Electrostatic potential of the DNA binding surface of hApe1 and the corresponding surface area of Crc

<p>. The color coded electrostatic surface potential of hApe1 (<b>A</b>) and Crc (<b>B</b>) was drawn using the Adaptive Poisson-Boltzmann Solver package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064609#pone.0064609-Baker1" target="_blank">[37]</a> within PYMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064609#pone.0064609-DeLano1" target="_blank">[36]</a>. The electrostatic potential ranges from −5 (red) to +5 (blue) kT/e. The path of the DNA is shown in orange for hApe1 (<b>A</b>) and superimposed on Crc (<b>B</b>).</p

Electrophoretic mobility shift assays using CrcŹ RNA.

<p>Electrophoretic mobility shift assay of 10 nM 5′-end labeled CrcZ′ RNA with increasing amounts of His-Crc purified from the <i>E. coli</i> strain Rosetta™ (DE3)(pLysS pETM14lic-His₆Crc) by one-step NAC (<b>A</b>) and by one-step NAC followed by SEC (<b>B</b>), respectively. EMSA assay employing the His-Crc protein purified from the <i>P. aeruginosa</i> strain PAO1(pME9670) by one-step NAC (<b>C</b>), the protein eluate obtained after one-step NAC from strain Rosetta™ (DE3)(pLysS, pETM14lic) (mock; no Crc protein) (<b>D</b>) and the His-Crc protein from the <i>E. coli hfq-</i> strain JW4130(pME9670) by one-step NAC (<b>E</b>). Lane 1, no protein was added to labeled <i>CrcZ`</i> RNA. Lanes 2-4, the protein fractions were added in 50, 100 and 200-fold molar excess over labeled RNA. In the case of the mock preparation (<b>D</b>), the same amount of protein was added to RNA as in the experiments shown in panels <b>A</b>, <b>B</b> and <b>C</b>.</p

Structural comparison of Crc with AP endonucleases.

<p>(<b>A</b>), Superposition of the ribbon diagrams of Crc and its ortholog hApe1. Crc and hApe1 are colored in green and cyan, respectively. The positions of amino acid residues corresponding to active site residues in AP proteins are depicted in orange. (<b>B</b>), The catalytically active site of hApe1 (PDB accession code 1DE8) (cyan) is superposed with the corresponding area of Crc (green). (<b>C</b>), Sequence alignment of Crc with hApe1 (<i>Homo sapiens</i>) and Nape (<i>Neisseria meningitidis</i>). The four highly conserved residues located at the catalytically active site are highlighted in pink.</p

Data collection and refinement statistics.

a<p>Values in parentheses are for the highest resolution shell.</p>b<p></p>c<p></p>d<p></p><p>Where is the mean intensity of multiple observations of the symmetry-related reflections, N is the redundancy.</p>e<p></p>f<p>R_free is the cross-validation R_factor computed for the test set of reflections (5%) which are omitted in the refinement process.</p

Morbidity and mortality after anaesthesia in early life: results of the European prospective multicentre observational study, neonate and children audit of anaesthesia practice in Europe (NECTARINE)

Background: Neonates and infants requiring anaesthesia are at risk of physiological instability and complications, but triggers for peri-anaesthetic interventions and associations with subsequent outcome are unknown. Methods: This prospective, observational study 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. The primary aim was to identify thresholds of pre-determined physiological variables that triggered a medical intervention. The secondary aims were to evaluate morbidities, mortality at 30 and 90 days, or both, and associations with critical events. Results: Infants (n=5609) born at mean (standard deviation [sd]) 36.2 (4.4) weeks postmenstrual age (35.7% preterm) underwent 6542 procedures within 63 (48) days of birth. Critical event(s) requiring intervention occurred in 35.2% of cases, mainly hypotension (&gt;30% decrease in blood pressure) or reduced oxygenation (SpO2 &lt;85%). Postmenstrual age influenced the incidence and thresholds for intervention. Risk of critical events was increased by prior neonatal medical conditions, congenital anomalies, or both (relative risk [RR]=1.16; 95% confidence interval [CI], 1.04-1.28) and in those requiring preoperative intensive support (RR=1.27; 95% CI, 1.15-1.41). Additional complications occurred in 16.3% of patients by 30 days, and overall 90-day mortality was 3.2% (95% CI, 2.7-3.7%). Co-occurrence of intraoperative hypotension, hypoxaemia, and anaemia was associated with increased risk of morbidity (RR=3.56; 95% CI, 1.64-7.71) and mortality (RR=19.80; 95% CI, 5.87-66.7). Conclusions: Variability in physiological thresholds that triggered an intervention, and the impact of poor tissue oxygenation on patient's outcome, highlight the need for more standardised perioperative management guidelines for neonates and infants

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31â127 anaesthetic procedures in 30â874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0â5·5) with an incidence of respiratory critical events of 3·1% (2·9â3·3). Cardiovascular instability occurred in 1·9% (1·7â2·1), with an immediate poor outcome in 5·4% (3·7â7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10â000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86â0·90; p<0·0001), medical history, and physical condition (1·60, 1·40â1·82; p<0·0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0·99, 0·981â0·997; p<0·0048 for respiratory critical events, and 0·98, 0·97â0·99; p=0·0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia. Funding European Society of Anaesthesiology