Synthesis of precursor maltose-binding protein with proline in the + 1 position of the cleavage site interferes with the activity of Escherichia coli signal peptidase I in vivo

The residues occupying the -3 and -1 positions relative to the cleavage site of secretory precursor proteins are usually amino acids with small, neutral side chains that are thought to constitute the recognition site for the processing enzyme, signal peptidase. No restrictions have been established for residues positioned +1 to the cleavage site, although there have been several indications that mutant precursor proteins with a proline at +1 cannot be processed by Escherichia coli signal peptidase I (also called leader peptidase). A maltose-binding protein (MBP) species with proline at +1, designated MBP27-P, was translocated efficiently but not processed when expressed in E. coli cells. Unexpectedly, induced expression of MBP27-P was found to have an adverse effect on the processing kinetics of five different nonlipoprotein precursors analyzed, but not precursor Lpp (the major outer membrane lipoprotein) processed by a different enzyme, signal peptidase II. Cell growth also was inhibited following induction of MBP27-P synthesis. Substitutions in the MBP27-P signal peptide that blocked MBP translocation across the cytoplasmic membrane and, hence, access to the processing enzyme or that altered the signal peptidase I recognition site at position -1 restored both normal growth and processing of other precursors. Since overproduction of signal peptidase I also restored normal growth and processing to cells expressing unaltered MBP27-P, it was concluded that precursor MBP27-P interferes with the activity of the processing enzyme, probably by competing as a noncleavable substrate for the enzyme's active site. Thus, although signal peptidase I, like many other proteases, is unable to cleave an X-Pro bond, a proline at +1 does not prevent the enzyme from recognizing the normal processing site. When the RBP signal peptide was substituted for the MBP signal peptide of MBP27-P, the resultant hybrid protein was processed somewhat inefficiently at an alternate cleavage site and elicited a much reduced effect on cell growth and signal peptidase I activity. Although the MBP signal peptide also has an alternate cleavage site, the different properties of the RBP and MBP signal peptides with regard to the substitution of proline at +1 may be related to their respective secondary structures in the processing site region

Folding of maltose-binding protein : evidence for the identity of the rate-determining step in vivo and in vitro

The folding of maltose-binding protein, a periplasmic protein in Escherichia coli, was shown to proceed through the same rate-limiting step whether folding occurred in the cell under physiological conditions or in vitro in the absence of other proteins. Four species of maltose-binding protein containing aminoacyl substitutions identified as decreasing the rate of folding of the protein in vivo were purified, and their denaturant-induced folding transitions were analyzed by monitorin8 the intrinsic fluorescence of tryptophan. In all four cases the rate of folding in vitro was slower than that of the wild-type maltose-binding protein; thus the same step determines the rate of folding in vivo and in vitro.The folding of maltose-binding protein, a periplasmic protein in Escherichia coli, was shown to proceed through the same rate-limiting step whether folding occurred in the cell under physiological conditions or in vitro in the absence of other proteins. Four species of maltose-binding protein containing aminoacyl substitutions identified as decreasing the rate of folding of the protein in vivo were purified, and their denaturant-induced folding transitions were analyzed by monitorin8 the intrinsic fluorescence of tryptophan. In all four cases the rate of folding in vitro was slower than that of the wild-type maltose-binding protein; thus the same step determines the rate of folding in vivo and in vitro

Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein.

Maltose-binding protein (MBP) is essential for maltose transport and chemotaxis in Escherichia coli. To perform these functions it must interact with two sets of cytoplasmic membrane proteins, the MalFGK transport complex and the chemotactic signal transducer Tar. MBP is present at high concentrations, on the order of 1 mM, in the periplasm of maltose-induced or malTc constitutive cells. To determine how the amount of MBP affects transport and taxis, we utilized a series of malE signal-sequence mutations that interfere with export of MBP. The MBP content in shock fluid from cells carrying the various mutations ranged from 4 to 23% of the malE+ level. The apparent Km for maltose transport varied by less than a factor of 2 among malE+ and mutant strains. At a saturating maltose concentration 9% (approximately 90 microM) of the malE+ amount of MBP was required for half-maximal uptake rates. Transport exhibited a sigmoidal dependence on the amount of periplasmic MBP, indicating that MBP may be involved in a cooperative interaction at some stage of the transport process. The chemotactic response to a saturating maltose stimulus exhibited a first-order dependence on the amount of periplasmic MBP. Thus, interaction of a single substrate-bound MBP with Tar appears sufficient to initiate a chemotactic signal from the transducer. A half-maximal chemotactic response occurred at 25% of the malE+ MBP level, suggesting that in vivo the KD for binding of maltose-loaded MBP to Tar is quite high (approximately 250 microM)

Active transport of maltose in membrane vesicles obtained from Escherichia coli cells producing tethered maltose-binding protein.

Attempts to reconstitute periplasmic binding protein-dependent transport activity in membrane vesicles have often resulted in systems with poor and rather inconsistent activity, possibly because of the need to add a large excess of purified binding protein to the vesicles. We circumvented this difficulty by using a mutant which produces a precursor maltose-binding protein that is translocated across the cytoplasmic membrane but is not cleaved by the signal peptidase (J. D. Fikes and P. J. Bassford, Jr., J. Bacteriol. 169:2352-2359, 1987). The protein remains tethered to the cytoplasmic membrane, presumably through the hydrophobic signal sequence, and we show here that the spheroplasts and membrane vesicles prepared from this mutant catalyze active maltose transport without the addition of purified maltose-binding protein. In vesicles, the transport requires electron donors, such as ascorbate and phenazine methosulfate or D-lactate. However, inhibition by dicyclohexylcarbodiimide and stimulation of transport by the inculsion of ADP or ATP in the intravesicular space suggest that ATP (or compounds derived from it) is involved in the energization of the transport. The transport activity of intact cells can be recovered without much inactivation in the vesicles, and their high activity and ease of preparation will be useful in studies of the mechanism of the binding protein-dependent transport process

Effect of early vasopressin vs norepinephrine on kidney failure in patients with septic shock. The VANISH Randomized Clinical Trial

IMPORTANCE: Norepinephrine is currently recommended as the first-line vasopressor in septic shock; however, early vasopressin use has been proposed as an alternative. OBJECTIVE: To compare the effect of early vasopressin vs norepinephrine on kidney failure in patients with septic shock. DESIGN, SETTING, AND PARTICIPANTS: A factorial (2×2), double-blind, randomized clinical trial conducted in 18 general adult intensive care units in the United Kingdom between February 2013 and May 2015, enrolling adult patients who had septic shock requiring vasopressors despite fluid resuscitation within a maximum of 6 hours after the onset of shock. INTERVENTIONS: Patients were randomly allocated to vasopressin (titrated up to 0.06 U/min) and hydrocortisone (n = 101), vasopressin and placebo (n = 104), norepinephrine and hydrocortisone (n = 101), or norepinephrine and placebo (n = 103). MAIN OUTCOMES AND MEASURES: The primary outcome was kidney failure-free days during the 28-day period after randomization, measured as (1) the proportion of patients who never developed kidney failure and (2) median number of days alive and free of kidney failure for patients who did not survive, who experienced kidney failure, or both. Rates of renal replacement therapy, mortality, and serious adverse events were secondary outcomes. RESULTS: A total of 409 patients (median age, 66 years; men, 58.2%) were included in the study, with a median time to study drug administration of 3.5 hours after diagnosis of shock. The number of survivors who never developed kidney failure was 94 of 165 patients (57.0%) in the vasopressin group and 93 of 157 patients (59.2%) in the norepinephrine group (difference, -2.3% [95% CI, -13.0% to 8.5%]). The median number of kidney failure-free days for patients who did not survive, who experienced kidney failure, or both was 9 days (interquartile range [IQR], 1 to -24) in the vasopressin group and 13 days (IQR, 1 to -25) in the norepinephrine group (difference, -4 days [95% CI, -11 to 5]). There was less use of renal replacement therapy in the vasopressin group than in the norepinephrine group (25.4% for vasopressin vs 35.3% for norepinephrine; difference, -9.9% [95% CI, -19.3% to -0.6%]). There was no significant difference in mortality rates between groups. In total, 22 of 205 patients (10.7%) had a serious adverse event in the vasopressin group vs 17 of 204 patients (8.3%) in the norepinephrine group (difference, 2.5% [95% CI, -3.3% to 8.2%]). CONCLUSIONS AND RELEVANCE: Among adults with septic shock, the early use of vasopressin compared with norepinephrine did not improve the number of kidney failure-free days. Although these findings do not support the use of vasopressin to replace norepinephrine as initial treatment in this situation, the confidence interval included a potential clinically important benefit for vasopressin, and larger trials may be warranted to assess this further. TRIAL REGISTRATION: clinicaltrials.gov Identifier: ISRCTN 20769191

Pre-hospital assessment of the role of adrenaline : measuring the effectiveness of drug administration in cardiac arrest (PARAMEDIC-2) : trial protocol

Despite its use since the 1960s, the safety or effectiveness of adrenaline as a treatment for cardiac arrest has never been comprehensively evaluated in a clinical trial. Although most studies have found that adrenaline increases the chance of return of spontaneous circulation for short periods, many studies found harmful effects on the brain and raise concern that adrenaline may reduce overall survival and/or good neurological outcome. The PARAMEDIC-2 trial seeks to determine if adrenaline is safe and effective in out-of-hospital cardiac arrest. This is a pragmatic, individually randomised, double blind, controlled trial with a parallel economic evaluation. Participants will be eligible if they are in cardiac arrest in the out-of-hospital environment and advanced life support is initiated. Exclusions are cardiac arrest as a result of anaphylaxis or life threatening asthma, and patient known or appearing to be under 16 or pregnant. 8000 participants treated by 5 UK ambulance services will be randomised between December 2014 and August 2017 to adrenaline (intervention) or placebo (control) through opening pre-randomised drug packs. Clinical outcomes are survival to 30 days (primary outcome), hospital discharge, 3, 6 and 12 months, health related quality of life, and neurological and cognitive outcomes (secondary outcomes). Trial registration (ISRCTN73485024)

Vitamin D deficiency contributes directly to the acute respiratory distress syndrome (ARDS)

Rationale: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and intensive therapy unit mortality but has not been assessed as a risk factor for acute respiratory distress syndrome (ARDS). Causality of these associations has never been demonstrated. Objectives: To determine if ARDS is associated with vitamin D deficiency in a clinical setting and to determine if vitamin D deficiency in experimental models of ARDS influences its severity. Methods: Human, murine and in vitro primary alveolar epithelial cell work were included in this study. Findings: Vitamin D deficiency (plasma 25(OH)D levels 600 genes. In a clinical setting, pharmacological repletion of vitamin D prior to oesophagectomy reduced the observed changes of in vivo measurements of alveolar capillary damage seen in deficient patients. Conclusions: Vitamin D deficiency is common in people who develop ARDS. This deficiency of vitamin D appears to contribute to the development of the condition, and approaches to correct vitamin D deficiency in patients at risk of ARDS should be developed

Biomarker-guided antibiotic stewardship in suspected ventilator-associated pneumonia (VAPrapid2) : a randomised controlled trial and process evaluation

Background Ventilator-associated pneumonia is the most common intensive care unit (ICU)-acquired infection, yet accurate diagnosis remains difficult, leading to overuse of antibiotics. Low concentrations of IL-1β and IL-8 in bronchoalveolar lavage fluid have been validated as effective markers for exclusion of ventilator-associated pneumonia. The VAPrapid2 trial aimed to determine whether measurement of bronchoalveolar lavage fluid IL-1β and IL-8 could effectively and safely improve antibiotic stewardship in patients with clinically suspected ventilator-associated pneumonia. Methods VAPrapid2 was a multicentre, randomised controlled trial in patients admitted to 24 ICUs from 17 National Health Service hospital trusts across England, Scotland, and Northern Ireland. Patients were screened for eligibility and included if they were 18 years or older, intubated and mechanically ventilated for at least 48 h, and had suspected ventilator-associated pneumonia. Patients were randomly assigned (1:1) to biomarker-guided recommendation on antibiotics (intervention group) or routine use of antibiotics (control group) using a web-based randomisation service hosted by Newcastle Clinical Trials Unit. Patients were randomised using randomly permuted blocks of size four and six and stratified by site, with allocation concealment. Clinicians were masked to patient assignment for an initial period until biomarker results were reported. Bronchoalveolar lavage was done in all patients, with concentrations of IL-1β and IL-8 rapidly determined in bronchoalveolar lavage fluid from patients randomised to the biomarker-based antibiotic recommendation group. If concentrations were below a previously validated cutoff, clinicians were advised that ventilator-associated pneumonia was unlikely and to consider discontinuing antibiotics. Patients in the routine use of antibiotics group received antibiotics according to usual practice at sites. Microbiology was done on bronchoalveolar lavage fluid from all patients and ventilator-associated pneumonia was confirmed by at least 104 colony forming units per mL of bronchoalveolar lavage fluid. The primary outcome was the distribution of antibiotic-free days in the 7 days following bronchoalveolar lavage. Data were analysed on an intention-to-treat basis, with an additional per-protocol analysis that excluded patients randomly assigned to the intervention group who defaulted to routine use of antibiotics because of failure to return an adequate biomarker result. An embedded process evaluation assessed factors influencing trial adoption, recruitment, and decision making. This study is registered with ISRCTN, ISRCTN65937227, and ClinicalTrials.gov, NCT01972425. Findings Between Nov 6, 2013, and Sept 13, 2016, 360 patients were screened for inclusion in the study. 146 patients were ineligible, leaving 214 who were recruited to the study. Four patients were excluded before randomisation, meaning that 210 patients were randomly assigned to biomarker-guided recommendation on antibiotics (n=104) or routine use of antibiotics (n=106). One patient in the biomarker-guided recommendation group was withdrawn by the clinical team before bronchoscopy and so was excluded from the intention-to-treat analysis. We found no significant difference in the primary outcome of the distribution of antibiotic-free days in the 7 days following bronchoalveolar lavage in the intention-to-treat analysis (p=0·58). Bronchoalveolar lavage was associated with a small and transient increase in oxygen requirements. Established prescribing practices, reluctance for bronchoalveolar lavage, and dependence on a chain of trial-related procedures emerged as factors that impaired trial processes

Cumulative Prognostic Score Predicting Mortality in Patients Older Than 80 Years Admitted to the ICU.

OBJECTIVES: To develop a scoring system model that predicts mortality within 30 days of admission of patients older than 80 years admitted to intensive care units (ICUs). DESIGN: Prospective cohort study. SETTING: A total of 306 ICUs from 24 European countries. PARTICIPANTS: Older adults admitted to European ICUs (N = 3730; median age = 84 years [interquartile range = 81-87 y]; 51.8% male). MEASUREMENTS: Overall, 24 variables available during ICU admission were included as potential predictive variables. Multivariable logistic regression was used to identify independent predictors of 30-day mortality. Model sensitivity, specificity, and accuracy were evaluated with receiver operating characteristic curves. RESULTS: The 30-day-mortality was 1562 (41.9%). In multivariable analysis, these variables were selected as independent predictors of mortality: age, sex, ICU admission diagnosis, Clinical Frailty Scale, Sequential Organ Failure Score, invasive mechanical ventilation, and renal replacement therapy. The discrimination, accuracy, and calibration of the model were good: the area under the curve for a score of 10 or higher was .80, and the Brier score was .18. At a cut point of 10 or higher (75% of all patients), the model predicts 30-day mortality in 91.1% of all patients who die. CONCLUSION: A predictive model of cumulative events predicts 30-day mortality in patients older than 80 years admitted to ICUs. Future studies should include other potential predictor variables including functional status, presence of advance care plans, and assessment of each patient's decision-making capacity