[Prospective Cohort Study of In-Hospital Patients Undergoing Ophthalmic Surgery for the Validation of ClassIntra: Classification of Intraoperative Adverse Events]. / Prospektive Kohortenstudie bei stationären augenchirurgischen Patienten für die Validierung der ClassIntra-Klassifikation für unerwünschte intraoperative Ereignisse.

BACKGROUND: To the best of our knowledge, there is no validated classification to assess intraoperative adverse events (iAEs) in ophthalmic surgery. ClassIntra is a newly developed classification for surgery- and anaesthesia-related iAEs that has been recently validated in various surgical disciplines, but not in ophthalmic surgery. We aim to assess the validity and practicability of ClassIntra in patients undergoing ophthalmic surgery. METHODS: A consecutive sample of in-hospital patients undergoing any type of ophthalmic surgery was included in this single-centre prospective cohort study. iAEs were classified using ClassIntra, consisting of 5 severity grades according to the symptoms of the patient and the required treatment. All patients were followed for two weeks to record all postoperative adverse events according to Clavien-Dindo. The primary endpoint was the risk-adjusted association between the most severe iAE and the weighted sum of all postoperative adverse events within the two-week follow-up using the Comprehensive Complication Index (CCI). In addition, ophthalmologists and anaesthesiologists were asked to complete an online survey assessing the severity of iAEs for 10 fictitious clinical case scenarios. Reliability was assessed by comparing the clinicians' ratings to the prespecified benchmark rating of the study team. RESULTS: In this study, 100 in-hospital patients with an average age of 64 years (SD 15) were included. The majority of all patients were ASA II (n = 53, 53%) or III (n = 42, 42%). Thirty-two iAEs were recorded in 22 patients (17 grade I, 12 grade II, 3 grade III). Ninety-four postoperative adverse events occurred in 50 patients (44 grade I, 36 grade II, 14 grade IIIa). We found a mean difference in CCI of 2,1 (95% confidence interval [CI] - 2,5 to 6,8) per one unit increase in severity grades of ClassIntra. Fifty ophthalmologists and anaesthesiologists completed the online survey (response rate 54%). The intraclass correlation coefficient was 0,79 (95% CI 0,64 to 0,94). CONCLUSIONS: The application of ClassIntra during daily routine in ophthalmic surgery showed the usefulness and practicability of this classification for the standardised assessment of intraoperative adverse events. Although construct validity could not be demonstrated, the good reliability in the survey's rating underlines the criterion validity of this newly developed classification in ophthalmic surgery.

Structural biology: analysis of 'downhill' protein folding.

There is controversy as to whether homologues from the peripheral subunit binding domain family of small proteins fold 'downhill' (that is, non-cooperatively, in the absence of free-energy barriers between conformations) and whether they modulate their size for biological function. Sadqi et al. claim that Naf-BBL--a naphthylalanine-labelled, truncated version of this domain--folds in this way, on the grounds that they recorded a wide spread of melting temperatures of individual atoms measured by proton nuclear magnetic resonance (NMR) during their thermal denaturation. But their data are not of adequate quality to distinguish, within experimental error, between downhill folding and folding with a cooperative transition. Accordingly, their results offer no compelling evidence that Naf-BBL folds downhill, particularly as non-truncated, unmodified peripheral subunit binding domains seem to fold cooperatively.

Ultra-fast barrier-limited folding in the peripheral subunit-binding domain family.

We have determined the solution structures, equilibrium properties and ultra-fast folding kinetics for three bacterial homologues of the peripheral subunit-binding domain (PSBD) family. The mesophilic homologue, BBL, was less stable than the thermophilic and hyper-thermophilic variants (E3BD and POB, respectively). The broad unfolding transitions of each PSBD, when probed by different techniques, were essentially superimposable, consistent with co-operative denaturation. Temperature-jump and continuous-flow fluorescence methods were used to measure the folding kinetics for E3BD, POB and BBL. E3BD folded fairly rapidly at 298K (folding half-time approximately 25 micros) and BBL and POB folded even faster (folding half-times approximately 3-5 micros). The variations in equilibrium and kinetic behaviour observed for the PSBD family resembles that of the homeodomain family, where the folding pattern changes from apparent two-state transitions to multi-state kinetics as the denatured state becomes more structured. The faster folding of POB may be a consequence of its higher propensity to form helical structure in the region corresponding to the folding nucleus of E3BD. The ultra-fast folding of BBL appears to be a consequence of residual structure in the denatured ensemble, as with engrailed homeodomain. We discuss issues concerning "one-state", downhill folding, and find no evidence for, and strong evidence against, it occurring in these PSBDs. The shorter construct used previously for BBL was destabilized significantly and the stability further perturbed by the introduction of fluorescent probes. Thermal titrations for 11 side-chains scattered around the protein, when probed by (13)C-NMR experiments, could be fit globally to a common co-operative transition.

Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines.

The NATs (arylamine N-acetyltransferases) are a well documented family of enzymes found in both prokaryotes and eukaryotes. NATs are responsible for the acetylation of a range of arylamine, arylhydrazine and hydrazine compounds. We present here an investigation into the catalytic triad of residues (Cys-His-Asp) and other structural features of NATs using a variety of methods, including site-directed mutagenesis, X-ray crystallography and bioinformatics analysis, in order to investigate whether each of the residues of the catalytic triad is essential for catalytic activity. The catalytic triad of residues, Cys-His-Asp, is a well defined motif present in several families of enzymes. We mutated each of the catalytic residues in turn to investigate the role they play in catalysis. We also mutated a key residue, Gly126, implicated in acetyl-CoA binding, to examine the effects on acetylation activity. In addition, we have solved the structure of a C70Q mutant of Mycobacterium smegmatis NAT to a resolution of 1.45 A (where 1 A=0.1 nm). This structure confirms that the mutated protein is correctly folded, and provides a structural model for an acetylated NAT intermediate. Our bioinformatics investigation analysed the extent of sequence conservation between all eukaryotic and prokaryotic NAT enzymes for which sequence data are available. This revealed several new sequences, not yet reported, of NAT paralogues. Together, these studies have provided insight into the fundamental core of NAT enzymes, and the regions where sequence differences account for the functional diversity of this family. We have confirmed that each of the three residues of the triad is essential for acetylation activity.

One-state downhill versus conventional protein folding.

Classical protein folding invokes a cooperative transition between distinct thermodynamic states that are individually populated at equilibrium and separated by an energy barrier. It has been proposed, however, that the small protein, BBL, undergoes one-step downhill folding whereby it folds non-cooperatively to its native state without encountering an appreciable energy barrier. Only a single conformational ensemble is populated under given conditions, and so the denatured state ensemble progressively changes into the native structure. A wide dispersion of thermal denaturation midpoints that was observed for an extrinsically labelled fragment of BBL is proposed to be evidence for its one-state, downhill folding, a phenomenon that is also suggested to be functionally important for BBL and its homologues. We found, however, that thermal denaturation of unlabelled wild-type BBL was highly cooperative, with very similar transition midpoints for the melting of secondary and tertiary interactions, as well as for individual residues when monitored by NMR. Similar results were also observed for two other homologues, E3BD and POB. Further, the extrinsic fluorophores perturbed the unfolding energetics of labelled BBL, and complicated its equilibrium behaviour. One-step downhill folding may well occur for some proteins that do not have distinct folded states but not for BBL and its well-folded homologues.