Epidemiology and Outcomes of Critically Ill Children at Risk for Pediatric Acute Respiratory Distress Syndrome:A Pediatric Acute Respiratory Distress Syndrome Incidence and Epidemiology Study

OBJECTIVES: Interventional trials aimed at pediatric acute respiratory distress syndrome prevention require accurate identification of high-risk patients. In this study, we aimed to characterize the frequency and outcomes of children meeting "at risk for pediatric acute respiratory distress syndrome" criteria as defined by the Pediatric Acute Lung Injury Consensus Conference. DESIGN: Planned substudy of the prospective multicenter, international Pediatric Acute Respiratory Distress Syndrome Incidence and Epidemiology study conducted during 10 nonconsecutive weeks (May 2016-June 2017). SETTING: Thirty-seven international PICUs. PATIENTS: Three-hundred ten critically ill children meeting Pediatric Acute Lung Injury Consensus Conference "at-risk for pediatric acute respiratory distress syndrome" criteria. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We evaluated the frequency of children at risk for pediatric acute respiratory distress syndrome and rate of subsequent pediatric acute respiratory distress syndrome diagnosis and used multivariable logistic regression to identify factors associated with subsequent pediatric acute respiratory distress syndrome. Frequency of at risk for pediatric acute respiratory distress syndrome was 3.8% (95% CI, 3.4-5.2%) among the 8,122 critically ill children who were screened and 5.8% (95% CI, 5.2-6.4%) among the 5,334 screened children on positive pressure ventilation or high-flow oxygen. Among the 310 at-risk children, median age was 2.1 years (interquartile range, 0.5-7.3 yr). Sixty-six children (21.3%) were subsequently diagnosed with pediatric acute respiratory distress syndrome, a median of 22.6 hours (interquartile range, 9.8-41.0 hr) later. Subsequent pediatric acute respiratory distress syndrome was associated with increased mortality (21.2% vs 3.3%; p < 0.001) and longer durations of invasive ventilation and PICU care. Subsequent pediatric acute respiratory distress syndrome rate did not differ by respiratory support modality at the time of meeting at risk criteria but was independently associated with lower initial saturation:FIO2 ratio, progressive tachycardia, and early diuretic administration. CONCLUSIONS: The Pediatric Acute Lung Injury Consensus Conference "at-risk for pediatric acute respiratory distress syndrome" criteria identify critically ill children at high risk of pediatric acute respiratory distress syndrome and poor outcomes. Interventional trials aimed at pediatric acute respiratory distress syndrome prevention should target patients early in their illness course and include patients on high-flow oxygen and positive pressure ventilation

The state of the Martian climate

60°N was +2.0°C, relative to the 1981–2010 average value (Fig. 5.1). This marks a new high for the record. The average annual surface air temperature (SAT) anomaly for 2016 for land stations north of starting in 1900, and is a significant increase over the previous highest value of +1.2°C, which was observed in 2007, 2011, and 2015. Average global annual temperatures also showed record values in 2015 and 2016. Currently, the Arctic is warming at more than twice the rate of lower latitudes

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to &lt;90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], &gt;300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of &lt;15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P&lt;0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P&lt;0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Viral Glycoprotein Complex Formation, Essential Function and Immunogenicity in the Guinea Pig Model for Cytomegalovirus

<div><p>Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (<i>GP55</i> for gB, <i>GP75</i> for gH, <i>GP115</i> for gL, <i>GP100</i> for gM, <i>GP73</i> for gN and <i>GP74</i> for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene knockout results were similar to HCMV, except in the case of the gO homolog, which was non-essential in epithelial tropic virus but essential in lab adapted GPCMV. Overall, the findings demonstrate the similarity between HCMV and GPCMV glycoproteins and strengthen the relevance of this model for development of CMV intervention strategies.</p></div

GPCMV gM /gN complex formation and immunoprecipitation (IP) assays.

<p>All IPs were performed with GFP-Trap (ChromoTek) as described in materials and methods to immunopreciptiate proteins that interacted with gMGFP or GFP control. <b>(i)</b> gMGFP and gNmCherry co-expression and IP. Lanes 1 and 4 are total cell lysate of gMGFP and gNmCherry, respectively. Lanes 3 and 6, IP reactions. Lanes 2 and 5, control mock (MI) cell lysate. For gM detection, anti-GFP antibody (lanes 1–3). For gN detection, anti-mCherry antibody (lanes 4–6). <b>(ii)</b> Control GFP IP. GP84 protein tagged with GFP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135567#pone.0135567.ref051" target="_blank">51</a>] was co-expressed with gN(f)FLAG. Lanes 1 and 4 total cell lysate for GP84GFP and gN(f)FLAG respectively. Lanes 3 and 6, IP reactions for GP84GFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock control (MI). <b>(iii)</b> gMGFP and gN(f)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysates of gMGFP and gN(f)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock (MI) control cell lysate. 6. Detection for gN(f)FLAG by anti-FLAG antibody. <b>(iv)</b>. gMGFP and gN(s)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysate of gMGFP and gN(s)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(s)FLAG transfected cells. Lanes 2 and 5, mock control (MI). Detection for gN(s)FLAG by anti-FLAG antibody. Specific protein bands are indicated by an arrow. In gMGFP expressing cells a second higher MW protein (100 kDa) was detected and labelled x. All gels (4–20%) SDS-PAGE included a lane for a kDa ladder (MagicMark Protein Standard, Life Technologies). Ladder lanes not shown.</p

Transient expression of GPCMV gM and gN homologs and analysis of tagged proteins by western blot.

<p><b>A</b>. Predicted amino acid sequence for GP73 (gN) with potential glycosylation sites shaded in green. A truncated version of gN was generated by deletion of the first 40 codons, which included the majority of the predicted signal peptide sequence (underlined, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135567#pone.0135567.s004" target="_blank">S4 Fig</a>). The truncated gN(s) initiated from the first internal methionine (shaded in red). <b>B</b>. Figure of gN and gM expression constructs. Full length gN tagged with C-terminal mCherry was designated gNmCherry, B(i). Full length gN with C-terminal FLAG epitope tag was designated gN(f), B(ii), and truncated gN designated gN(s), B(iii). Full length gM was C-terminal tagged with GFP and designated gMGFP, B(iv). Size of predicted MW of tagged proteins indicated (kDa). <b>C</b>. Western blots were performed on transient plasmid expression of gN and gM tagged proteins in GPL cells. C(i) gNmCherry expression detected using anti-mCherry antibody. Lanes: (1) kDa ladder; (2) and (3) gNmcherry; (4) mock untransfected GPL cell lysate. C(ii) gN(f)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(f)FLAG. C(iii) gN(s)FLAG expression detected using anti-FLAG antibody. Lanes: (1) kDa ladder; (2) and (3) gN(s)FLAG (4) mock untransfected GPL cell lysate. C(iv) gMGFP expression detected using anti-GFP antibody. Lanes: (1) kDa ladder; (2) and (3) gM; (4) mock untransfected GPL cell lysate. <b>D</b>. gN and gM expression in the presence or absence of glycosylation inhibitor. D(i) Western blot of gN(f)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(ii) Western blot of gN(s)FLAG in the presence of tunicamycin (lane 2) or absence (lane 1). D(iii) Western blot of gMGFP in the presence of tunicamycin (lane 2) or absence (lane 1). Control mock untransfected GPL cell lysate lane 3 D(i)-(iii).</p

Transient expression of wild type or mutant gO in the presence or absence of GPCMV.

<p>The cellular location and molecular weight of gO protein was investigated by transient expression studies. Panels E, J and O are western blots for wild type or mutant gO using anti-FLAG antibody. Other panels are immunofluorescene images of wild type and mutant gO protein cellular localization by transient plasmid expression in GPL cells (A-D); GPL cells the presence of tunicamycin (glycosylation inhibitor, 2.5 ug/ml) (F-I); GPL cells plus GPCMV (K-N). Matched paired panels for gO (FITC) or gO (FITC) and DAPI (merged): A and B; C and D; F and G; H and I; K and L; M and N. Western blots: for wild type or gO mutant (E); wild type or gO mutant in the presence of tunicamycin (J); wild type or gO mutant in the presence of GPCMV (O).</p

Transfection of glycoprotein mutant KO GPCMV BACs onto GPL cells.

<p>Individual mutant GPCMV BACs were separately transfected onto GPL fibroblast cells to regenerate virus. GFP reporter gene encoded in the viral genome enabled real time tracking of the development of virus from individual transfected cells. Glycoprotein mutant GPCMV BACs were either transfected individually (panels A, C, E, G, I, K and L) or in combination with a rescue plasmid encoding a wild type locus to restore the mutant back to wild type phenotype where GFP virus could be detected spreading across the cell monolayer (panels B, D, F and H). The gH mutant was also transfected onto a cell line expressing gH <i>in trans</i> to support virus growth (panel J). A gH rescue virus was also generated by co-transfection with a rescue locus plasmid (data not shown). Panels K and L show the outcome for a gO knockout mutant based on the back drop of a virus carrying (L) or lacking (K) epithelial cell tropism. Only gO mutant GPCMV with epithelial tropism could grow on GPL cells. A rescue virus of panel K was generated by co-transfection of the gO GPCMV mutant with a wild type locus plasmid to restore wild type virus phenotype (data not shown). Images taken between day 16–18 post transfection.</p

GPCMV gM /gN complex formation and immunoprecipitation (IP) assays.

<p>All IPs were performed with GFP-Trap (ChromoTek) as described in materials and methods to immunopreciptiate proteins that interacted with gMGFP or GFP control. <b>(i)</b> gMGFP and gNmCherry co-expression and IP. Lanes 1 and 4 are total cell lysate of gMGFP and gNmCherry, respectively. Lanes 3 and 6, IP reactions. Lanes 2 and 5, control mock (MI) cell lysate. For gM detection, anti-GFP antibody (lanes 1–3). For gN detection, anti-mCherry antibody (lanes 4–6). <b>(ii)</b> Control GFP IP. GP84 protein tagged with GFP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135567#pone.0135567.ref051" target="_blank">51</a>] was co-expressed with gN(f)FLAG. Lanes 1 and 4 total cell lysate for GP84GFP and gN(f)FLAG respectively. Lanes 3 and 6, IP reactions for GP84GFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock control (MI). <b>(iii)</b> gMGFP and gN(f)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysates of gMGFP and gN(f)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock (MI) control cell lysate. 6. Detection for gN(f)FLAG by anti-FLAG antibody. <b>(iv)</b>. gMGFP and gN(s)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysate of gMGFP and gN(s)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(s)FLAG transfected cells. Lanes 2 and 5, mock control (MI). Detection for gN(s)FLAG by anti-FLAG antibody. Specific protein bands are indicated by an arrow. In gMGFP expressing cells a second higher MW protein (100 kDa) was detected and labelled x. All gels (4–20%) SDS-PAGE included a lane for a kDa ladder (MagicMark Protein Standard, Life Technologies). Ladder lanes not shown.</p