Alpha class glutathione S-transferase, isozyme A1-1, is a dimer (51 kDa) of identical subunits. Using the crystal structure, two main areas of subunit interaction were chosen for study: (1) the hydrophobic ball and socket comprised of Phe52 from one subunit fitting into a socket formed on the other subunit by Met94, Phe136, and Val139 and (2) the Arg/Glu region consisting of Arg69 and Glu97 from both subunits. We introduced substitutions of these residues, by site-directed mutagenesis, to evaluate the importance of each at the subunit interface and to determine if monomeric enzymes could be generated using single mutations. Mutating each residue of the socket region to alanine results in little change in the kinetic parameters, and all are dimeric enzymes. In contrast, when Phe52, the ball residue, is replaced with alanine, the enzyme has very low activity and a weight average molecular mass of 31.9 kDa, as determined by sedimentation equilibrium experiments. Substitutions for Glu97 which eliminate the charge cause no appreciable changes in the kinetic parameters or molecular mass. Eliminating the charge on Arg69 (as in R69Q) results in a dimeric enzyme; however, when the charge is reversed (as in R69E), the weight average molecular mass is greatly shifted toward that of the monomer (33 kDa) and the changes in kinetic parameters are reasonably small. We determined the molecular masses in the presence of glutathione for F52A and R69E to ascertain whether the monomeric species retains activity. For R69E, it appears that the monomer is active, albeit less so than the dimer, while for F52A, the monomer and dimer both appear to exhibit very low activity. The dimeric species is needed to obtain high specific activity. We conclude that, of the residues that were studied, Phe52 and Arg69 are the major determinants of dimer formation and a single mutation at either position substantially hinders dimerization. The use of a mutant glutathione S-transferase which retains activity yet has a greatly weakened tendency to dimerize (such as R69E) may be advantageous for certain applications of GST fusion proteins.

ObjectiveTo describe senior paediatric emergency clinician perspectives on the optimal frequency of and preferred modalities for practising critical paediatric procedures.MethodsMulticentre multicountry cross-sectional survey of senior paediatric emergency clinicians working in 96 EDs affiliated with the Pediatric Emergency Research Network.Results1332/2446 (54%) clinicians provided information on suggested frequency of practice and preferred learning modalities for 18 critical procedures. Yearly practice was recommended for six procedures (bag valve mask ventilation, cardiopulmonary resuscitation (CPR), endotracheal intubation, laryngeal mask airway insertion, defibrillation/direct current (DC) cardioversion and intraosseous needle insertion) by at least 80% of respondents. 16 procedures were recommended for yearly practice by at least 50% of respondents. Two procedures (venous cutdown and ED thoracotomy) had yearly practice recommended by <40% of respondents. Simulation was the preferred learning modality for CPR, bag valve mask ventilation, DC cardioversion and transcutaneous pacing. Practice in alternative clinical settings (eg, the operating room) was the preferred learning modality for endotracheal intubation and laryngeal mask insertion. Use of models/mannequins for isolated procedural training was the preferred learning modality for all other invasive procedures. Free-text responses suggested the utility of cadaver labs and animal labs for more invasive procedures (thoracotomy, intercostal catheter insertion, open surgical airways, venous cutdown and pericardiocentesis).ConclusionsPaediatric ED clinicians suggest that most paediatric critical procedures should be practised at least annually. The preferred learning modality depends on the skill practised; alternative clinical settings are thought to be most useful for standard airway manoeuvres, while simulation-based experiential learning is applicable for most other procedures.

Critical procedures in children occur infrequently. Clinical exposure in the ED is therefore unreliable as the sole source of experience for critical procedures.

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Background: Children rarely experience critical illness, resulting in low exposure of emergency physicians (EPs) to critical procedures. Our primary objective was to describe senior EP confidence, most recent performance, and/or supervision of critical nonairway procedures. Secondary objectives were to compare responses between those who work exclusively in PEM and those who do not and to determine whether confidence changed for selected procedures according to increasing patient age. Methods: Survey of senior EPs working in 96 emergency departments (EDs) affiliated with the Pediatric Emergency Research Networks. Questions assessed training, performance, supervision, and confidence in 11 nonairway critical procedures, including cardiopulmonary resuscitation (CPR), vascular access, chest decompression, and cardiac procedures.Results: Of 2446 physicians, 1503 (61%) responded to the survey. Within the previous year, only CPR and insertion of an intraosseous needle had been performed by at least 50% of respondents: over 20% had performed defibrillation/direct current cardioversion. More than 50% of respondents had never performed or supervised ED thoracotomy, pericardiocentesis, venous cutdown, or transcutaneous pacing. Self-reported confidence was high for all patient age groups for CPR, needle thoracocentesis, tube thoracostomy, intraosseous needle insertion, and defibrillation/DC cardioversion. Confidence levels increased with increasing patient age for central venous and arterial line insertion. Respondents working exclusively in PEM were more likely to report being at least somewhat confident in defibrillation/DC cardioversion, intraosseous needle insertion, and central venous line insertion in particular age groups; however, they were less likely to be at least somewhat confident in ED thoracotomy and transcutaneous pacing.Conclusions: Cardiopulmonary resuscitation and intraosseous needle insertion were the only critical nonairway procedures performed by at least half of EPs within the previous year. Confidence was higher for these procedures, and needle and tube thoracostomy. These data may inform the development of continuing medical education activities to maintain pediatric procedural skills for emergency physicians.