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

Glucagon-like peptide-1 enhances cardiac L-type Ca2+ currents via activation of the cAMP-dependent protein kinase A pathway

<p>Abstract</p> <p>Background</p> <p>Glucagon-like peptide-1 (GLP-1) is a hormone predominately synthesized and secreted by intestinal L-cells. GLP-1 modulates multiple cellular functions and its receptor agonists are now used clinically for diabetic treatment. Interestingly, preclinical and clinical evidence suggests that GLP-1 agonists produce beneficial effects on dysfunctional hearts via acting on myocardial GLP-1 receptors. As the effects of GLP-1 on myocyte electrophysiology are largely unknown, this study was to assess if GLP-1 could affect the cardiac voltage-gated L-type Ca²⁺current (I_Ca).</p> <p>Methods</p> <p>The whole-cell patch clamp method was used to record I_Caand action potentials in enzymatically isolated cardiomyocytes from adult canine left ventricles.</p> <p>Results</p> <p>Extracellular perfusion of GLP-1 (7-36 amide) at 5 nM increased I_Caby 23 ± 8% (<it>p </it>< 0.05, n = 7). Simultaneous bath perfusion of 5 nM GLP-1 plus 100 nM Exendin (9-39), a GLP-1 receptor antagonist, was unable to block the GLP-1-induced increase in I_Ca; however, the increase in I_Cawas abolished if Exendin (9-39) was pre-applied 5 min prior to GLP-1 administration. Intracellular dialysis with a protein kinase A inhibitor also blocked the GLP-1-enhanced I_Ca. In addition, GLP-1 at 5 nM prolonged the durations of the action potentials by 128 ± 36 ms (<it>p </it>< 0.01) and 199 ± 76 ms (<it>p </it>< 0.05) at 50% and 90% repolarization (n = 6), respectively.</p> <p>Conclusions</p> <p>Our data demonstrate that GLP-1 enhances I_Cain canine cardiomyocytes. The enhancement of I_Cais likely via the cAMP-dependent protein kinase A mechanism and may contribute, at least partially, to the prolongation of the action potential duration.</p

A Subgroup Analysis of Legal Needs Among Older Adults in Rural Communities

A legal needs assessment of older adults in Maine was conducted by surveying key populations of older adults who are often hard to reach through traditional outreach and service provision methods. The focus of this assessment was on basic demographics, use of LSE services, legal issues, preferred methods for receiving legal information, and the utility of various legal service options. Surveys were distributed via area agencies on aging, and concluded that the ley legal issues included financial scams, home repair problems, obtaining or retaining government benefits, debt collection, and accessing medical services. Sixty-seven percent of individuals aged 70 or older reported having legal issues in the previous year. Providing resources to local area agencies on aging is a critical component to allowing for information about resources to be distributed

Novel Characteristics of Valveless Pumping

This study investigates the occurrence of valveless pumping in a fluidfilled system consisting of two open tanks connected by an elastic tube. We show that directional flow can be achieved by introducing a periodic pinching applied at an asymmetrical location along the tube, and that the flow direction depends on the pumping frequency. We propose a relation between wave propagation velocity, tube length, and resonance frequencies associated with shifts in the pumping direction using numerical simulations. The eigenfrequencies of the system are estimated from the linearized system, and we show that these eigenfrequencies constitute the resonance frequencies and the horizontal slope frequencies of the system; 'horizontal slope frequency' being a new concept. A simple model is suggested, explaining the effect of the gravity driven part of the oscillation observed in response to the tank and tube diameter changes. Results are partly compared with experimental findings.Art. no. 22450

Contraceptive practices of women visiting a gynecology clinic in Beijing, China

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/135321/1/ijgo64.pd

An Analysis of Current and Projected Rural Older Adult Legal Services Needs

Maine is now the oldest state in the nation, and is one of the most rural states. A legal needs assessment of older adults in Maine was conducted by analyzing the findings from recent research conducted in six other states and service data from Maine Legal Services for the Elderly over a one-year time period. The six states analyzed were Kentucky, Michigan, Nevada, North Dakota, Ohio, and Utah. There were a total of over 7,300 older adults that responded to the legal needs surveys. The assessment concluded that the high-level service needs included the following: health insurance, government benefits, estate planning, and personal finances and consumer issues. Other needs included help with housing, abuse, employment, and family matters. This assessment was a crucial project in establishing means to plan to distribute a legal needs survey in Maine

NEXAFS and XPS of p-Aminobenzoic Acid Polymorphs: The Influence of Local Environment

Nitrogen K-edge XPS and NEXAFS of the two polymorphic forms of para- aminobenzoic acid (PABA) are significantly different reflecting variation in hydrogen bonding. Alteration in hydrogen bonding at the amino group leads to a shift to high energy for both the XPS N 1s core level and the 3π* NEXAFS resonance with β-PABA. Participation of the amine group in the aromatic system causes the 1π* resonance to be sensitive to the nature of the intermolecular bonding at the para-carboxylic acid group, and a shift to low energy for α- PABA is observed due to hydrogen-bonded carboxylic acid dimer formation. FEFF calculations also successfully reproduce both the energy and intensity variations observed for the σ* shape resonance associated with the C-N bond, with the majority of the decrease in energy observed for b-PABA arising from the longer C-N bond