Code Blue

Aliquots (100 µg) of the protein extracts from three replicates for each cellular condition were fractionated on a preparative by 10% SDS-PAGE, 16 × 20 cm. The protein electrophoretic patterns were stained using Gel Code Blue Stain Reagent (Thermo Fisher Scientific, Waltham, MA, USA). Each gel lane was cut into 5 mm slices and these later were excised from gel. An in situ trypsin digestion of the slices was carried out [37 (link),38 (link),39 (link),40 (link)]. Peptide mixtures were resuspended in 0.2% HCOOH and an MS analysis was performed using a LTQ-Orbitrap XL (Thermo Scientific, Bremen, Germany) coupled with nanoEASY II, Nanoseparations chromatographic system (75 µm–l 20 cm, column, Thermo Scientific, Bremen, Germany). The peptide mixture was concentrated and desalted onto a 2 cm trapping column (C18, ID 100 μm, 5 μm) and then fractionated onto 20 cm C18 reverse phase silica capillary column (ID 75 μm, 5 μm) (Nanoseparations). The peptides were eluted by a nonlinear gradient—4% B solvent (A eluent: 0.1% formic acid; B eluent: 80% acetonitrile, 0.08% formic acid) during 5 min, from 4 to 40% B in 45 min, and from 40 to 90% B in 1 min at flow rate of 250 nL/min [17 (link)]. An MS analysis was performed with a resolution set to 30000, and mass range from m/z 400 to 1800 Da. The three most intense doubly, triply, and fourthly charged ions were selected and fragmented using Collision Induced Dissociation (CID) fragmentation. A proteomic analysis was performed using a Proteome Discoverer™ platform (version 1.3.0.339; Thermo Scientific, Bremen, Germany), interfaced with an in-house Mascot server (version 2.3, Matrix Science, London, UK) for protein identifications. All of the peak lists were processed using the following parameters: (I) Spectrum Selector. Min Precursor Mass: 350 Da, Max. Precursor Mass: 5000 Da, Minimum Peak Count: 1; (II) Mascot: 1. Input Data. Protein Database: SwissProt, Enzyme: Trypsin, Maximum Missed Cleavage Sites: 2, Instrument: ESI-FTICR, Taxonomy: Homo sapiens. 2. Tolerances. Precursor Mass Tolerance: 5 ppm, Fragment Mass Tolerance: 0.8 Da. 3. Dynamic Modification. Methionine Oxidation, N-terminal Glutamine cyclization to Pyroglutamic Acid, N-terminal protein Acetylation. 4. Static modification. Cysteine Carboamidomethylation. Proteins identified by a minimum of two peptides were accepted [20 (link)].

Clinical data were grouped into 1-hour blocks for 24 hours ending at the event for case patients or at the end of 12 hours of data collection for control patients. Where there were missing data, the most recent recorded data were used, consistent with the approach used for other scores in critically ill children [21 (link)]. The greatest subscore for each item within each hour was identified and used to calculate the Bedside PEWS score for that hour. We then calculated the maximum PEWS score for the 12 hours ending 1 hour before the clinical deterioration event and in the six 4-hour blocks preceding ICU admission in patients urgently admitted to the ICU.
The primary analysis evaluated the hypothesis that the Bedside PEWS score can identify children at risk for cardiopulmonary arrest with at least one hour's notice. Logistic regression was used to compare the maximum Bedside PEWS score of case and control patients using all 12 hours of data in control patients and the 12 hours of data ending 1 hour before either urgent ICU admission or a code blue event in the case patients. The AUCROC curve was determined from the c-statistic calculated by logistic regression, and the 95% confidence interval (95% CI) was calculated using an accepted algorithm [22 ]. The ROC curve was represented graphically, and the sensitivity and specificity of the score at thresholds of 7 and 8 were calculated based on our previous work [6 (link)].
Repeated measures linear regression was used to evaluate the temporal evolution of scores preceding urgent ICU admission and code blue events in case patients. The dependent variable was the maximum Bedside PEWS score for each of the six 4-hour time periods preceding the clinical deterioration event. The independent variable was the midpoint of the time interval expressed in hours from the time of ICU admission. Linear regression was used to evaluate the relationships between the maximum Bedside PEWS score and the number of risk factors for cardiac arrest. Separate analyses were performed for case and control patients.
The association between the retrospective rating of nurses and the case or control status of patients was evaluated using logistic regression. We used clinical data from the 12 hours ending 1 hour before the clinical deterioration event and for 12 hours in control patients to calculate the maximum Bedside PEWS score. These data were paired with corresponding survey data from frontline nurses. When more than one nurse was surveyed in this time period, we used the data from the nurse who had last cared for the patient. The responses of the frontline nurses were represented on a numerical scale from 1 to 5. We tabulated the maximum Bedside PEWS score for each level of nurse rating in case and control patients. Logistic regression was used to evaluate the performance of nurse rating, the Bedside PEWS score, and the nurse rating with the Bedside PEWS score. We used the c-statistic as a measure of the AUCROC curve and calculated the 95% CI. Comparison of the AUCROC curve for the nurse rating and the maximum Bedside PEWS score was carried out as described by DeLong et al. [23 (link)].
Subgroup analyses described score performance in the following patient categories: urgent ICU patients, code blue patients, those who fell within any of the five age categories of the Bedside PEWS score, across institutions, patients with chronic conditions (bone marrow or organ transplantation, cardiac disease, severe cerebral palsy), patients with medical devices that might have place them at increased risk (tracheostomy, enterostomy feeding device, home oxygen), patients with acute illness (diabetic ketoacidosis, seizures), patients whose conditions had increased complexity (> 3 services involved in care, > 10 medications), patients with an administrative risk (recent transfer of primary service, ICU transfer, postoperative, off-service patient), and patients who had cardiopulmonary arrest. Power calculations based on our previous work suggested that differences between means could be shown with 30 patients per group. Given that our objectives were to evaluate score performance within specified subgroups and at each hospital, we sought to maximise the numbers of cases and controls from participating hospitals. Numbers were thus determined by the duration of the study at each hospital. The protocol was reviewed and approved by the research ethics boards at participating hospitals. All research ethics boards required consent for staff participation in the surveys and waived the need for patient consent.

The recombinant proteins were purified using a denaturing protocol: bacterial cells transformed by L1, L2, E4, and E7 plasmids were lysed in a Phosphate buffer (100 mM Na₂HPO₄ 300 mM NaCl, 10 mM Tris, 1% Triton X-100, pH 8), containing 8 M urea, while bacterial cells transformed by E6 plasmids were lysed in Phosphate buffer, containing 6 M Guanidine-HCl. Proteins were purified by affinity chromatography on Ni-NTA resin and eluted by a pH gradient. The fractions containing the proteins were pooled, adjusted to a neutral pH by adding Tris pH 8.8 and stored in urea buffer at -30°C, until use. Protein concentration was determined by standard method (BC protein assay, BIORAD). To evaluate the purity, each protein samples of L1, L2, E4, E6 and E7 were denatured in SDS-loading buffer (25 mM Tris-HCl pH 6.8, 5 % β-Mercapto-ethanol, 2% SDS, 50% glycerol), separated in pre-cast gel Nu-PAGE MES-SDS (INVITROGEN), stained by Gel Code Blue Stain Reagent (PIERCE). The proteins were identified by Western blot using the monoclonal anti poly-Histidine antibody Clone HIS1 (Sigma-Aldrich). A peroxidase-conjugate goat anti-mouse IgG (H+L) (SBA-INC USA) was used as secondary antibody. The immune-complexes were revealed by chemiluminescence (Amersham Bioscience).

The quantitative analysis of intermolecular interactions in the crystal structure is obtained by HS analysis using Crystal Explorer 3.1 software.³⁶ The HS analysis involves the partition of electron density (ED) of a molecule into atomic fragments such that a molecule within a crystal is given by a weighting functionwhere ρ(r) is a spherically averaged Hartree–Fock atomic ED function of i^th nucleus. The cutoff of the weight function is 0.5 Å.
Further, the visualization of different interactions in the crystal structure is performed using different functions such as d_e, d_i, d_norm, shape index and curvedness mapped on the HS of the molecules. The normalized contact distance (d_norm) in the HS analysis is given byd_e denotes HS distance from nearest nucleus situated outside surface, d_i is the HS distance from nearest nucleus situated inside surface and r^vdw corresponds to van der Waals radius. The d_norm parameter on the HS of the molecule is visualized in a red-white-blue color code. The corresponding intermolecular contacts which are less than their vdW radii are indicated by red regions on the HS. Whereas the blue regions show intermolecular contacts of distance longer than their vdW radii. White regions signify that the contact distance is identical to the summation of vdW radii. Complementary nature of intermolecular interactions during the packing of molecules is identified by shape index. The curvedness is also useful parameter to understand the interactions such as π⋯π interactions. The flat regions of a surface are highlighted by a low value of curvedness. Similarly, the 2D fingerprint plot associated with the HS give quantitative information on the percentage contribution of individual interactions to the supramolecular assembly.

The bicistronic constructs containing Ku70-Strep or Ku70^C85Y-Strep and Ku80-6xHis were designed as previously described (65 (link)). In particular, the genes were chemically synthetized (GenScript, Piscataway, NJ, USA) and cloned into the pET21a vector. Ku heterodimers were recombinantly produced in Escherichia coli BL21 (DE3) cells and purified. Briefly, the Ku70–Ku80 and Ku70^C85Y–Ku80 heterodimers were produced in ZYM-5052 medium (66 (link)) supplemented with ampicillin (100 mg/L), extracted and purified by immobilized ion metal affinity chromatography (ABT, Torrejon de Ardoz, Madrid, Spain), followed by Strep purification on Strep-Tactin resin (IBA Lifesciences, Gottingen, Germany). Fractions containing the highest amount of protein were pooled and buffer-exchanged with HEPES buffer (HEPES 25 mM, NaCl 100 mM, pH 7) by gel filtration on PD-10 columns (GE Healthcare, Little Chalfont, UK). Protein concentration was determined with the Bradford assay (Bio-Rad, Hercules, USA), using bovine serum albumin as a standard. SDS-PAGE was performed on 12% polyacrylamide gels and stained with Gel-Code Blue (Pierce, Rockford, USA) after electrophoresis. Broad-range, pre-stained molecular-mass markers (GeneSpin, Milan, Italy) were used as standards.

GFP production was assessed on the soluble protein fraction by monitoring fluorescence emission at 528 nm with a Cary Eclipse (Varian Inc., Palo Alto, CA, USA) spectrofluorometer using an excitation wavelength of 475 nm. The specific fluorescence was calculated using the following equation: $$\text{Specific}\text{fluorescence}(\text{a.u}./\text{mg})=\frac{\text{Fluorescence}\text{at}528\text{nm}(\text{a.u}.)}{P_{\mathit{tot}}(\text{mg})}{10}^{-3}$$
GFP production was also tested on whole cells by flow cytometry with a CytoFlex S cytofluorimeter equipped with argon ion laser at 488 nm (Beckman Coulter, Life Sciences, Indianapolis, IN, USA). Analyses were carried out on cell suspension acquiring 10,000 events.
At the end of the induction phase (48 h), GFP production was analyzed by SDS-PAGE. The crude extracts, soluble and insoluble protein fractions were denatured by adding Laemmli buffer (60 mM Tris-Cl pH 6.8, 2% w/v SDS, 10% v/v glycerol, 5% v/v β-mercaptoethanol, 0.02‰ w/v bromophenol blue) and boiling the samples at 99 °C for 5 min. Balanced amounts of these samples, corresponding to a OD₆₀₀ ~ 0.15, were loaded on 14% SDS-PAGE and proteins visualized after staining with Gel-Code Blue (ThermoFisher, Waltham, MA, USA).