A destabilized bacterial luciferase for dynamic gene expression studies

Fusions of genetic regulatory elements with reporter genes have long been used as tools for monitoring gene expression and have become a major component in synthetic gene circuit implementation. A major limitation of many of these systems is the relatively long half-life of the reporter protein(s), which prevents monitoring both the initiation and the termination of transcription in real-time. Furthermore, when used as components in synthetic gene circuits, the long time constants associated with reporter protein decay may significantly degrade circuit performance. In this study, short half-life variants of LuxA and LuxB from Photorhabdus luminescens were constructed in Escherichia coli by inclusion of an 11-amino acid carboxy-terminal tag that is recognized by endogenous tail-specific proteases. Results indicated that the addition of the C-terminal tag affected the functional half-life of the holoenzyme when the tag was added to luxA or to both luxA and luxB, but modification of luxB alone did not have a significant effect. In addition, it was also found that alteration of the terminal three amino acid residues of the carboxy-terminal tag fused to LuxA generated variants with half-lives of intermediate length in a manner similar to that reported for GFP. This report is the first instance of the C-terminal tagging approach for the regulation of protein half-life to be applied to an enzyme or monomer of a multi-subunit enzyme complex and will extend the utility of the bacterial luciferase reporter genes for the monitoring of dynamic changes in gene expression

The clinical relevance of oliguria in the critically ill patient : Analysis of a large observational database

Funding Information: Marc Leone reports receiving consulting fees from Amomed and Aguettant; lecture fees from MSD, Pfizer, Octapharma, 3 M, Aspen, Orion; travel support from LFB; and grant support from PHRC IR and his institution. JLV is the Editor-in-Chief of Critical Care. The other authors declare that they have no relevant financial interests. Publisher Copyright: © 2020 The Author(s). Copyright: Copyright 2020 Elsevier B.V., All rights reserved.Background: Urine output is widely used as one of the criteria for the diagnosis and staging of acute renal failure, but few studies have specifically assessed the role of oliguria as a marker of acute renal failure or outcomes in general intensive care unit (ICU) patients. Using a large multinational database, we therefore evaluated the occurrence of oliguria (defined as a urine output 16 years) patients in the ICON audit who had a urine output measurement on the day of admission were included. To investigate the association between oliguria and mortality, we used a multilevel analysis. Results: Of the 8292 patients included, 2050 (24.7%) were oliguric during the first 24 h of admission. Patients with oliguria on admission who had at least one additional 24-h urine output recorded during their ICU stay (n = 1349) were divided into three groups: transient - oliguria resolved within 48 h after the admission day (n = 390 [28.9%]), prolonged - oliguria resolved > 48 h after the admission day (n = 141 [10.5%]), and permanent - oliguria persisting for the whole ICU stay or again present at the end of the ICU stay (n = 818 [60.6%]). ICU and hospital mortality rates were higher in patients with oliguria than in those without, except for patients with transient oliguria who had significantly lower mortality rates than non-oliguric patients. In multilevel analysis, the need for RRT was associated with a significantly higher risk of death (OR = 1.51 [95% CI 1.19-1.91], p = 0.001), but the presence of oliguria on admission was not (OR = 1.14 [95% CI 0.97-1.34], p = 0.103). Conclusions: Oliguria is common in ICU patients and may have a relatively benign nature if only transient. The duration of oliguria and need for RRT are associated with worse outcome.publishersversionPeer reviewe

Temporal changes in the epidemiology, management, and outcome from acute respiratory distress syndrome in European intensive care units: a comparison of two large cohorts

Background: Mortality rates for patients with ARDS remain high. We assessed temporal changes in the epidemiology and management of ARDS patients requiring invasive mechanical ventilation in European ICUs. We also investigated the association between ventilatory settings and outcome in these patients. Methods: This was a post hoc analysis of two cohorts of adult ICU patients admitted between May 1–15, 2002 (SOAP study, n = 3147), and May 8–18, 2012 (ICON audit, n = 4601 admitted to ICUs in the same 24 countries as the SOAP study). ARDS was defined retrospectively using the Berlin definitions. Values of tidal volume, PEEP, plateau pressure, and FiO2 corresponding to the most abnormal value of arterial PO2 were recorded prospectively every 24 h. In both studies, patients were followed for outcome until death, hospital discharge or for 60 days. Results: The frequency of ARDS requiring mechanical ventilation during the ICU stay was similar in SOAP and ICON (327[10.4%] vs. 494[10.7%], p = 0.793). The diagnosis of ARDS was established at a median of 3 (IQ: 1–7) days after admission in SOAP and 2 (1–6) days in ICON. Within 24 h of diagnosis, ARDS was mild in 244 (29.7%), moderate in 388 (47.3%), and severe in 189 (23.0%) patients. In patients with ARDS, tidal volumes were lower in the later (ICON) than in the earlier (SOAP) cohort. Plateau and driving pressures were also lower in ICON than in SOAP. ICU (134[41.1%] vs 179[36.9%]) and hospital (151[46.2%] vs 212[44.4%]) mortality rates in patients with ARDS were similar in SOAP and ICON. High plateau pressure (> 29 cmH2O) and driving pressure (> 14 cmH2O) on the first day of mechanical ventilation but not tidal volume (> 8 ml/kg predicted body weight [PBW]) were independently associated with a higher risk of in-hospital death. Conclusion: The frequency of and outcome from ARDS remained relatively stable between 2002 and 2012. Plateau pressure > 29 cmH2O and driving pressure > 14 cmH2O on the first day of mechanical ventilation but not tidal volume > 8 ml/kg PBW were independently associated with a higher risk of death. These data highlight the continued burden of ARDS and provide hypothesis-generating data for the design of future studies

Influence of molecular and supramolecular factors on sensor properties of Langmuir-Blodgett films of tert-buthyl-substituted copper azaporphyrines towards hydrocarbons

Langmuir-Blodgett (LB) films of tert-butyl-substituted copper azaporphyrines, namely binuclear phthalocyanine (Cu2Pc2') and porphyrazine (CuPaz'), were formed from the floating layers differently structured. Effect of the film structure on sorption of benzene and hexane by the LB films was studied microgravimetrically. The greatest benzene/hexane selectivity was found for the homogeneous film of Cu2Pc2'