Biosynthesis and chemical diversity of ß-lactone natural products.

Covering: up to 2018 ß-Lactones are strained rings that are useful organic synthons and pharmaceutical warheads. Over 30 core scaffolds of ß-lactone natural products have been described to date, many with potent bioactivity against bacteria, fungi, or human cancer cell lines. ß-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 ß-lactone biosynthesis. Here we review recent discoveries in enzymatic ß-lactone ring closure via ATP-dependent synthetases, intramolecular cyclization from seven-membered rings, and thioesterase-mediated cyclization during release from nonribosomal peptide synthetase assembly lines. We also comprehensively cover the diversity and taxonomy of source organisms for ß-lactone natural products including their isolation from bacteria, fungi, plants, insects, and marine sponges. This work identifies computational and experimental bottlenecks and highlights future directions for genome-based discovery of biosynthetic gene clusters that may produce novel compounds with ß-lactone rings.

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. The primary outcome was survival to hospital discharge; the secondary outcome was favorable neurologic function at discharge. The per-protocol (primary analysis) population included all randomly assigned participants who met eligibility criteria and received any dose of a trial drug and whose initial cardiac-arrest rhythm of ventricular fibrillation or pulseless ventricular tachycardia was refractory to shock. RESULTS: In the per-protocol population, 3026 patients were randomly assigned to amiodarone (974), lidocaine (993), or placebo (1059); of those, 24.4%, 23.7%, and 21.0%, respectively, survived to hospital discharge. The difference in survival rate for amiodarone versus placebo was 3.2 percentage points (95% confidence interval [CI], -0.4 to 7.0; P=0.08); for lidocaine versus placebo, 2.6 percentage points (95% CI, -1.0 to 6.3; P=0.16); and for amiodarone versus lidocaine, 0.7 percentage points (95% CI, -3.2 to 4.7; P=0.70). Neurologic outcome at discharge was similar in the three groups. There was heterogeneity of treatment effect with respect to whether the arrest was witnessed (P=0.05); active drugs were associated with a survival rate that was significantly higher than the rate with placebo among patients with bystander-witnessed arrest but not among those with unwitnessed arrest. More amiodarone recipients required temporary cardiac pacing than did recipients of lidocaine or placebo. CONCLUSIONS: Overall, neither amiodarone nor lidocaine resulted in a significantly higher rate of survival or favorable neurologic outcome than the rate with placebo among patients with out-of-hospital cardiac arrest due to initial shock-refractory ventricular fibrillation or pulseless ventricular tachycardia. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT01401647.).

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

Enzyme-catalyzed ß-lactone formation from ß-hydroxy acids is a crucial step in bacterial biosynthesis of ß-lactone natural products and membrane hydrocarbons. We developed a novel, continuous assay for ß-lactone synthetase activity using synthetic ß-hydroxy acid substrates with alkene or alkyne moieties. ß-Lactone formation is followed by rapid decarboxylation to form a conjugated triene chromophore for real-time evaluation by UV/Vis spectroscopy. The assay was used to determine steady-state kinetics of a long-chain ß-lactone synthetase, OleC, from the plant pathogen Xanthomonas campestris. Site-directed mutagenesis was used to test the involvement of conserved active site residues in Mg2+ and ATP binding. A previous report suggested OleC adenylated the substrate hydroxy group. Here we present several lines of evidence, including hydroxylamine trapping of the AMP intermediate, to demonstrate the substrate carboxyl group is adenylated prior to making the ß-lactone final product. A panel of nine substrate analogues were used to investigate the substrate specificity of X. campestris OleC by HPLC and GC-MS. Stereoisomers of 2-hexyl-3hydroxyoctanoic acid were synthesized and OleC preferred the (2R,3S) diastereomer consistent with the stereo-preference of upstream and downstream pathway enzymes. This biochemical knowledge was used to guide phylogenetic analysis of the ß-lactone synthetases to map their functional diversity within the acyl-CoA synthetase, NRPS adenylation domain, and luciferase superfamily.

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. Patients presenting with initial shock-refractory VF/VT were previously reported. The current study was a prespecified analysis in a separate cohort that initially presented with nonshockable OHCA and was randomized on subsequently developing shock-refractory VF/VT. The primary outcome was survival to hospital discharge. Secondary outcomes included discharge functional status and adverse drug-related effects. RESULTS: Of 37 889 patients with OHCA, 3026 with initial VF/VT and 1063 with initial nonshockable-turned-shockable rhythms were treatment-eligible, were randomized, and received their assigned drug. Baseline characteristics among patients with nonshockable-turned-shockable rhythms were balanced across treatment arms, except that recipients of a placebo included fewer men and were less likely to receive bystander cardiopulmonary resuscitation. Active-drug recipients in this cohort required fewer shocks, supplemental doses of their assigned drug, and ancillary antiarrhythmic drugs than recipients of a placebo (P<0.05). In all, 16 (4.1%) amiodarone, 11 (3.1%) lidocaine, and 6 (1.9%) placebo-treated patients survived to hospital discharge (P=0.24). No significant interaction between treatment assignment and discharge survival occurred with the initiating OHCA rhythm (asystole, pulseless electric activity, or VF/VT). Survival in each of these categories was consistently higher with active drugs, although the trends were not statistically significant. Adjusted absolute differences (95% confidence interval) in survival from nonshockable-turned-shockable arrhythmias with amiodarone versus placebo were 2.3% (-0.3, 4.8), P=0.08, and for lidocaine versus placebo 1.2% (-1.1, 3.6), P=0.30. More than 50% of these survivors were functionally independent or required minimal assistance. Drug-related adverse effects were infrequent. CONCLUSIONS: Outcome from nonshockable-turned-shockable OHCA is poor but not invariably fatal. Although not statistically significant, point estimates for survival were greater after amiodarone or lidocaine than placebo, without increased risk of adverse effects or disability and consistent with previously observed favorable trends from treatment of initial shock-refractory VF/VT with these drugs. Together the findings may signal a clinical benefit that invites further investigation. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01401647.

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. We used Cox and logistic regression modeling, adjusting for Utstein covariates, to estimate the probability of ROSC, survival, and favorable neurological outcomes as a function of resuscitation time, and applied these models to estimate the number of missed survivors in those who had TR (prior to 20, 30, or 40 minutes). We determined the time juncture at which (1) the likelihood of survival fell below 1%, and (2) the proportion of survivors who had achieved ROSC exceeded 99%. RESULTS: Of 5674 adult EMS-treated cases, 46% achieved ROSC, and 12% survived. The median time of TR was 27.0 minutes (IQR 19.0-35.0). Continuing resuscitation until 40 minutes yielded an estimated 17 additional survivors (95% CI 13-21), 10 (95% CI 7-13) with favorable neurological outcomes. The probability of survival of those in refractory arrest decreased below 1% at 28 minutes (95% CI 24-30 minutes). At 36 minutes (95% CI 34-38 minutes) >99% of survivors had achieved ROSC. CONCLUSION: We identified possible deaths due to intra-arrest prognostication. Resuscitation should be continued for a minimum of 30 minutes in all patients, however for those with initial shockable rhythms 40 minutes appears to be warranted. Interventional trials and observational studies should standardize or adjust for duration of resuscitation prior to TR.

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, oleABCD encodes the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A (CoA) molecules to form a ß-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modeling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-CoA substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating that Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is affected by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer.

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 Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive ß-lactone intermediate produced by the OleC-catalyzed reaction.IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.

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

OleB is an α/ß-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 ß-lactone decarboxylation reaction, to complete cis-olefin biosynthesis. OleB reactions monitored by 1H nuclear magnetic resonance spectroscopy revealed a selectivity for decarboxylating cis-ß-lactones and no discernible activity with trans-ß-lactones, consistent with the known configuration of pathway intermediates. Protein sequence analyses showed OleB proteins were most related to haloalkane dehalogenases (HLDs) and retained the canonical Asp-His-Asp catalytic triad of HLDs. Unexpectedly, it was determined that an understudied subfamily, denoted as HLD-III, is comprised mostly of OleB proteins encoded within oleABCD gene clusters, suggesting a misannotation. OleB from X. campestris showed very low dehalogenase activity only against haloalkane substrates with long alkyl chains. A haloalkane substrate mimic alkylated wild-type X. campestris OleB but not OleBD114A, implicating this residue as the active site nucleophile as in HLDs. A sequence-divergent OleB, found as part of a natural OleBC fusion and classified as an HLD-III, from the Gram-positive bacterium Micrococcus luteus was demonstrated to have the same activity, stereochemical preference, and dependence on the proposed Asp nucleophile. H218O studies with M. luteus OleBC suggested that the canonical alkyl-enzyme intermediate of HLDs is hydrolyzed differently by OleB enzymes, as 18O is not incorporated into the nucleophilic aspartic acid. This work defines a previously unrecognized reaction in nature, functionally identifies some HLD-III enzymes as ß-lactone decarboxylases, and posits an enzymatic mechanism of ß-lactone decarboxylation.

ß-Lactone Synthetase Found in the Olefin Biosynthesis Pathway.

The first ß-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-ß-hydroxy acid substrates to yield cis- and trans-ß-lactones, respectively. Protein sequence comparisons reveal that enzymes homologous to OleC are encoded in natural product gene clusters that generate ß-lactone rings, suggesting a common mechanism of biosynthesis.

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. Patients were classified as low risk if they had an EDACS <16, no new ischaemia on ECG and non-elevated serial 0-hour and 2-hour cardiac troponin concentrations. The primary outcome was the number of patients who had a predetermined major adverse cardiac event (MACE) at 30 days after presentation. RESULTS: Of the 763 patients, 317 (41.6%) were classified as low risk by the EDACS-ADP. The sensitivity, specificity, negative predictive value and positive predictive value of the EDACS-ADP for 30-day MACE were 100% (95% CI 94.2% to 100%), 46.4% (95% CI 42.6% to 50.2%), 100% (95% CI 98.5% to 100.0%) and 17.5% (95% CI 14.1% to 21.3%), respectively. CONCLUSIONS: This study validated the EDACS-ADP in a novel context and supports its safe use in a North American population. It confirms that EDACS-ADP can facilitate progression to early outpatient investigation in up to 40% of ED chest pain patients within 2 hours.

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. We calculated adjusted odds ratios for survival to hospital discharge, 1-day survival, and any return of circulation. We included 9136 adult patients from 9 US and Canadian cities with a mean age of 67.5 years, mean compression depth of 41.9 mm, and a return of circulation of 31.3%, 1-day survival of 22.8%, and survival to hospital discharge of 7.3%. For survival to discharge, the adjusted odds ratios were 1.04 (95% CI, 1.00-1.08) for each 5-mm increment in compression depth, 1.45 (95% CI, 1.20-1.76) for cases within 2005 depth range (>38 mm), and 1.05 (95% CI, 1.03-1.08) for percentage of minutes in depth range (10% change). Covariate-adjusted spline curves revealed that the maximum survival is at a depth of 45.6 mm (15-mm interval with highest survival between 40.3 and 55.3 mm) with no differences between men and women. CONCLUSIONS: This large study of out-of-hospital cardiac arrest patients demonstrated that increased cardiopulmonary resuscitation compression depth is strongly associated with better survival. Our adjusted analyses, however, found that maximum survival was in the depth interval of 40.3 to 55.3 mm (peak, 45.6 mm), suggesting that the 2010 American Heart Association cardiopulmonary resuscitation guideline target may be too high. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00394706.

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. Patients in the early-analysis group were assigned to receive 30 to 60 seconds of EMS-administered CPR and those in the later-analysis group were assigned to receive 180 seconds of CPR, before the initial electrocardiographic analysis. The primary outcome was survival to hospital discharge with satisfactory functional status (a modified Rankin scale score of ≤3, on a scale of 0 to 6, with higher scores indicating greater disability). RESULTS: We included 9933 patients, of whom 5290 were assigned to early analysis of cardiac rhythm and 4643 to later analysis. A total of 273 patients (5.9%) in the later-analysis group and 310 patients (5.9%) in the early-analysis group met the criteria for the primary outcome, with a cluster-adjusted difference of -0.2 percentage points (95% confidence interval, -1.1 to 0.7; P=0.59). Analyses of the data with adjustment for confounding factors, as well as subgroup analyses, also showed no survival benefit for either study group. CONCLUSIONS: Among patients who had an out-of-hospital cardiac arrest, we found no difference in the outcomes with a brief period, as compared with a longer period, of EMS-administered CPR before the first analysis of cardiac rhythm. (Funded by the National Heart, Lung, and Blood Institute and others; ROC PRIMED ClinicalTrials.gov number, NCT00394706.).

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. SETTING: Seven U.S. and Canadian urban regions. PATIENTS: We studied emergency medical services treated out-of-hospital cardiac arrest patients from the Resuscitation Outcomes Consortium Epistry-Cardiac Arrest for whom electronic cardiopulmonary resuscitation compression depth data were available, from May 2006 to June 2009. MEASUREMENTS: We calculated anterior chest wall depression in millimeters and the period of active cardiopulmonary resuscitation (chest compression fraction) for each minute of cardiopulmonary resuscitation. We controlled for covariates including compression rate and calculated adjusted odds ratios for any return of spontaneous circulation, 1-day survival, and hospital discharge. MAIN RESULTS: We included 1029 adult patients from seven U.S. and Canadian cities with the following characteristics: Mean age 68 yrs; male 62%; bystander witnessed 40%; bystander cardiopulmonary resuscitation 37%; initial rhythms: Ventricular fibrillation/ventricular tachycardia 24%, pulseless electrical activity 16%, asystole 48%, other nonshockable 12%; outcomes: Return of spontaneous circulation 26%, 1-day survival 18%, discharge 5%. For all patients, median compression rate was 106 per minute, median compression fraction 0.65, and median compression depth 37.3 mm with 52.8% of cases having depth <38 mm and 91.6% having depth <50 mm. We found an inverse association between depth and compression rate ( p < .001). Adjusted odds ratios for all depth measures (mean values, categories, and range) showed strong trends toward better outcomes with increased depth for all three survival measures. CONCLUSIONS: We found suboptimal compression depth in half of patients by 2005 guideline standards and almost all by 2010 standards as well as an inverse association between compression depth and rate. We found a strong association between survival outcomes and increased compression depth but no clear evidence to support or refute the 2010 recommendations of >50 mm. Although compression depth is an important component of cardiopulmonary resuscitation and should be measured routinely, the most effective depth is currently unknown.

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. Details are given for strategies to optimize protein expression and to design a multi-affinity tag purification scheme for large multienzyme assemblies. These insights are drawn from our study of bacterial hydrocarbon biosynthesis.

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. Methods and Results We performed a post hoc analysis of nontraumatic paramedic-treated out-of-hospital cardiac arrests. The primary outcome was favorable neurological status at hospital discharge, with a secondary outcome of survival to discharge. We fit a logistic regression analysis to determine the association of early compared with delayed DTT , dichotomized by the median DTT duration, and outcomes. Of 3805 patients enrolled in the CCC (Continuous Chest Compressions) Trial in British Columbia, 570 were included in this analysis. There was substantial variation in DTT among patients receiving TTM . The median DTT duration was 122 minutes (interquartile range 35-218). Favorable neurological outcomes in the early and delayed DTT groups were 48% and 38%, respectively. Compared with delayed DTT (interquartile range 167-319 minutes), early DTT (interquartile range 20-81 minutes) was associated with survival (adjusted odds ratio 1.56, 95% CI 1.02-2.38) but not with favorable neurological outcomes (adjusted odds ratio 1.45, 95% CI , 0.94-2.22) at hospital discharge. Conclusions There was wide variability in the initiation of TTM among comatose out-of-hospital cardiac arrest survivors. Initiation of TTM within 122 minutes of hospital admission was associated with improved survival. These results support in-hospital efforts to achieve early DTT among out-of-hospital cardiac arrest patients admitted to the hospital.

BIPHASIC Trial: a randomized comparison of fixed lower versus escalating higher energy levels for defibrillation in out-of-hospital cardiac arrest.

BACKGROUND: There is little clear evidence as to the optimal energy levels for initial and subsequent shocks in biphasic waveform defibrillation. The present study compared fixed lower- and escalating higher-energy regimens for out-of-hospital cardiac arrest. METHODS AND RESULTS: The Randomized Controlled Trial to Compare Fixed Versus Escalating Energy Regimens for Biphasic Waveform Defibrillation (BIPHASIC Trial) was a multicenter, randomized controlled trial of 221 out-of-hospital cardiac arrest patients who received > or = 1 shock given by biphasic automated external defibrillator devices that were randomly programmed to provide, blindly, fixed lower-energy (150-150-150 J) or escalating higher-energy (200-300-360 J) regimens. Patient mean age was 66.0 years; 79.6% were male. The cardiac arrest was witnessed in 63.8%; a bystander performed cardiopulmonary resuscitation in 23.5%; and initial rhythm was ventricular fibrillation/ventricular tachycardia in 92.3%. The fixed lower- and escalating higher-energy regimen cases were similar for the 106 multishock patients and for all 221 patients. In the primary analysis in multishock patients, conversion rates differed significantly (fixed lower, 24.7%, versus escalating higher, 36.6%; P=0.035; absolute difference, 11.9%; 95% CI, 1.2 to 24.4). Ventricular fibrillation termination rates also were significantly different between groups (71.2% versus 82.5%; P=0.027; absolute difference, 11.3%; 95% CI, 1.6 to 20.9). For the secondary analysis of first shock success, conversion rates were similar between the fixed lower and escalating higher study groups (38.4% versus 36.7%; P=0.92), as were ventricular fibrillation termination rates (86.8% versus 88.8%; P=0.81). There were no distinguishable differences between regimens for survival outcomes or adverse effects. CONCLUSIONS: This is the first randomized trial to compare fixed lower and escalating higher biphasic energy regimens in out-of-hospital cardiac arrest, and it demonstrated higher rates of ventricular fibrillation conversion and termination with an escalating higher-energy regimen for patients requiring multiple shocks. These results suggest that patients in ventricular fibrillation benefit from higher biphasic energy levels if multiple defibrillation shocks are required.

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 ß-keto acid. Here, the mechanistic role of the conserved His285 is investigated through mutagenesis, activity assays, and X-ray crystallography. Our data demonstrate that His285 is required for product formation, influences the thiolase nucleophile Cys143 and the acyl-enzyme intermediate before and after transesterification, and orchestrates substrate coordination as a defining component of an oxyanion hole. As a consequence, His285 plays a key role in enabling a mechanistic strategy in OleA that is distinct from other thiolases.

Aminophylline in bradyasystolic cardiac arrest: a randomised placebo-controlled trial.

BACKGROUND: Endogenous adenosine might cause or perpetuate bradyasystole. Our aim was to determine whether aminophylline, an adenosine antagonist, increases the rate of return of spontaneous circulation (ROSC) after out-of-hospital cardiac arrest. METHODS: In a double-blind trial, we randomly assigned 971 patients older than 16 years with asystole or pulseless electrical activity at fewer than 60 beats per minute, and who were unresponsive to initial treatment with epinephrine and atropine, to receive intravenous aminophylline (250 mg, and an additional 250 mg if necessary) (n=486) or placebo (n=485). The patients were enrolled between January, 2001 and September, 2003, from 1886 people who had had cardiac arrests. Standard resuscitation measures were used for at least 10 mins after the study drug was administered. Analysis was by intention-to-treat. This trial is registered with the ClinicalTrials.gov registry with the number NCT00312273. FINDINGS: Baseline characteristics and survival predictors were similar in both groups. The median time from the arrival of the advanced life-support paramedic team to study drug administration was 13 min. The proportion of patients who had an ROSC was 24.5% in the aminophylline group and 23.7% in the placebo group (difference 0.8%; 95% CI -4.6% to 6.2%; p=0.778). The proportion of patients with non-sinus tachyarrhythmias after study drug administration was 34.6% in the aminophylline group and 26.2% in the placebo group (p=0.004). Survival to hospital admission and survival to hospital discharge were not significantly different between the groups. A multivariate logistic regression analysis showed no evidence of a significant subgroup or interactive effect from aminophylline. INTERPRETATION: Although aminophylline increases non-sinus tachyarrhythmias, we noted no evidence that it significantly increases the proportion of patients who achieve ROSC after bradyasystolic cardiac arrest.

Tissue plasminogen activator in cardiac arrest with pulseless electrical activity.

BACKGROUND: Coronary thrombosis and pulmonary thromboembolism are common causes of cardiac arrest. We assessed whether the administration of tissue plasminogen activator (t-PA) during cardiopulmonary resuscitation would benefit patients with cardiac arrest and pulseless electrical activity of unknown or presumed cardiovascular cause. METHODS: Patients who were older than 16 years of age and who had more than one minute of pulseless electrical activity that was unresponsive to initial therapy outside the hospital or in the emergency department were eligible. Patients were randomly assigned to receive 100 mg of t-PA or placebo intravenously over a 15-minute period in a double-blind fashion. Standard resuscitation was then continued for at least 15 minutes. The primary outcome was survival to hospital discharge. RESULTS: During the study period, 1583 patients with cardiac arrest were treated and 233 patients were enrolled (117 in the t-PA group and 116 in the placebo group). The characteristics of the patients in the two groups were similar. One patient in the t-PA group survived to hospital discharge, as compared with none in the placebo group (absolute difference between groups, 0.9; 95 percent confidence interval, -2.6 to 4.8; P=0.99). The proportion of patients with return of spontaneous circulation was 21.4 percent in the t-PA group and 23.3 percent in the placebo group (absolute difference between groups, -1.9; 95 percent confidence interval, -12.6 to 8.8; P=0.85). CONCLUSIONS: We found no evidence of a beneficial effect of fibrinolysis in patients with cardiac arrest and pulseless electrical activity of unknown or presumed cardiovascular cause. Our study had limited statistical power, and it remains unknown whether there is a small treatment effect or whether selected subgroups may benefit.

The relationship between shocks and survival in out-of-hospital cardiac arrest patients initially found in PEA or asystole.

OBJECTIVE: To describe survival rates from out-of-hospital cardiac arrest for patients who present with pulseless electrical activity or asystole according to whether they remained in a non-shockable rhythm or converted to ventricular fibrillation and were shocked appropriately. RESULTS: Observational analysis of a cardiac arrest registry collected as part of a randomized trial. SETTING: Five urban/suburban cities in the United States and Canada. PARTICIPANTS: Trial subjects (adult, treated, non-traumatic) whose first documented heart rhythm/state following cardiac arrest was asystole or pulseless electrical activity. INTERVENTION: Periodic pauses to assess for shockable rhythm. MAIN OUTCOME MEASURE: Survival to hospital discharge. RESULTS: Of 1377 cardiac arrest patients, 738 presented with an initial arrest rhythm/state of either pulseless electrical activity or asystole. Of the 738, 78% (n=574) subsequently remained in a non-shockable rhythm/state at each evaluation throughout the resuscitation (No-Shock group) while 22% (n=164) converted to ventricular fibrillation and were shocked by emergency medical service (Shock group). Survival to hospital discharge was significantly greater in the No-Shock group (4.9% versus 0.6%, p=0.01). Shock group remained a predictor (odds ratios=0.18, p=0.036) of death after adjustment for potential confounders. CONCLUSIONS: These results suggest that patients with cardiac arrest who develop VF during the course of treatment for initially observed pulseless electrical activity or asystole do not benefit from conventional approaches to treatment such as defibrillation. Further study is warranted to define the optimal treatment of this patient cohort.