Improving the Brightness of Pyronin Fluorophore Systems through Quantum-Mechanical Predictions.

The pyronin class of fluorophores serves a critical role in numerous imaging applications, particularly involving preferential staining of RNA through base pair intercalation. Despite this important role in molecular staining applications, the same set of century-old pyronins (i.e., pyronin Y (PY) and pyronin B (PB)), which possess relatively low fluorophore brightness, are still predominantly being used due to the lack of methodology for generating enhanced variants. Here, we use TD-DFT calculations of interconversion energies between structures on the S1 surface as a preliminary means to evaluate fluorophore brightness for a proposed set of pyronins containing variable substitution patterns at the 2, 3, 6, and 7 positions. Using a nucleophilic aromatic substitution/hydride addition approach, we synthesized the same set of pyronins and demonstrate that quantum-mechanical computations are useful for predicting fluorophore performance. We produced the brightest series of pyronin fluorophores described to date, which possess considerable gains over PY and PB.

Synthesis of a fluorinated pyronin that enables blue light to rapidly depolarize mitochondria.

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Brain Emergency Management Initiative for Optimizing Hub-Helicopter Emergency Medical Systems-Spoke Transfer Networks.

OBJECTIVE: Embolectomy is standard for select occlusions up to 24 hours. Transfer patients may have worse outcomes than those originating in embolectomy centers. We developed the Brain Emergency Management Initiative (BEMI) protocol to streamline this transfer process and mimic the urgency that surrounds ST-elevation myocardial infarction cardiac evaluations. METHODS: We conducted an exploratory assessment of consecutive acute telestroke patients transferred for potential intervention in pre-BEMI versus BEMI periods. Times included spoke in, spoke out, hub in, and groin puncture. Outcomes included discharge destination and symptomatic intracranial hemorrhage. RESULTS: Overall, 68 transfers were assessed. There was a higher National Institute of Neurological Disorders and Stroke in BEMI (11 pre-BEMI vs. 20 B.M., P = .01). There were shorter spoke door in to door out (143 vs. 118 minutes, P = .01) and spoke door out to hub door in times (23 minutes pre-BEMI vs. 21 minutes BEMI, P = .001). For embolectomy patients, there was shorter hub door in to reperfusion (83 minutes pre-BEMI vs. 74 minutes BEMI, P = .04) and recombinant tissue plasminogen decision to groin puncture (155 minutes pre-BEMI vs. 130 minutes BEMI; P = .01). There were no symptomatic intracranial hemorrhage or discharge differences. CONCLUSION: In our hub-helicopter emergency medical services-spoke telestroke network, BEMI led to improved evaluation times. BEMI may serve as a model for future rapid stroke transfer pathways.