Huff Mcdowell (tradesyrup91)

Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISact with FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. AG-221 By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner. Opioid overdose and abuse have reached epidemic rates in the United States. Medical prescriptions are a large source of opioid misuse. Our quality improvement initiative aimed to reduce opioid exposure from the pediatric emergency department (ED). Objective was to reduce opioid doses prescribed weekly from our ED by 50% within 4 months. Three categories of interventions were implemented in Plan-Do-Study-Act cycles guidelines and education, electronic medical record optimization, and provider-specific feedback. Primary measures were opioid doses prescribed weekly from the ED and opioid doses per 100 ED visits. Process measures were opioid prescriptions, opioid doses per prescription, and opioid prescriptions for unspecified abdominal pain, headache, and viral upper respiratory infection. Balancing measures were phone calls and return visits for poor pain control in patients prescribed opioids and reports of poor pain control in call backs to orthopedic reduction patients. We used statistical process control to examine changes in measures over time. Opioid doses decreased from 153 to 14 per week and from 8 to 0.7 doses per 100 ED visits in 10 months, sustained for 9 months. Opioid prescriptions, opioid doses per prescription, and prescriptions for unspecified abdominal pain, headache, and viral upper respiratory infection decreased. Phone calls and return visits in patients prescribed opioids did not increase. There were 2 reports of poor pain control among 152 orthopedic reduction patients called back. We decreased opioid doses prescribed weekly from the pediatric ED by 91% while minimizing return visits and reports of poor pain control. We decreased opioid doses prescribed weekly from the pediatric ED by 91% while minimizing return visits and reports of poor pain control. Pediatric ethics consultations are important but understudied, with little known about consultations' contextual attributes, which may influence how ethically problematic situations are perceived and addressed. We analyzed data regarding 245 pediatric clinical ethics consultations per