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

The use of intravascular ultrasound imaging to improve use of inferior vena cava filters in a high-risk bariatric population

ObjectivePulmonary embolism is the leading cause of death after gastric bypass procedures for obesity, approximating 0.5% to 4%. All bariatric patients, but especially the super-obese, which have a body mass index (BMI) >50 kg/m2, are at significant risk for postoperative venous thromboembolism (VTE). Visualization and weight limitations of fluoroscopy tables exclude most bariatric and all super-obese patients from inferior vena cava (IVC) filter placement using fluoroscopy. Intravascular ultrasound (IVUS)-guided IVC filter placement is the only modality that allows these high-risk patients to have an IVC filter placed.MethodsHospital and outpatient records of the 494 patients who underwent gastric bypass procedures from January 1, 2004, to May 31, 2006, were reviewed. All patients who had concurrent IVC filter placement with the use of IVUS guidance were selected. Comorbidities, outcomes, and complications were recorded.ResultsWe identified 27 patients with mean BMI of 70 ± 3 kg/m2; of these, 25 were super-obese (BMI >50 kg/m2). Procedures included five laparoscopic and 22 open gastric bypass operations. All patients underwent concurrent IVC filter placement using IVUS guidance. In addition to super-obesity, indications for IVC filter placement included history of VTE (n = 4), known hypercoagulable state (n = 2), and profound immobility (n = 21). Mean follow up was 293 ± 40 days. Technical success rate was 96.3%. There were no catheter site complications. In one surviving patient, a nonfatal pulmonary embolism was detected by computed tomography 2 months postoperatively. Two patients died, and autopsy excluded VTE as the cause of death in both.ConclusionThis study suggests efficacy of IVUS-guided IVC filter placement in preventing mortality from pulmonary embolism in high-risk bariatric patients, including the super-obese. IVUS-guided IVC filter placement can be safely performed with an excellent success rate in all bariatric patients, including the super-obese, who otherwise would not be candidates for IVC filter placement due to the limitations imposed by their large body habitus

Mid-term results with laser atherectomy in the treatment of infrainguinal occlusive disease

BackgroundLaser atherectomy offers a potential intervention for multivessel infrainguinal disease in patients with poor revascularization options. Despite promising early results reported in the literature, the proper patient population who might benefit from laser atherectomy has yet to be determined.MethodsFrom July 2004 to June 2006, patients undergoing laser atherectomy were retrospectively reviewed and assessed for comorbidities, operative and follow-up variables potentially associated with the end points of nondefinitive therapy, and limb salvage.ResultsDuring the study period, 40 patients (21 women, 19 men) underwent laser atherectomy, and the average follow-up was 461 ± 49 days (range, 17 to 1050 days). Their average age was 68 ± 2 years (range, 43 to 93 years). The indication for laser atherectomy was critical limb ischemia in 26 (65%) and lower limb claudication in 11 (35%). A total of 47 lesions were treated in the following arterial segments: 34 femoropopliteal and 13 infrapopliteal. Femoropopliteal distribution by the Trans-Atlantic Society Classification (TASC) was A in 3, B in 17, C in 10, D in 4, and infrapopliteal lesions distribution was A in 1, B in 3, C in 4, and D in 5. Adjunctive angioplasty was used in 75% of cases. The overall technical success rate (<50% residual stenosis) was 88%. Laser atherectomy–based treatment was the definitive therapy for 23 patients (58%), and the overall 12-month primary patency was 44%. The limb salvage rate at 12 months in 26 patients with critical limb ischemia was 55%. Renal failure was a risk factor for amputation (P < .001) and failed primary patency (P < .05), type 2 diabetes mellitus was a risk factor for amputation (P < .05), and poor tibial runoff was associated with failed primary patency and amputation (P < .05). Outcome was associated with the number of patent infrapopliteal runoff vessels.ConclusionThese data demonstrate that laser atherectomy can be used with high initial technical success rate. Chronic renal failure and diabetes are risk factors for a negative outcome. Poor results in patients with diabetes and renal failure necessitate careful case selection in this subgroup, in which laser atherectomy is less likely to provide a definitive revascularization result or limb salvage

Oxygen Recovery Kinetics in the Forearm Flexors of Multiple Ability Groups of Rock Climbers

Fryer, SM, Stoner, L, Dickson, TG, Draper, SB, McCluskey, MJ, Hughes, JD, How, SC, and Draper, N. Oxygen recovery kinetics in the forearm flexors of multiple ability groups of rock climbers. J Strength Cond Res 29(6): 1633-1639, 2015-The purpose of this study was to determine muscle tissue oxidative capacity and recovery in intermediate, advanced, and elite rock climbers. Forty-four male participants performed (a) sustained and (b) intermittent contractions at 40% of maximal volitional contraction (MVC) on a sport-specific fingerboard until volitional fatigue. Near-infrared spectroscopy was used to assess muscle tissue oxygenation during both the exercise and the 5-minutes passive recovery period, in the flexor digitorum profundus (FDP) and flexor carpi radialis (FCR). During the sustained contraction only, muscle tissue deoxygenation (O2 debt) in the FDP and FCR was significantly greater in elite climbers compared with the control, intermediate, and advanced groups (FDP: 32 vs. 15, 19, 22%; FCR: 19 vs. 11, 8, 15%, respectively). However, elite climbers had a significantly quicker time to half recovery (T1/2) than the control and intermediate groups in the FDP (8 vs. 95 and 47 seconds, respectively) and the FCR (7 vs. 30 and 97 seconds, respectively) because the O2% recovered per second being significantly greater (FDP: 4.2 vs. 0.7 and 0.3; FCR: 4.8 vs. 0.1 and 0.2, respectively). Furthermore, during the intermittent contraction, T1/2 in elite climbers was significantly quicker compared with the control and intermediate groups in the FDP (8 vs. 93 and 83 seconds, respectively) and FCR (16 vs. 76 and 50 seconds, respectively). Consequently, lower-level climbers should focus training on specific intermittent fatigue protocols. Competition or elite climbers should make use of appropriate rests on route to aid recovery and increase the chances of reaching the next hold

Through a Glass, Darkly:The CIA and Oral History

This article broaches the thorny issue of how we may study the history of the CIA by utilizing oral history interviews. This article argues that while oral history interviews impose particular demands upon the researcher, they are particularly pronounced in relation to studying the history of intelligence services. This article, nevertheless, also argues that while intelligence history and oral history each harbour their own epistemological perils and biases, pitfalls which may in fact be pronounced when they are conjoined, the relationship between them may nevertheless be a productive one. Indeed, each field may enrich the other provided we have thought carefully about the linkages between them: this article's point of departure. The first part of this article outlines some of the problems encountered in studying the CIA by relating them to the author's own work. This involved researching the CIA's role in US foreign policy towards Afghanistan since a landmark year in the history of the late Cold War, 1979 (i.e. the year the Soviet Union invaded that country). The second part of this article then considers some of the issues historians must confront when applying oral history to the study of the CIA. To bring this within the sphere of cognition of the reader the author recounts some of his own experiences interviewing CIA officers in and around Washington DC. The third part then looks at some of the contributions oral history in particular can make towards a better understanding of the history of intelligence services and the CIA

Mahomet Aquifer Protection Task Force: Findings and Recommendations

The Mahomet Aquifer is one of Illinois’ most important groundwater resources, serving as the primary source of drinking water for more than 500,000 people in 15 Illinois counties and providing an estimated 220 million gallons of water per day to communities, agriculture, industry, and rural wells. In 2017, the Illinois General Assembly created the Mahomet Aquifer Protection Task Force (Task Force) to identify gaps in existing aquifer-protection regulations and efforts, specifically by: Developing a state plan to maintain the groundwater quality of the Mahomet Aquifer; Identifying current and potential contamination threats to the water quality of the Mahomet Aquifer; Identifying actions that might be taken to ensure the long-term protection of the Mahomet Aquifer; and Making legislative recommendations for the protection of the Mahomet Aquifer. The Task Force investigated and considered various actions, including legislative actions, to ensure the long-term protection of the Mahomet Aquifer and makes the following prioritized recommendations to the General Assembly and the Governor: 1. Provide 19.8 million dollars to the Prairie Research Institute (PRI) to use helicopter-based time-domain electromagnetics (HTEM) technology to more accurately map and characterize the Mahomet Aquifer to aid in identifying the connections with other aquifers and surface waters. 2. Use HTEM and other techniques to identify areas where the Mahomet Aquifer is recharged. 3. Integrate data collected via HTEM into next-generation groundwater flow models. 4. Develop and implement source water protection plans pursuant to 35 Ill. Adm. Code 604 Subpart C, after the effective date of adoption, for the community water supplies determined to be susceptible to groundwater contamination. 5. Implement the recommendations outlined for each identified threat and potential threat (as detailed below and in Section III.A) and provide additional funding (1 million dollars for one-time equipment acquisition and an additional 2.3 million annually) to PRI to deploy state-of-the-art monitoring networks and create the analytical capability to identify emerging contaminants of concern. 6. Improve education and outreach regarding the Mahomet Aquifer such that all stakeholders are better informed about water resources, water demand, and water supply planning and management, particularly when plans are made, reviewed, and updated. 7. Develop a group with a mission similar to the Mahomet Aquifer Protection Task Force that is a blend of other select individuals that serve in a quasi-government or government capacity to provide leadership, administrative stature, or process for regional water supply. 8. Plan cooperative research and data collection, analysis, management, and exchange by academic institutions, units of government, the private sector, and other stakeholders. 9. Use the established water supply planning process to review and update regional and local water supply plans at least every five years. 10. Ensure comprehensive use reporting by consistently and fully funding the Illinois Water Inventory Program.Illinois General AssemblyOpe

Chimerism in Wild Adult Populations of the Broadcast Spawning Coral Acropora millepora on the Great Barrier Reef

Chimeras are organisms containing tissues or cells of two or more genetically distinct individuals, and are known to exist in at least nine phyla of protists, plants, and animals. Although widespread and common in marine invertebrates, the extent of chimerism in wild populations of reef corals is unknown.The extent of chimerism was explored within two populations of a common coral, Acropora millepora, on the Great Barrier Reef, Australia, by using up to 12 polymorphic DNA microsatellite loci. At least 2% and 5% of Magnetic Island and Pelorus Island populations of A. millepora, respectively, were found to be chimeras (3% overall), based on conservative estimates. A slightly less conservative estimate indicated that 5% of colonies in each population were chimeras. These values are likely to be vast underestimates of the true extent of chimerism, as our sampling protocol was restricted to a maximum of eight branches per colony, while most colonies consist of hundreds of branches. Genotypes within chimeric corals showed high relatedness, indicating that genetic similarity is a prerequisite for long-term acceptance of non-self genotypes within coral colonies.While some brooding corals have been shown to form genetic chimeras in their early life history stages under experimental conditions, this study provides the first genetic evidence of the occurrence of coral chimeras in the wild and of chimerism in a broadcast spawning species. We hypothesize that chimerism is more widespread in corals than previously thought, and suggest that this has important implications for their resilience, potentially enhancing their capacity to compete for space and respond to stressors such as pathogen infection

Improving outpatient warfarin use for hospitalized patients with atrial fibrillation.

Atrial fibrillation affects an estimated 5 million Americans and accounts for approximately 15% of all strokes. Few studies have successfully addressed patient screening, assessment, and introduction of appropriate antithrombotic therapy in patients with atrial fibrillation. Objective: To assess whether an intervention improved planned antithrombotic prescribing at the time of discharge in hospitalized patients. Methods: The study was a prospectively designed, retrospectively evaluated, non-blinded, historical control study of a pharmacist-initiated intervention. The intervention, consisting of pharmacist review and assessment of antithrombotic prescribing in patients with non-valvular atrial fibrillation, was conducted in an urban teaching hospital. Results: Although antithrombotic prescribing was not significantly higher at discharge in the 252 enrolled subjects (control 67.3% vs. intervention 70.8%; p = 0.58), a significantly greater number of patients had a written discharge plan for antithrombotic therapy (control 73.5% vs. intervention 88.3%; p < 0.01). The adjusted odds ratio that the study group was associated with an improvement in planned or actual warfarin use was 2.46 (95% CI 1.63-3.74). In addition, clinicians adhered to guidelines for antithrombotic therapy in patients with atrial fibrillation more frequently in the intervention group (control 70.4% vs. intervention 88.2%; p < 0.01). Conclusion: A program designed to identify hospitalized patients with non-valvular atrial fibrillation, assess their need for stroke prophylaxis, and initiate appropriate antithrombotic therapy led to an increase in planned antithrombotic, and most importantly, warfarin use upon discharge from the hospital. Confirmation that an increase in planned antithrombotic use upon discharge results in an actual increase in use after discharge is needed to determine the true effectiveness of this intervention