The crystal structure of the semireduced form of cyclic nucleotide phosphodiesterase (CPDase) from Arabidopsis thaliana has been solved by molecular replacement and refined at the resolution of 1.8 Å. We have previously reported the crystal structure of the native form of this enzyme, whose main target is ADP-ribose 1؆,2؆-cyclic phosphate, a product of the tRNA splicing reaction. CPDase possesses six cysteine residues, four of which are involved in forming two intra-molecular disulfide bridges. One of these bridges, between Cys-104 and Cys-110, is opened in the semireduced CPDase, whereas the other remains intact. This change of the redox state leads to a conformational rearrangement in the loop covering the active site of the protein. While the native structure shows this partially disordered loop in a coil conformation, in the semireduced enzyme the Nterminal lobe of this loop winds up and elongates the preceding ␣-helix. The semireduced state of CPDase also enabled co-crystallization with a putative inhibitor of its enzymatic activity, 2,3-cyclic uridine vanadate. The ligand is bound within the active site, and the mode of binding is in agreement with the previously proposed enzymatic mechanism. Selected biophysical properties of the oxidized and the semireduced CPDase are also discussed.

BackgroundCentral line-associated bloodstream infections (CLABSIs) are major contributors to preventable harm in the inpatient paediatric setting. Despite multiple guidelines to reduce CLABSI, sustaining reliable central line maintenance bundle compliance remains elusive. We identified frontline and family engagement as key drivers for this initiative. The baseline CLABSI rate for all our paediatric inpatient units (January 2016–January 2017) was 1.71/1000 central line days with maintenance bundle compliance at 87.9% (monthly range 44%–100%).ObjectiveTo reduce CLABSI by increasing central line maintenance bundle compliance to greater than 90% using kamishibai card (K-card) audits and family ‘key card’ education.MethodsWe transitioned our central line maintenance bundle audits from checklists to directly observed K-card audits. K-cards list the central line maintenance bundle elements to be reviewed with frontline staff. Key cards are cue cards developed using a plain-language summary of CLABSI K-cards and used by frontline staff to educate families. Key cards were distributed to families of children with central lines to simultaneously engage patients, families and frontline staff after a successful implementation of the K-card audit process. A survey was used to obtain feedback from families.ResultsIn the postintervention period (February 2017–December 2019), our CLABSI rate was 0.63/1000 central line days, and maintenance bundle compliance improved to 97.1% (monthly range 86%–100%, p<0.001). Of the 45 family surveys distributed, 20 (44%) were returned. Nineteen respondents (95%) reported being extremely satisfied with the key card programme and provided positive comments.ConclusionCombining the key card programme with K-card audits was associated with improved maintenance bundle compliance and a reduction in CLABSI. This programme has the potential for use in multiple healthcare improvement initiatives.

A series of allosteric effectors of hemoglobin, 2-(aryloxy)-2-alkanoic acids, was prepared to investigate the effect of the stereocenter on allosteric activity. The chiral analogues were based on the lead compound, RSR13 (3b), with different alkyl/alkanoic and cycloalkyl/cycloalkanoic groups positioned at the acidic chiral center. Of the 23 racemic molecules synthesized, 5 were selected for resolution based on structure-activity relationships. One chiral analogue, (-)-(1R,2R)-1-[4-[[(3, 5-dimethylanilino)carbonyl]methyl]phenoxy]-2-methylcyclopentane carbox ylic acid (11), exhibited greater in vitro activity in hemoglobin solutions than its antipode, racemate, and RSR13. Compound (-)-(1R, 2R)-11 was equipotent with RSR13 in whole blood, is a candidate for in vivo animal studies, and if efficacious and safe has a potential for use in humans. In general, it was found that chirality affects allosteric effector activity with measurable differences observed between enantiomers and the racemates.

We have compared selected biophysical properties of three phosphodiesterases, from Arabidopsis thaliana, Saccharomyces cerevisiae, and Escherichia coli. All of them belong to a recently identified family of cyclic nucleotide phosphodiesterases. Experiments elucidating folding stability, protein fluorescence, oligomerization behavior, and the effects of substrates were conducted, revealing differences between the plant and the yeast protein. According to CD spectroscopy, the latter protein exhibits an (␣ ؉ ␤) fold rather than an (␣/␤) fold as found with CPDase (A. thaliana). The redox-dependent structural reorganization recently found for the plant protein by X-ray crystallography could not be detected by CD spectroscopy due to its only marginal effect on the total percentage of helical content. However, in the present study a redoxdependent effect was also observed for the yeast CPDase. The enzymatic activity of wild type CPDase (A. thaliana) as well as of four mutants were characterized by isothermal titration calorimetry and the results prove the requirement of all four residues of the previously identified tandem signature motif for the catalytic function. Within the comparison of the three proteins in this study, the PDase Homolog/RNA ligase (E. coli) shares more similarities with the plant than with the yeast protein.

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