Recombinant Expression and Purification of Large Bacterial Multienzyme Assemblies for Biosynthetic Processes.

Many biosynthetic transformations have strict spatial and temporal requirements that necessitate the physical association of multiple enzymes for proper function. Here, we describe protocols for obtaining large multienzyme assemblies (>500 kDa) by recombinant expression in Escherichia coli. We focus on assemblies from stand-alone enzymes joined by intermolecular forces rather than multiple catalytic domains from a single polypeptide chain.

Door-to-Targeted Temperature Management Initiation Time and Outcomes in Out-of-Hospital Cardiac Arrest: Insights From the Continuous Chest Compressions Trial.

Background Targeted temperature management ( TTM ) is a recommended treatment modality to improve neurological outcomes in patients with out-of-hospital cardiac arrest. The impact of the duration from hospital admission to TTM initiation (door-to- TTM ; DTT ) on clinical outcomes has not been well elucidated. We hypothesized that shorter DTT initiation intervals would be associated with improved survival with favorable neurological outcome.

Mechanism of a Standalone β-Lactone Synthetase: New Continuous Assay for a Widespread ANL Superfamily Enzyme.

Enzyme-catalyzed β-lactone formation from β-hydroxy acids is a crucial step in bacterial biosynthesis of β-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for β-lactone synthetase activity using synthetic β-hydroxy acid substrates with alkene or alkyne moieties. β-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy.

Biosynthesis and chemical diversity of β-lactone natural products.

Covering: up to 2018 β-Lactones are strained rings that are useful organic synthons and pharmaceutical warheads. Over 30 core scaffolds of β-lactone natural products have been described to date, many with potent bioactivity against bacteria, fungi, or human cancer cell lines. β-Lactone natural products are chemically diverse and have high clinical potential, but production of derivatized drug leads has been largely restricted to chemical synthesis partly due to gaps in biochemical knowledge about β-lactone biosynthesis.

The role of OleA His285 in orchestration of long-chain acyl-coenzyme A substrates.

Renewable production of hydrocarbons is being pursued as a petroleum-independent source of commodity chemicals and replacement for biofuels. The bacterial biosynthesis of long-chain olefins represents one such platform. The process is initiated by OleA catalyzing the condensation of two fatty acyl-coenzyme A substrates to form a β-keto acid.

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis.

In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, encodes the enzymes necessary to produce -olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching.

Antiarrhythmic Drugs for Nonshockable-Turned-Shockable Out-of-Hospital Cardiac Arrest: The ALPS Study (Amiodarone, Lidocaine, or Placebo).

Background: Out-of-hospital cardiac arrest (OHCA) commonly presents with nonshockable rhythms (asystole and pulseless electric activity). It is unknown whether antiarrhythmic drugs are safe and effective when nonshockable rhythms evolve to shockable rhythms (ventricular fibrillation/pulseless ventricular tachycardia [VF/VT]) during resuscitation.

Methods: Adults with nontraumatic OHCA, vascular access, and VF/VT anytime after ≥1 shock(s) were prospectively randomized, double-blind, to receive amiodarone, lidocaine, or placebo by paramedics.

OleB from Bacterial Hydrocarbon Biosynthesis Is a β-Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases.

OleB is an α/β-hydrolase found in bacteria that biosynthesize long-chain olefinic hydrocarbons, but its function has remained obscure. We report that OleB from the Gram-negative bacterium Xanthomonas campestris performs an unprecedented β-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-β-lactones and no discernible activity with trans-β-lactones, consistent with the known configuration of pathway intermediates.

Gains of Continuing Resuscitation in Refractory Out-of-hospital Cardiac Arrest: A Model-based Analysis to Identify Deaths Due to Intra-arrest Prognostication.

Objective: Prognostication bias, in which a clinician predicts a negative outcome and terminates resuscitation (TR) thereby ensuring a poor outcome, is a rarely identified limitation of out-of-hospital cardiac arrest (OHCA) research. We sought to estimate the number of deaths due to intra-arrest prognostication in a cohort of OHCA's, and use this data to estimate the incremental benefit of continuing resuscitation.

Methods: This study examined a cohort of consecutive non-traumatic EMS-treated OHCAs from a provincial ambulance service, between 2007 and 2011 inclusive.

Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis.

Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from in and purification over His and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged.

β-Lactone Synthetase Found in the Olefin Biosynthesis Pathway.

The first β-lactone synthetase enzyme is reported, creating an unexpected link between the biosynthesis of olefinic hydrocarbons and highly functionalized natural products. The enzyme OleC, involved in the microbial biosynthesis of long-chain olefinic hydrocarbons, reacts with syn- and anti-β-hydroxy acid substrates to yield cis- and trans-β-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate β-lactone rings, suggesting a common mechanism of biosynthesis.

Unchanged pediatric out-of-hospital cardiac arrest incidence and survival rates with regional variation in North America.

Aim: Outcomes for pediatric out-of-hospital cardiac arrest (OHCA) are poor. Our objective was to determine temporal trends in incidence and mortality for pediatric OHCA.

Methods: Adjusted incidence and hospital mortality rates of pediatric non-traumatic OHCA patients from 2007-2012 were analyzed using the 9 region Resuscitation Outcomes Consortium-Epidemiological Registry (ROC-Epistry) database.

An Environmental Scan of Academic Emergency Medicine at the 17 Canadian Medical Schools: Why Does this Matter to Emergency Physicians?

Objective: We sought to conduct a major objective of the CAEP Academic Section, an environmental scan of the academic emergency medicine programs across the 17 Canadian medical schools.

Methods: We developed an 84-question questionnaire, which was distributed to academic heads. The responses were validated by phone by the lead author to ensure that the questions were answered completely and consistently.

External validation of the emergency department assessment of chest pain score accelerated diagnostic pathway (EDACS-ADP).

Objective: The emergency department assessment of chest pain score accelerated diagnostic pathway (EDACS-ADP) facilitates low-risk ED chest pain patients early to outpatient investigation. We aimed to validate this rule in a North American population.

Methods: We performed a retrospective validation of the EDACS-ADP using 763 chest pain patients who presented to St Paul's Hospital, Vancouver, Canada, between June 2000 and January 2003.

Amiodarone, Lidocaine, or Placebo in Out-of-Hospital Cardiac Arrest.

Background: Antiarrhythmic drugs are used commonly in out-of-hospital cardiac arrest for shock-refractory ventricular fibrillation or pulseless ventricular tachycardia, but without proven survival benefit.

Methods: In this randomized, double-blind trial, we compared parenteral amiodarone, lidocaine, and saline placebo, along with standard care, in adults who had nontraumatic out-of-hospital cardiac arrest, shock-refractory ventricular fibrillation or pulseless ventricular tachycardia after at least one shock, and vascular access. Paramedics enrolled patients at 10 North American sites.

Association of advanced airway device with chest compression fraction during out-of-hospital cardiopulmonary arrest.

Background: Select Emergency Medical Services (EMS) practitioners substitute endotracheal intubation (ETI) with supraglottic airway (SGA) insertion to minimize CPR chest compression interruptions, but the resulting effects upon chest compression fraction (CCF) are unknown. We sought to determine the differences in CCF between adult out-of-hospital cardiac arrest (OHCA) receiving ETI and those receiving SGA.

Methods: We studied adult, non-traumatic OHCA patients enrolled in the Resuscitation Outcomes Consortium (ROC) Prehospital Resuscitation using an Impedance valve and an Early vs.

A quantitative analysis of out-of-hospital pediatric and adolescent resuscitation quality--A report from the ROC epistry-cardiac arrest.

Aim: High-quality cardiopulmonary resuscitation (CPR) may improve survival. The quality of CPR performed during pediatric out-of-hospital cardiac arrest (p-OHCA) is largely unknown. The main objective of this study was to describe the quality of CPR performed during p-OHCA resuscitation attempts.

What is the optimal chest compression depth during out-of-hospital cardiac arrest resuscitation of adult patients?

Background: The 2010 American Heart Association guidelines suggested an increase in cardiopulmonary resuscitation compression depth with a target >50 mm and no upper limit. This target is based on limited evidence, and we sought to determine the optimal compression depth range.

Methods And Results: We studied emergency medical services-treated out-of-hospital cardiac arrest patients from the Resuscitation Outcomes Consortium Prehospital Resuscitation Impedance Valve and Early Versus Delayed Analysis clinical trial and the Epistry-Cardiac Arrest database.

Early coronary angiography and induced hypothermia are associated with survival and functional recovery after out-of-hospital cardiac arrest.

Background: The rate and effect of coronary interventions and induced hypothermia after out-of-hospital cardiac arrest (OHCA) are unknown. We measured the association of early (≤24h after arrival) coronary angiography, reperfusion, and induced hypothermia with favorable outcome after OHCA.

Methods: We performed a secondary analysis of a multicenter clinical trial (NCT00394706) conducted between 2007 and 2009 in 10 North American regions.

Endotracheal intubation versus supraglottic airway insertion in out-of-hospital cardiac arrest.

Objective: To simplify airway management and minimize cardiopulmonary resuscitation (CPR) chest compression interruptions, some emergency medical services (EMS) practitioners utilize supraglottic airway (SGA) devices instead of endotracheal intubation (ETI) as the primary airway adjunct in out-of-hospital cardiac arrest (OHCA). We compared the outcomes of patients receiving ETI with those receiving SGA following OHCA.

Methods: We performed a secondary analysis of data from the multicenter Resuscitation Outcomes Consortium (ROC) PRIMED trial.

What is the role of chest compression depth during out-of-hospital cardiac arrest resuscitation?

Background: The 2010 international guidelines for cardiopulmonary resuscitation recently recommended an increase in the minimum compression depth from 38 to 50 mm, although there are limited human data to support this. We sought to study patterns of cardiopulmonary resuscitation compression depth and their associations with patient outcomes in out-of-hospital cardiac arrest cases treated by the 2005 guideline standards.

Design: Prospective cohort.

Early versus later rhythm analysis in patients with out-of-hospital cardiac arrest.

Background: In a departure from the previous strategy of immediate defibrillation, the 2005 resuscitation guidelines from the American Heart Association-International Liaison Committee on Resuscitation suggested that emergency medical service (EMS) personnel could provide 2 minutes of cardiopulmonary resuscitation (CPR) before the first analysis of cardiac rhythm. We compared the strategy of a brief period of CPR with early analysis of rhythm with the strategy of a longer period of CPR with delayed analysis of rhythm.

Methods: We conducted a cluster-randomized trial involving adults with out-of-hospital cardiac arrest at 10 Resuscitation Outcomes Consortium sites in the United States and Canada.

Increased survival after EMS witnessed cardiac arrest. Observations from the Resuscitation Outcomes Consortium (ROC) Epistry-Cardiac arrest.

Background: Out of hospital cardiac arrest (OHCA) is common and lethal. It has been suggested that OHCA witnessed by EMS providers is a predictor of survival because advanced help is immediately available. We examined EMS witnessed OHCA from the Resuscitation Outcomes Consortium (ROC) to determine the effect of EMS witnessed vs.

A geospatial assessment of transport distance and survival to discharge in out of hospital cardiac arrest patients: Implications for resuscitation centers.

Objectives: National leaders have suggested that patients with an out of hospital cardiac arrest (OOHCA) may benefit from transport to specialized hospitals. We sought to assess the survival of OOHCA patients by transport distance and hospital proximity.

Methods: Prospective, cohort study of OOHCA patients in 11 Resuscitation Outcomes Consortium (ROC) sites across North America.