Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

BACKGROUND: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. METHODS: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. RESULTS: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1–6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2<90% for 60 s) was reported in 40%. No associated risk factors could be identified among co-morbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. CONCLUSIONS: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event

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

Assembly of D1/D2 complexes of photosystem II: binding of pigments and a network of auxiliary proteins

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high-light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A (RubA) and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls, pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound chlorophyll was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the chlorophylls and both pheophytins. We suggest that chlorophyll can be delivered to RCII from either monomeric PSI or Ycf39/Hlip complexes

The Ycf48 accessory factor occupies the site of the oxygen-evolving manganese cluster during photosystem II biogenesis

Robust oxygenic photosynthesis requires a suite of accessory factors to ensure efficient assembly and repair of the oxygen-evolving photosystem two (PSII) complex. The highly conserved Ycf48 assembly factor binds to the newly synthesized D1 reaction center polypeptide and promotes the initial steps of PSII assembly, but its binding site is unclear. Here we have used cryo-electron microscopy to determine the structure of a cyanobacterial PSII D1/D2 reaction center assembly complex with Ycf48 attached. Ycf48, a 7-bladed beta propeller, binds to the amino-acid residues of D1 that ultimately ligate the water-oxidising Mn4CaO5 cluster, thereby preventing the premature binding of Mn2+ and Ca2+ ions and protecting the site from damage. Interactions with D2 help explain how Ycf48 promotes assembly of the D1/D2 complex. Overall, our work provides valuable insights into the early stages of PSII assembly and the structural changes that create the binding site for the Mn4CaO5 cluster

The Photosystem II Assembly Factor Ycf48 from the Cyanobacterium Synechocystis sp. PCC 6803 Is Lipidated Using an Atypical Lipobox Sequence.

Photochemical energy conversion during oxygenic photosynthesis is performed by membrane-embedded chlorophyll-binding protein complexes. The biogenesis and maintenance of these complexes requires auxiliary protein factors that optimize the assembly process and protect nascent complexes from photodamage. In cyanobacteria, several lipoproteins contribute to the biogenesis and function of the photosystem II (PSII) complex. They include CyanoP, CyanoQ, and Psb27, which are all attached to the lumenal side of PSII complexes. Here, we show that the lumenal Ycf48 assembly factor found in the cyanobacterium Synechocystis sp. PCC 6803 is also a lipoprotein. Detailed mass spectrometric analysis of the isolated protein supported by site-directed mutagenesis experiments indicates lipidation of the N-terminal C29 residue of Ycf48 and removal of three amino acids from the C-terminus. The lipobox sequence in Ycf48 contains a cysteine residue at the -3 position compared to Leu/Val/Ile residues found in the canonical lipobox sequence. The atypical Ycf48 lipobox sequence is present in most cyanobacteria but is absent in eukaryotes. A possible role for lipoproteins in the coordinated assembly of cyanobacterial PSII is discussed

The photosystem II assembly factor Ycf48 from the cyanobacterium <i>Synechocystis</i> sp. PCC 6803 is lipidated using an atypical lipobox sequence

Photochemical energy conversion during oxygenic photosynthesis is performed by membrane-embedded chlorophyll-binding protein complexes. The biogenesis and maintenance of these complexes requires auxiliary protein factors that optimize the assembly process and protect nascent complexes from photodamage. In cyanobacteria, several lipoproteins contribute to the biogenesis and function of the photosystem II (PSII) complex. They include CyanoP, CyanoQ, and Psb27, which are all attached to the lumenal side of PSII complexes. Here, we show that the lumenal Ycf48 assembly factor found in the cyanobacterium Synechocystis sp. PCC 6803 is also a lipoprotein. Detailed mass spectrometric analysis of the isolated protein supported by site-directed mutagenesis experiments indicates lipidation of the N-terminal C29 residue of Ycf48 and removal of three amino acids from the C-terminus. The lipobox sequence in Ycf48 contains a cysteine residue at the −3 position compared to Leu/Val/Ile residues found in the canonical lipobox sequence. The atypical Ycf48 lipobox sequence is present in most cyanobacteria but is absent in eukaryotes. A possible role for lipoproteins in the coordinated assembly of cyanobacterial PSII is discusse