Pyruvate decarboxylase (PDC) is one of several enzymes that require thiamin diphosphate (ThDP) and a divalent cation as essential cofactors. Recently, the three-dimensional structures of the enzyme from two yeasts have been determined. While these structures shed light on the binding of the cofactors and the reaction mechanism, the interactions between the substrate pyruvate and the enzyme remain unclear. We have used PDC from Zynzomonas mobilis as a model for these enzymes in order to study substrate binding. The recombinant enzyme was expressed in Escherichia coli. High yield, simplicity of purification, high stability and simple kinetics make this model well suited for these studies. Activity measurements in the pH range between 5.8 and 8.5 indicated that a His residue may be involved in substrate binding. Analysis of an alignment of all known PDC protein sequences showed two invariant His residues (His113 and Hisll4) which, according to the crystal structure of yeast PDC, are in the vicinity of the active site. Here we demonstrate that replacement of His114 by Gln does not have a great effect on cofactor and substrate binding. However, the k,,, is decreased indicating that Hisl 14 may assist in catalysis. In contrast, substitution of His113 by Gln renders the enzyme completely inactive. This mutant has decreased affinity for both cofactors, as revealed by measurements of tryptophan fluorescence quenching. However, this decreased affinity is insufficient to account for the complete loss of activity. Despite its inability to support overall catalysis, this [Glnl13]PDC mutant is capable of releasing acetaldehyde from 2-( 1 -hydroxyethyl)thiamin diphosphate supplied exogenously. It is proposed that upon substrate binding, His113 is placed close to C2 of the thiazole ring. Subsequent deprotonation of this atom leads to a conformational change that allows a flexible loop (residues 105-112) that precedes Hisl 13 to close over the active site. Hence, replacement of His113 by another residue interferes with this closure of the active site and thus disrupts the catalytic process.

Background: Multi-institutional, international practice variation of pediatric anaphylaxis management by healthcare providers has not been reported.Objective: Characterize variability in epinephrine administration for pediatric anaphylaxis across institutions, including frequency and types of medication errors. Methods:A prospective, observational, study using a standardized in situ simulated anaphylaxis scenario was performed across 28 healthcare institutions in six countries. The on-duty healthcare team was called for a child (patient simulator) in anaphylaxis. Real medications and supplies were obtained from their actual locations. Demographic data about team members, institutional protocols for anaphylaxis, timing of epinephrine delivery, medication errors, and systems safety issues discovered during the simulation were collected.Results: Thirty-seven in situ simulations were performed. Anaphylaxis guidelines existed in 41% (15/37) of institutions. Teams used a cognitive aid for medication dosing 41% (15/37) of the time and 32% (12/37) for preparation. Epinephrine auto injectors (EAIs) were not available in 54% (20/37) of institutions and were used in only 14% (5/37) simulations. Median time to epinephrine administration was 95 seconds (IQR 77, 252) for EAI and 263 seconds (IQR 146, 407.5) for manually prepared epinephrine (p=.12). At least one medication error occurred in 68% (25/37) of simulations. Prior nursing experience with epinephrine administration for anaphylaxis was associated with fewer preparation (p=.04) and administration (p=.01) errors.Latent safety threats (LSTs) were reported by 30% (11/37) of institutions, more than half of these (6/11) involved a cognitive aid. Conclusion and Relevance:A multicenter, international study of simulated pediatric anaphylaxis reveals: 1) variation in management between institutions in usage of protocols,

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An intractable problem that characterizes the contemporary philosophical discussion of emotion is whether emotions are fundamentally cognitive or noncognitive. In this article, I will establish that this problem arises from the influence of an underlying philosophical anthropology that entails a mind/body “split” ultimately inherited from Cartesianism, and further show that it can be fruitfully addressed by adopting a contemporary construal of the self and emotions derived from the philosophy of a key critic of Descartes’ dualism, Spinoza.

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