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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.

Despite the transformative potential of the ‘New Wars’ paradigm, what might be labeled the ‘first generation’ of scholarship has been subject to a range of cross-disciplinary conceptual and empirical challenges that have called into question many of its key findings. While there is much that is valid in these critiques of the New Wars thesis, ultimately it is premature to conclude that they have fatally undermined the New Wars paradigm as a heuristically useful framework for inquiring into the (changing) character of violent political conflict. All of this suggests that what is needed now is not the abandonment of the New Wars paradigm, but a longer-term historical perspective that can help us avoid many of the conceptual errors that plague the extant literature.

Background In a departure from the previous strategy of immediate defibrillation, the 2005 resuscitation guidelines from the American Heart Association–International Liaison Com mittee 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.)

Cationic antimicrobial peptides are an evolutionarily ancient and essential element of innate immunity in higher organisms. The precise mechanism by which these peptides exert their antimicrobial activity on bacteria is not well understood. Decapeptides based on the C-terminus of human beta-defensin-3 were designed and evaluated to study the role of charge in defining the antimicrobial activity and selectivity of these peptides against Escherichia coli. Acetylated derivatives of these peptides were prepared in order to further evaluate how positively charged primary amines contribute to potency in these small antimicrobial peptides. These peptides enabled us to explore the relationship between net charge, charge distribution and antimicrobial activity. While the results indicate that net charge is a major factor in antimicrobial activity in these peptides, the actual relationship between charge and potency appears to be more complex.

The PAD Trial is a prospective, multicenter, randomized clinical study testing whether volunteer, non-medical responders can improve survival from out-of-hospital cardiac arrest (OOH-CA) by using automated external defibrillators (AEDs). These lay volunteers, who have no traditional responsibility to respond to a medical emergency as part of their primary job description, will form part of a comprehensive, integrated community approach to the treatment of OOH-CA. The study is being conducted at 24 field centers in the United States and Canada. Approximately 1000 community units (e.g. apartment or office buildings, gated communities, sports facilities, senior centers, shopping malls, etc.) were randomized to treatment by trained laypersons who will provide either cardiopulmonary resuscitation (CPR) alone or CPR plus use of an AED, while awaiting arrival of the community's emergency medical services responders. The primary endpoint is the number of OOH-CA victims who survive to hospital discharge. Secondary endpoints include neurological status, health-related quality of life (HRQL), cost, and cost-effectiveness. Data collection will last approximately 15 months and is expected to be completed in September 2003.

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.

Cationic antimicrobial peptides are an evolutionarily ancient and essential element of innate immunity in higher organisms. The precise mechanism by which these peptides exert their antimicrobial activity on bacteria is not well understood. Decapeptides based on the C-terminus of human beta-defensin-3 were designed and evaluated to study the role of charge in defining the antimicrobial activity and selectivity of these peptides against Escherichia coli. Acetylated derivatives of these peptides were prepared in order to further evaluate how positively charged primary amines contribute to potency in these small antimicrobial peptides. These peptides enabled us to explore the relationship between net charge, charge distribution and antimicrobial activity. While the results indicate that net charge is a major factor in antimicrobial activity in these peptides, the actual relationship between charge and potency appears to be more complex.

Introduction : In 2005, the American Heart Association recommended increased “hands-on time” during cardiopulmonary resuscitation (CPR) based on animal research and small case series. This study estimated the effect of increasing CPR fraction (proportion of resuscitation time with active CPR) on survival in a cohort of patients with out-of hospital ventricular fibrillation or ventricular tachycardia (VF/VT). Methods: Patients were selected from the ROC Epistry who had a confirmed VF/VT cardiac arrest that was not witnessed by Emergency Medical Services (EMS), received no public access defibrillation shock prior to EMS arrival, and had impedance recordings of CPR before the first shock. The proportion of each minute with active CPR, from defibrillator pad application until the first shock, was measured from the electronic resuscitation record by skilled readers who were blinded to hospital discharge outcome. The effect of increasing CPR fraction on survival to hospital discharge was adjusted for age, sex, bystander CPR, public location, interval from 911 call to defibrillator activation, chest compression rate, and ROC community. Results: Of 7963 EMS-treated cases of cardiac arrest without public access defibrillation, 1893 had an initial rhythm of VF/VT and 283 of those had electronic tracings and confirmed outcome. Mean age was 63 years and 81% were male. Bystanders performed CPR on 51% and 41% arrested in a public location. Outcomes and odds ratios (OR) with 95% confidence intervals (CI) of survival are shown from lowest to highest category of CPR fraction. Conclusions: This study provides preliminary evidence that increasing CPR fraction is associated with increased survival from VF/VT cardiac arrest. Though the study was observational, relatively small, and able only to measure CPR fraction after pad application, these findings suggest that provision of minimally interrupted CPR has direct clinical impact on survival after VF cardiac arrest.

Triage reliability studies typically use hypothetical scenarios and weighted kappa scores where agreement within one level is considered satisfactory. But if triage category is used to help define ED case-mix groups for comparative or benchmarking processes, agreement on exact triage level and major system involved is important. Our hypothesis was that a computerized menu that links presenting complaints to preferred triage levels (PC-linked triage) would provide high triage reliability.Our objective was to assess inter-rater reliability of PC-linked triage using the Canadian Emergency Department Triage and Acuity Scale (CTAS) in a real-time clinical setting, considering agreement on exact triage level and primary body system involved.On duty triage nurses entered patient presenting complaint and PC-linked triage level as per standard procedure. In a convenience sample of patients, a second nurse, blinded to triage assignment, observed the triage interaction and independently entered presenting complaint and triage level on a dummy terminal.During the study, 15 nurse pairs triaged 266 patients. Study patients matched actual emergency department case mix closely. Triage nurses agreed exactly in 74% of cases and within one level in 94% of cases. The unweighted kappa value was 0.66 (95% confidence interval [CI], 0.60-0.73) and the quadratic weighted kappa value was 0.75 (95% CI, 0.68-0.81). Kappa for agreement on major system involved was 0.80 (95% CI, 0.69-0.91).PC-linked triage has high inter-rater reliability in a real-time clinical setting. PC-linked triage may be useful as one factor in defining case-mix groups for benchmarking and comparative purposes.

Fibrinolytic therapy has become the standard therapy for acute myocardial infarction with ST segment elevation. Many clinical trials have established that early administration of therapy correlates with improved outcomes. However, there are very few published data analyzing time to treatment in Canadian hospitals.To examine all time intervals from onset of symptoms to treatment with fibrinolytic therapy in patients in Canada.Using the FASTRAK II database, time intervals in 11,574 patients treated with fibrinolytic therapy in 106 contributing institutions across Canada from 1998 to 2000 were studied. Variables contributing to long delays in starting fibrinolytic therapy were analyzed.The mean time from onset of symptoms to arrival at hospital was 162 min (2.7 h). Only 6.3% of patients received fibrinolytic therapy within 1 h of symptom onset. Time from hospital arrival to acquisition of first 12 lead electrocardiogram was 14 min. Mean time from diagnostic electrocardiogram to decision to treat was 29.7 min, and time from decision to treat to administration of fibrinolytic therapy was 11 min. The overall average time from arrival at hospital to administration of fibrinolytic therapy was 69 min.In Canada, time from onset of symptoms to hospital presentation precludes early fibrinolytic therapy administration in the majority of cases. Time intervals from arrival in the emergency department to administration of fibrinolytic therapy are longer than the published and accepted standards. Strategies to alter health care seeking behaviour and to minimize in-hospital delays are needed.

The PAD Trial is a prospective, multicenter, randomized clinical study testing whether volunteer, non-medical responders can improve survival from out-of-hospital cardiac arrest (OOH-CA) by using automated external defibrillators (AEDs). These lay volunteers, who have no traditional responsibility to respond to a medical emergency as part of their primary job description, will form part of a comprehensive, integrated community approach to the treatment of OOH-CA. The study is being conducted at 24 field centers in the United States and Canada. Approximately 1000 community units (e.g. apartment or office buildings, gated communities, sports facilities, senior centers, shopping malls, etc.) were randomized to treatment by trained laypersons who will provide either cardiopulmonary resuscitation (CPR) alone or CPR plus use of an AED, while awaiting arrival of the community's emergency medical services responders. The primary endpoint is the number of OOH-CA victims who survive to hospital discharge. Secondary endpoints include neurological status, health-related quality of life (HRQL), cost, and cost-effectiveness. Data collection will last approximately 15 months and is expected to be completed in September 2003.