Proteolytic Processing of Nlrp1b Is Required for Inflammasome Activity

Nlrp1b is a NOD-like receptor that detects the catalytic activity of anthrax lethal toxin and subsequently co-oligomerizes into a pro-caspase-1 activation platform known as an inflammasome. Nlrp1b has two domains that promote oligomerization: a NACHT domain, which is a member of the AAA+ ATPase family, and a poorly characterized Function to Find Domain (FIIND). Here we demonstrate that proteolytic processing within the FIIND generates N-terminal and C-terminal cleavage products of Nlrp1b that remain associated in both the auto-inhibited state and in the activated state after cells have been treated with lethal toxin. Functional significance of cleavage was suggested by the finding that mutations that block processing of Nlrp1b also prevent the ability of Nlrp1b to activate pro-caspase-1. By using an uncleaved mutant of Nlrp1b, we established the importance of cleavage by inserting a heterologous TEV protease site into the FIIND and demonstrating that TEV protease processed this site and induced inflammasome activity. Proteolysis of Nlrp1b was shown to be required for the assembly of a functional inflammasome: a mutation within the FIIND that abolished cleavage had no effect on self-association of a FIIND-CARD fragment, but did reduce the recruitment of pro-caspase-1. Our work indicates that a post-translational modification enables Nlrp1b to function

The tenth data release of the Sloan digital sky survey: First spectroscopic data from the SDSS-iii apache point observatory galactic evolution experiment

The Sloan Digital Sky Survey (SDSS) has been in operation since 2000 April. This paper presents the tenth public data release (DR10) from its current incarnation, SDSS-III. This data release includes the first spectroscopic data from the Apache Point Observatory Galaxy Evolution Experiment (APOGEE), along with spectroscopic data from the Baryon Oscillation Spectroscopic Survey (BOSS) taken through 2012 July. The APOGEE instrument is a near-infrared R ~ 22,500 300-fiber spectrograph covering 1:514-1:696 μm. The APOGEE survey is studying the chemical abundances and radial velocities of roughly 100,000 red giant star candidates in the bulge, bar, disk, and halo of the Milky Way. DR10 includes 178,397 spectra of 57,454 stars, each typically observed three or more times, from APOGEE. Derived quantities from these spectra (radial velocities, effective temperatures, surface gravities, and metallicities) are also included. DR10 also roughly doubles the number of BOSS spectra over those included in the ninth data release. DR10 includes a total of 1,507,954 BOSS spectra, comprising 927,844 galaxy spectra; 182,009 quasar spectra; and 159,327 stellar spectra, selected over 6373.2 deg2.This is an author-created, un-copyedited version of an article accepted for publication in The Astrophysical Journal Supplement Series. The publisher is not responsible for any errors or omissions in this version of the manuscript or any version derived from it. The Version of Record is available online at http://dx.doi.org/10.1088/0067-0049/211/2/17. The accepted version will be under embargo until the 18th March 2015

The Eleventh and Twelfth Data Releases of the Sloan Digital Sky Survey: Final Data from SDSS-III

The third generation of the Sloan Digital Sky Survey (SDSS-III) took data from 2008 to 2014 using the original SDSS wide-field imager, the original and an upgraded multi-object fiber-fed optical spectrograph, a new near-infrared high-resolution spectrograph, and a novel optical interferometer. All of the data from SDSS-III are now made public. In particular, this paper describes Data Release 11 (DR11) including all data acquired through 2013 July, and Data Release 12 (DR12) adding data acquired through 2014 July (including all data included in previous data releases), marking the end of SDSS-III observing. Relative to our previous public release (DR10), DR12 adds one million new spectra of galaxies and quasars from the Baryon Oscillation Spectroscopic Survey (BOSS) over an additional 3000 deg2 of sky, more than triples the number of H-band spectra of stars as part of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE), and includes repeated accurate radial velocity measurements of 5500 stars from the Multi-object APO Radial Velocity Exoplanet Large-area Survey (MARVELS). The APOGEE outputs now include the measured abundances of 15 different elements for each star. In total, SDSS-III added 5200 deg2 of ugriz imaging; 155,520 spectra of 138,099 stars as part of the Sloan Exploration of Galactic Understanding and Evolution 2 (SEGUE-2) survey; 2,497,484 BOSS spectra of 1,372,737 galaxies, 294,512 quasars, and 247,216 stars over 9376 deg2; 618,080 APOGEE spectra of 156,593 stars; and 197,040 MARVELS spectra of 5513 stars. Since its first light in 1998, SDSS has imaged over 1/3 of the Celestial sphere in five bands and obtained over five million astronomical spectra. © 2015. The American Astronomical Society

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

The Eleventh and Twelfth Data Releases of the Sloan Digital Sky Survey: Final Data from SDSS-III

The third generation of the Sloan Digital Sky Survey (SDSS-III) took data from 2008 to 2014 using the original SDSS wide-field imager, the original and an upgraded multi-object fiber-fed optical spectrograph, a new near-infrared high-resolution spectrograph, and a novel optical interferometer. All of the data from SDSS-III are now made public. In particular, this paper describes Data Release 11 (DR11) including all data acquired through 2013 July, and Data Release 12 (DR12) adding data acquired through 2014 July (including all data included in previous data releases), marking the end of SDSS-III observing. Relative to our previous public release (DR10), DR12 adds one million new spectra of galaxies and quasars from the Baryon Oscillation Spectroscopic Survey (BOSS) over an additional 3000 deg2 of sky, more than triples the number of H-band spectra of stars as part of the Apache Point Observatory (APO) Galactic Evolution Experiment (APOGEE), and includes repeated accurate radial velocity measurements of 5500 stars from the Multi-object APO Radial Velocity Exoplanet Large-area Survey (MARVELS). The APOGEE outputs now include the measured abundances of 15 different elements for each star. In total, SDSS-III added 5200 deg2 of ugriz imaging; 155,520 spectra of 138,099 stars as part of the Sloan Exploration of Galactic Understanding and Evolution 2 (SEGUE-2) survey; 2,497,484 BOSS spectra of 1,372,737 galaxies, 294,512 quasars, and 247,216 stars over 9376 deg2; 618,080 APOGEE spectra of 156,593 stars; and 197,040 MARVELS spectra of 5513 stars. Since its first light in 1998, SDSS has imaged over 1/3 of the Celestial sphere in five bands and obtained over five million astronomical spectra. \ua9 2015. The American Astronomical Society

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Restoration of cleavage in the Nlrp1b1 V988D mutant rescues inflammasome activity.

<p>(A and D) Cells were transfected with pcDNA3-pro-IL-1β-HA and pcDNA3-pro-caspase-1-T7 in addition to the indicated combinations of pNTAP plasmids encoding Nlrp1b proteins and pcDNA3-TEV-protease-T7. Approximately 24 h after transfection, cells were treated with LeTx for 3 h. Cell lysates were collected and probed for HA-tagged pro-IL-1β and β-actin; cell medium was collected and immunoprecipitated with anti-HA antibodies and probed for HA-tagged IL-1β by immunoblotting. (B) HT1080 cells were transfected with pNTAP plasmids encoding Nlrp1b proteins, pcDNA3-pro-IL-1β-HA, and pcDNA3-TEV-protease-T7 as indicated. Approximately 24 h after transfection, cells were treated with LeTx (10⁻⁸ M LF and 10⁻⁸ M PA) for 3 h. Cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin and immunoblotted using an Nlrp1b antibody. (C) Cells were transfected with pNTAP plasmids encoding the indicated Nlrp1b proteins and pcDNA3-TEV-protease-T7 as indicated. Approximately 24 h following transfection, cells were lysed and TAP-tagged proteins were precipitated with streptavidin resin and immunoblotted using an Nlrp1b antibody. Arrow indicates cleavage product. Blots are representative of three independent experiments.</p

The V988D mutation in Nlrp1b1_720–1233 reduces its ability to associate with pro-caspase-1.

<p>(A) HT1080 cells were transfected with pcDNA3-pro-caspase-1-T7 and pcDNA3-pro-IL-1β-HA and co-transfected as indicated with pNTAP-Nlrp1b1_720–1233-HA, pNTAP-Nlrp1b1_720–1233-T7, pNTAP-Nlrp1b1_720–1233-V988D-HA, and pNTAP-Nlrp1b1_720–1233-V988D. Approximately 24 h after transfection, cell lysates were collected and probed for HA-tagged pro-IL-1β and β-actin; cell supernatants were collected and immunoprecipitated with anti-HA antibodies and probed for HA-tagged IL-1β by immunoblotting. (B) Cells were transfected with indicated combinations of pNTAP-Nlrp1b1_720–1233-HA and pNTAP-Nlrp1b1_720–1233-T7 WT or V988D as well as with pcDNA3-pro-caspase-1-C284A-FLAG and pcDNA3-pro-IL-1β-HA. Approximately 24 h following transfection, cells were lysed and immunoprecipitated using an anti-T7 antibody. Proteins were immunoblotted with antibodies against HA, caspase-1, T7 and β-actin. Blots are representative of three independent experiments.</p

Mutation of amino acids 1100–1106 impairs FIIND self-association, FIIND cleavage, and Nlrp1b1 activity.

<p>(A) Nlrp1b1_1100–1233 or Nlrp1b1_1100–1233-7A (containing alanine substitution mutations of amino acids 1100–1106) was expressed with pro-caspase-1 and pro-IL-1β-HA in HT1080 cells. Cells were lysed 24 h after transfection. Cell lysates were probed for T7-tagged Nlrp1b1 constructs and for β-actin by immunoblotting; supernatants were immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β. (B) T7-tagged and HA-tagged Nlrp1b1_1100–1233 or Nlrp1b1_1100–1233-7A were expressed in HT1080 cells. HA-tagged proteins were immunoprecipitated from cell lysates and the immunprecipitates were probed for T7- and HA-tagged proteins. (C) HA-tagged Nlrp1b1_1100–1233, Nlrp1b1_1100–1233-7A and Nlrp1b1_1142–1233 were co-expressed with pro-caspase-1-C284A-T7. Immunoprecipitations were performed with anti-T7 or control anti-GFP antibodies and immunoblotted for HA and T7 epitopes. Cell lysates (right panel) were immunoblotted as indicated. (D) Nrlp1b1 and Nrlp1b1-7A were expressed with pro-caspase-1 and pro-IL-1β-HA in HT1080 cells. Cells were treated with LeTx for 3 h and supernatants were probed for IL-1β-HA as above. (E) Cell lysates from (D) were probed for Nlrp1b1. Blots are representative of three independent experiments.</p