Cysteine Nucleophiles in Glycosidase Catalysis : Application of a Covalent β-L-Arabinofuranosidase Inhibitor

The recent discovery of zinc-dependent retaining glycoside hydrolases (GHs), with active sites built around a Zn(Cys)(3)(Glu) coordination complex, has presented unresolved mechanistic questions. In particular, the proposed mechanism, depending on a Zn-coordinated cysteine nucleophile and passing through a thioglycosyl enzyme intermediate, remains controversial. This is primarily due to the expected stability of the intermediate C-S bond. To facilitate the study of this atypical mechanism, we report the synthesis of a cyclophellitol-derived beta-l-arabinofuranosidase inhibitor, hypothesised to react with the catalytic nucleophile to form a non-hydrolysable adduct analogous to the mechanistic covalent intermediate. This beta-l-arabinofuranosidase inhibitor reacts exclusively with the proposed cysteine thiol catalytic nucleophiles of representatives of GH families 127 and 146. X-ray crystal structures determined for the resulting adducts enable MD and QM/MM simulations, which provide insight into the mechanism of thioglycosyl enzyme intermediate breakdown. Leveraging the unique chemistry of cyclophellitol derivatives, the structures and simulations presented here support the assignment of a zinc-coordinated cysteine as the catalytic nucleophile and illuminate the finely tuned energetics of this remarkable metalloenzyme clan.Medical BiochemistryBio-organic Synthesi

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Diagnostic Concordance of Echocardiography and Cardiac Magnetic Resonance-Based Tissue Tracking for Differentiating Constrictive Pericarditis From Restrictive Cardiomyopathy

Background\u2014Variations in longitudinal deformation of the left ventricle have been suggested to be useful for differentiating chronic constrictive pericarditis (CP) and restrictive cardiomyopathy (RCM). We assessed left ventricular mechanics derived from cardiac magnetic resonance (CMR) cine\u2013based and 2-dimensional echocardiography\u2013based tissue tracking to determine intermodality consistency of diagnostic information for differentiating CP from RCM. Methods and Results\u2014We retrospectively identified 92 patients who underwent both CMR and 2-dimensional echocardiography and who had a final diagnosis of CP (n=28), RCM (n=30), or no structural heart disease (n=34). Global longitudinal strain from long-axis views and circumferential strain from short-axis views were measured on 2-dimensional echocardiographic and CMR cine images using the same offline software. Logistic regression models with receiver operating characteristics curves, continuous net reclassification improvement, and the integrated discrimination improvement (IDI) were used for assessing the incremental predictive performance. Global longitudinal strain was higher in patients with CP than in those with RCM (P<0.001), and both techniques were found to have similar diagnostic value (area under the curve, 0.84 versus 0.88 for CMR and echocardiography, respectively). For echocardiography, the addition of global longitudinal strain to respiratory septal shift and early diastolic mitral annular velocity resulted in improved continuous net reclassification improvement (P<0.001 for both) and integrated discrimination improvement (P=0.005 and 0.024) for both models. Similarly, for CMR, the addition of global longitudinal strain to septal shift and pericardial thickness resulted in improved continuous net reclassification improvement (P<0.001 for both) and integrated discrimination improvement (P=0.003 and <0.001). Conclusions\u2014CMR and echocardiography tissue tracking\u2013derived left ventricular mechanics provide comparable diagnostic information for differentiating CP from RCM