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 involvement of cancer patients in the four stages of decision-making preceding continuous sedation until death: A qualitative study

BACKGROUND: Involving patients in decision-making is considered to be particularly appropriate towards the end of life. Professional guidelines emphasize that the decision to initiate continuous sedation should be made in accordance with the wishes of the dying person and be preceded by their consent. AIM: To describe the decision-making process preceding continuous sedation until death with particular attention to the involvement of the person who is dying. DESIGN: Qualitative case studies using interviews. SETTING/PARTICIPANTS: Interviews with 26 physicians, 30 nurses and 24 relatives caring for 24 patients with cancer who received continuous sedation until death in Belgium, the United Kingdom and the Netherlands. RESULTS: We distinguished four stages of decision-making: initiation, information exchange, deliberation and the decision to start continuous sedation until death. There was wide variation in the role the patient had in the decision-making process. At one end of the spectrum (mostly in the United Kingdom), the physician discussed the possible use of sedation with the patient, but took the decision themselves. At the other end (mostly in Belgium and the Netherlands), the patient initiated the conversation and the physician's role was largely limited to evaluating if and when the medical criteria were met. CONCLUSION: Decision-making about continuous sedation until death goes through four stages and the involvement of the patient in the decision-making varies. Acknowledging the potential sensitivity of raising the issue of end-of-life sedation, we recommend building into clinical practice regular opportunities to discuss the goals and preferences of the person who is dying for their future medical treatment and care

Post-Enron Implicit Audit Reporting Standards: Sifting through the Evidence

Auditor reporting, qualified/modified opinion, Enron, Andersen Data availability: All data are publicly available, G3, M4,