Prior data from our institution suggested that our paramedics can accurately interpret ST-segment elevation myocardial infarction (STEMI) on prehospital 12-lead electrocardiograms (ECGs), and that activation of the cardiac catheterization laboratory by paramedics immediately upon diagnosing STEMI at the scene could potentially decrease door-to-balloon (D2B) times. A "field activation" protocol was thus initiated in May 2010. This study examined D2B times and compliance with the national 90-minute D2B performance benchmark in the first 14 months. Hypothesis. We hypothesized that D2B times would be shorter, and 90-minute compliance better, when the catheterization laboratory was activated by emergency medical services (EMS), compared with when either EMS failed to activate the catheterization laboratory or when the STEMI patient arrived by means other than EMS. METHODS: For this prospective, observational study, EMS and hospital data were reviewed for consecutive STEMI patients at a single hospital between May 2010 and July 2011. Patients were categorized as: 1) EMS field activations, 2) patients transported by EMS without EMS catheterization laboratory activation (e.g., ambulance from outside our area, paramedic missed STEMI/protocol violation), or 3) walk-in STEMI patient. Data were manipulated in Excel, means with standard deviations (SDs) and 95% confidence intervals (95% CIs) were determined, and analysis of variance (ANOVA) with Dunnett's correction was used to compare groups. RESULTS: There were 38 EMS field activations, 47 nonactivation EMS STEMI arrivals, and 28 walk-in STEMI patients. The mean (±SD) D2B times were 37 (±17), 87 (±40), and 80 (±23) minutes, respectively. D2B time was better for the EMS field activations than for either nonactivation EMS transports (difference of means 35.3 min, 95% CI 22.3-48.3 min, p < 0.001) or walk-in patients (difference of means 37.0 min, 95% CI 21.8-52.2 min, p < 0.001). Compliance with the 90-minute D2B benchmark was 100%, 72%, and 68%, respectively, and was better for the EMS field activations than for either of the other groups (p < 0.001). CONCLUSIONS: In the system studied, EMS field activation of the catheterization laboratory for patients with STEMI is associated with shorter D2B times and better compliance with 90-minute benchmarks than ED activation for either walk-in STEMI patients or STEMI patients arriving by EMS without field activation. Improvements are needed in compliance with the field activation protocol to maximize these benefits. Key words: emergency medical services; emergency medical technicians; electrocardiography; myocardial infarction; heart catheterization.

A descriptive study of the "lift-assist" call.

INTRODUCTION: Responses for "lift assists" (LAs) are common in many emergency medical services (EMS) systems, and result when a person dials 9-1-1 because of an inability to get up, is subsequently determined to be uninjured, and is not transported for further medical attention. Although LAs often involve recurrent calls and are generally not reimbursable, little is known of their operational effects on EMS systems. We hypothesized that LAs present an opportunity for earlier treatment of subtle-onset medical conditions and injury prevention interventions in a population at high risk for falls. Objectives. To quantify LA calls in one community, describe EMS returns to the same address within 30 days following an index LA call, and characterize utilization of EMS by LA patients. METHODS: Data from the computer-aided dispatch (CAD) system of a suburban fire-based EMS system were retrospectively reviewed. All LAs from 2004 to 2009 were identified using "exit codes" transmitted by paramedics after each call. The number and nature of return visits to the same address within 30 days were examined. RESULTS: From 2004 through 2009, there were 1,087 LA responses (4.8% of EMS incidents) to 535 different addresses. Two-thirds of the LA calls (726; 66.8%) were to one-third of these addresses (174 addresses; 32.5%); 563 of the return calls to the same address occurred within 30 days after the index LA. For 214 of these return visits, it was possible to compare patient age and sex with those associated with the initial LA, revealing that 85% of return visits were likely for the same patients. Of these, 38.5% were for another LA/refusal of transport, 8.2% for falls and other injuries, and 47.3% for medical complaints. Hospital transport was required in 55.5% of these return visits. The EMS crews averaged 21.5 minutes out of service per LA call. CONCLUSION: Lift-assist calls are associated with substantial subsequent utilization of EMS, and should trigger fall prevention and other safety interventions. Based on our data, these calls may be early indicators of medical problems that require more aggressive evaluation.

Prehospital electronic patient care report systems: early experiences from emergency medical services agency leaders.

BACKGROUND: As the United States embraces electronic health records (EHRs), improved emergency medical services (EMS) information systems are also a priority; however, little is known about the experiences of EMS agencies as they adopt and implement electronic patient care report (e-PCR) systems. We sought to characterize motivations for adoption of e-PCR systems, challenges associated with adoption and implementation, and emerging implementation strategies. METHODS: We conducted a qualitative study using semi-structured in-depth interviews with EMS agency leaders. Participants were recruited through a web-based survey of National Association of EMS Physicians (NAEMSP) members, a didactic session at the 2010 NAEMSP Annual Meeting, and snowball sampling. Interviews lasted approximately 30 minutes, were recorded and professionally transcribed. Analysis was conducted by a five-person team, employing the constant comparative method to identify recurrent themes. RESULTS: Twenty-three interviewees represented 20 EMS agencies from the United States and Canada; 14 EMS agencies were currently using e-PCR systems. The primary reason for adoption was the potential for e-PCR systems to support quality assurance efforts. Challenges to e-PCR system adoption included those common to any health information technology project, as well as challenges unique to the prehospital setting, including: fear of increased ambulance run times leading to decreased ambulance availability, difficulty integrating with existing hospital information systems, and unfunded mandates requiring adoption of e-PCR systems. Three recurring strategies emerged to improve e-PCR system adoption and implementation: 1) identify creative funding sources; 2) leverage regional health information organizations; and 3) build internal information technology capacity. CONCLUSION: EMS agencies are highly motivated to adopt e-PCR systems to support quality assurance efforts; however, adoption and implementation of e-PCR systems has been challenging for many. Emerging strategies from EMS agencies and others that have successfully implemented EHRs may be useful in expanding e-PCR system use and facilitating this transition for other EMS agencies.

Metabolic syndrome and ectopic fat deposition: what can CT and MR provide?

OBJECTIVES: Metabolic syndrome affects 20-30% of adults and is increasing in prevalence, making it a leading public health issue. Radiologists often encounter images of obese patients during routine studies and are in a unique position to address the importance of excess fat and need to be aware of the spectrum of pathologic consequences in different organ systems. In this review, the role of CT and MR imaging in assessment of patients with metabolic syndrome will be reviewed and the constellation of structural and functional changes in the major affected organ systems due to ectopic fatty deposition will be discussed. METHODS: We specifically discuss the pathophysiology of metabolic syndrome, visceral versus subcutaneous obesity, cardiac lipomatosis, nonalcoholic fatty liver disease, nonalcoholic fatty pancreas disease, and fat deposition in other organs. CONCLUSION: Many of the multisystem manifestations of metabolic syndrome can be visualized on routine CT and MR images and radiologists can provide clinicians with important data regarding anatomic and pathologic distribution of fat in different organs. Perhaps the visualization of the fatty changes will provide tangible evidence to motivate patients to begin lifestyle modification.

Early cardiac catheterization laboratory activation by paramedics for patients with ST-segment elevation myocardial infarction on prehospital 12-lead electrocardiograms.

BACKGROUND: Prompt reperfusion in ST-segment elevation myocardial infarction (STEMI) saves lives. Although studies have shown that paramedics can reliably interpret STEMI on prehospital 12-lead electrocardiograms (p12ECGs), prehospital activation of the cardiac catheterization laboratory by emergency medical services (EMS) has not yet gained widespread acceptance. OBJECTIVE: To quantify the potential reduction in time to percutaneous coronary intervention (PCI) by early prehospital activation of the cardiac catheterization laboratory in STEMI. METHODS: This prospective, observational study enrolled all patients diagnosed with STEMI by paramedics in a mid-sized regional EMS system. Patients were enrolled if: 1) the paramedic interpreted STEMI on the p12ECG, 2) the Acute Cardiac Ischemia Time-Insensitive Predictive Instrument (ACI-TIPI) score was 75% or greater, and 3) the patient was transported to either of two area PCI centers. Data recorded included the time of initial EMS "STEMI alert" from the scene, time of arrival at the emergency department (ED), and time of actual catheterization laboratory activation by the ED physician, all using synchronized clocks. The primary outcome measure was the time difference between the STEMI alert and the actual activation (i.e., potential time savings). The false-positive rate (patients incorrectly diagnosed with STEMI by paramedics) was also calculated and compared with a locally accepted false-positive rate of 10%. RESULTS: Twelve patients were enrolled prior to early termination of the study. The mean and median potential time reductions were 15 and 11 minutes, respectively (range 7-29 minutes). There was one false STEMI alert (8.3% false-positive rate) for a patient with a right bundle branch block who subsequently had a non-ST-segment elevation myocardial infarction. The study was terminated when our cardiologists adopted a prehospital catheterization laboratory activation protocol based on our initial data. CONCLUSION: Important reductions in time to reperfusion seem possible by activation of the catheterization laboratory by EMS from the scene, with an acceptably low false-positive rate in this small sample. This type of clinical research can inform multidisciplinary policies and bring about meaningful clinical practice changes.

Severe angioedema in myxedema coma: a difficult airway in a rare endocrine emergency.

Myxedema coma is the most lethal manifestation of hypothyroidism. It is a true medical emergency and can result in profound hemodynamic instability and airway compromise. Myxedema coma currently remains a diagnostic challenge due to the rarity of cases seen today, and failure to promptly initiate therapy with replacement thyroid hormone can be fatal. As thyroid hormone therapy can take days or weeks to reverse the manifestations of myxedema coma, interim supportive therapy is critical while awaiting clinical improvement. Some patients will require endotracheal intubation in the emergency department (ED), and physicians should be aware that unanticipated posterior pharyngeal edema in myxedema coma could severely complicate airway management. Although mechanical ventilation is a well-described adjunctive therapy for myxedema coma, reports of the potential difficulty in securing a definitive airway in these patients are rare. We describe a case of an unidentified woman who presented to the ED with myxedema coma requiring urgent endotracheal intubation and was found to have extensive posterior pharyngeal angioedema inconsistent with her relatively benign external examination. This case highlights the typical features of myxedema coma and discusses our necessity for a rescue device in definitive endotracheal tube placement. Emergency physicians should anticipate a potentially difficult airway in all myxedema coma patients regardless of the degree of external facial edema present.

Advanced cardiac life support and defibrillation in severe hypothermic cardiac arrest.

The application of Advanced Cardiac Life Support (ACLS) in severe hypothermic cardiac arrest remains controversial. While the induction of mild hypothermia has been shown to improve outcomes in patients already resuscitated from cardiac arrest, it is unknown whether ACLS protocols are effective during the resuscitation of the severely hypothermic cardiac arrest patient. We describe a case of a 47-year-old man who was successfully resuscitated from a ventricular fibrillation (VF) arrest with a core body temperature of 26.4 degrees C. The patient had been found unresponsive in a bathtub of cold water following an apparent suicide attempt. An incorrect pronouncement of death by the fire department delayed his transport to the hospital by more than four hours. Once in the emergency department (ED), the patient sustained a VF cardiac arrest and was successfully defibrillated using ACLS protocols. He ultimately survived his hospitalization with near-complete neurologic recovery. In this case report, we discuss the application of ACLS to the resuscitation of the hypothermic cardiac arrest patient as well as the issues involved in the prehospital determination of death.

A novel model of blunt thoracic aortic injury: a mechanism confirmed?

BACKGROUND: There is no consensus on the mechanism of traumatic injury to the thoracic aorta and no reproducible animal model. Advances in injury scene analysis suggest that lateral and oblique force vectors cause aortic injury. We hypothesized that the spectrum of aortic injury could be reproduced in an animal model by application of an obliquely directed load to the pressurized aorta. METHODS: Graded air impulses of 80, 100, 110, and 120 pounds per square inch (PSI) were delivered to the descending thoracic aorta of 19 swine with a novel pneumatic device. Aortic isthmus strain was recorded with microminiature probes. Gross and microscopic injury was recorded with digital photography. RESULTS: The spectrum of human aortic injury was reproduced in this model. Deep injuries to the aortic media were common. The majority of injuries occurred within the region of the isthmus. Impulse pressure of 120 PSI caused transections, whereas lower impulse pressure resulted in less severe injuries. Aortic isthmus strain was greater in the animals exposed to 120 PSI than those receiving lower PSI (19.6 +/- 4.9% vs. 8.7 +/- 2.5%, p = 0.067). CONCLUSIONS: Direct loading of the pressurized descending thoracic aorta causes isthmus injury secondary to aortic wall strain. Deep medial lesions are common and could propagate soon after injury to form pseudoaneurysms. A critical load is required to cause complete uncontained transection with exsanguination, which may have relevance to injury scene death.

Predictors of complications after a prospective evaluation of diagnostic and therapeutic endovascular procedures.

OBJECTIVE: To prospectively evaluate complications after diagnostic and therapeutic endovascular procedures (DTEPs) and determine what factors are predictive. METHODS: From December 2002 to December 2003, all patients undergoing DTEPs performed by university vascular surgeons in a catheterization laboratory were prospectively evaluated. Medical demographics, procedure-related details, and type and severity of complications were recorded at the time of the procedure, during the first 24 hours, and at 2 to 4 weeks. Complications were classified as local vascular (LV), local nonvascular (LNV), systemic remote (SR), and major, minor, and nonsignificant. RESULTS: Three hundred-three DTEPs were performed (54.5% DEPs, 45.5% TEPs). At the time of DTEP, 28 complications occurred in 23 patients: 10 LV (3.3%), 15 LNV (5.0%), and 3 SR (1.0%). At 24 hours, 26 complications occurred in 25 patients: 5 LV (1.7%), 7 LNV (2.3%), and 14 SR (4.7%). At 2 to 4 weeks, 26 complications occurred 25 patients: 5 LV (1.7%), 7 LNV (2.3%), and 14 SR (4.7%). The combined major (7.3%) and minor (4.3%) complication rate attributed to DTEPs was 11.6%. Significant predictors (P < .05) by multivariate analysis included thrombolysis, prior stroke, an additional procedure during the study period, and diabetes mellitus (odds ratios: 9.1, 3.2, 2.7, and 2.4, respectively). CONCLUSION: According to newly applied reporting standards, the prospective evaluation of DTEPs reveals that complications are uniformly distributed by type and follow-up period. Just over 1 in 10 patients will suffer either a major or minor complication. Potential predictors have been identified that may assist in patient selection and treatment plans to lower complications resulting from DTEPs.

A phase space spline smoother for fitting trajectories.

This paper presents a phase space spline smoother, which is especially useful for finding a best-fit trajectory from multiple examples of a given physical motion. Unlike conventional spline smoothers, the phase space spline smoother can simultaneously fit position and velocity information. The use of velocity information is important for modeling the dynamic motion of physical systems because the state space of these systems typically includes both position and velocity variables. A detailed description of the computational procedure is presented, along with a discussion of computational expense and practical guidelines for variance estimation, preprocessing of the target dataset, smoothing of multidimensional datasets, and cross-validation for selection of smoothing weights. The smoother is demonstrated on a dataset of handwriting motions.