An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: Rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (<1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is =2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins

Evaluating multisite multiprofessional simulation training for a hyperacute stroke service using the behaviour change wheel

Background Stroke is a clinical priority requiring early specialist assessment and treatment. A London (UK) stroke strategy was introduced in 2010, with Hyper Acute Stroke Units (HASUs) providing specialist and high dependency care. To support increased numbers of specialist staff, innovative multisite multiprofessional simulation training under a standard protocol-based curriculum took place across London. This paper reports on an independent evaluation of the HASU training programme. The main aim was to evaluate mechanisms for behaviour change within the training design and delivery, and impact upon learners including potential transferability to the clinical environment. Methods The evaluation utilised the Behaviour Change Wheel framework. Procedures included: mapping training via the framework; examination of course material; direct and video-recorded observations of courses; pre-post course survey sheet; and follow up in-depth interviews with candidates and faculty. Results Patient management skills and trainee confidence were reportedly increased post-course (post-course median 6 [IQ range 5–6.33]; pre-course median 5 [IQ range 4.67–5.83]; z = 6.42, P &#60;.001). Thematic analysis showed that facilitated ‘debrief’ was the key agent in supporting both clinical and non-clinical skills. Follow up interviews in practice showed some sustained effects such as enthusiasm for role, and a focus on situational awareness, prioritization and verbalising thoughts. Challenges in standardising a multi-centre course included provision for local context/identity. Conclusions Pan-London simulation training under the London Stroke Model had positive outcomes in terms of self-reported skills and motivation. These effects persisted to an extent in practice, where staff could recount applications of learning. The evaluation demonstrated that a multiple centre simulation programme congruent with clinical practice can provide valuable standard training opportunities that support patient care

Resilience engineering as a quality improvement method in healthcare

Current approaches to quality improvement rely on the identification of past problems through incident reporting and audits or the use of Lean principles to eliminate waste, to identify how to improve quality. In contrast, Resilience Engineering (RE) is based on insights from complexity science, and quality results from clinicians’ ability to adapt safely to difficult situations, such as a surge in patient numbers, missing equipment or difficult unforeseen physiological problems. Progress in applying these insights to improve quality has been slow, despite the theoretical developments. In this chapter we describe a study in the Emergency Department of a large hospital in which we used RE principles to identify opportunities for quality improvement interventions. In depth observational fieldwork and interviews with clinicians were used to gather data about the key challenges faced, the misalignments between demand and capacity, adaptations that were required, and the four resilience abilities: responding, monitoring, anticipating and learning. Data were transcribed and used to write extended resilience narratives describing the work system. The narratives were analysed thematically using a combined deductive/inductive approach. A structured process was then used to identify potential interventions to improve quality. We describe one intervention to improve monitoring of patient flow and organisational learning about patient flow interventions. The approach we describe is challenging and requires close collaboration with clinicians to ensure accurate results. We found that using RE principles to improve quality is feasible and results in a focus on strengthening processes and supporting the challenges that clinicians face in their daily work

Capturing skin properties from dynamic mechanical analyses

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2012.Cataloged from PDF version of thesis.Includes bibliographical references (p. 76-79).Existing skin mechanical testing devices focus on measuring skin elasticity and are not tailored to assess the dynamic behavior of skin. The mathematical techniques used to analyze data collected using these devices are often not optimal. A new dynamic mechanical device that measures the linear dynamics of skin was developed and tested. The mechanical properties of skin were evaluated in experiments in which the stiffness and damping parameter were measured at different locations on the arm and hand, when stratum corneum hydration was varied by controlled changes in environmental humidity, and following the application of film-forming polymers. Parallel measurements were made with the Cutometer® so that the two devices could be compared. The findings revealed that reliable and valid measurements of skin mechanical properties can be obtained from the device. The stiffness of the skin was shown to vary significantly as a function of skin site, changes in stratum corneum hydration, and following the application of the polymer films. Changes in the damping parameter were less consistently associated with varying the condition of the skin. The high reliability and speed of measurement make this device and analytic procedure an attractive option for testing skin mechanics.by Erika Sandford.S.M