Correction: Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection.

[This corrects the article DOI: 10.1371/journal.pgen.1003007.].

Regulation of ATG4B stability by RNF5 limits basal levels of autophagy and influences susceptibility to bacterial infection.

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with-and regulation of its ubiquitination and stability by-RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5(-/-) MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5(-/-) using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5(-/-) mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5(-/-) macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.

Twelve-month, randomized, controlled trial of bimatoprost 0.01%, 0.0125%, and 0.03% in patients with glaucoma or ocular hypertension.

PURPOSE: To evaluate the intraocular pressure (IOP)-lowering efficacy and safety of ophthalmic formulations of bimatoprost 0.01% and 0.0125% compared with bimatoprost 0.03%. DESIGN: Prospective, randomized, double-masked, multicenter clinical trial. METHODS: Patients with glaucoma or ocular hypertension were randomized to receive once-daily bimatoprost 0.01% (n = 186), bimatoprost 0.0125% (n = 188), or bimatoprost 0.03% (n = 187) for 12 months. The primary efficacy measure was IOP. Safety measures included adverse events and an objective assessment of conjunctival hyperemia. RESULTS: Baseline mean IOPs were similar among treatment groups. Differences in mean IOP between the bimatoprost 0.01% or 0.0125% groups and the bimatoprost 0.03% group were less than 0.9 mm Hg throughout follow-up. Bimatoprost 0.01%, but not bimatoprost 0.0125%, was equivalent in efficacy to bimatoprost 0.03% based on predetermined criteria (limits of the 95% confidence interval of the between-group difference in mean IOP within +/- 1.5 mm Hg at all time points and within +/- 1 mm Hg at most time points). The overall incidence of treatment-related adverse events was reduced significantly in the bimatoprost 0.01% and bimatoprost 0.0125% groups compared with the bimatoprost 0.03% group (P < or = .034). The percentage of patients with a moderate to severe increase from the baseline macroscopic hyperemia score was: bimatoprost 0.01%, 3.2%; bimatoprost 0.0125%, 9.0%; bimatoprost 0.03%, 9.1% (P = .019 for bimatoprost 0.01% vs 0.03%). CONCLUSIONS: Bimatoprost 0.01% was equivalent to bimatoprost 0.03% in lowering IOP throughout 12 months of treatment and demonstrated improved tolerability, including less frequent and severe conjunctival hyperemia. Bimatoprost 0.01% demonstrated a better benefit-to-risk ratio than bimatoprost 0.0125%.

Versatile assays for high throughput screening for activators or inhibitors of intracellular proteases and their cellular regulators.

BACKGROUND: Intracellular proteases constitute a class of promising drug discovery targets. Methods for high throughput screening against these targets are generally limited to in vitro biochemical assays that can suffer many technical limitations, as well as failing to capture the biological context of proteases within the cellular pathways that lead to their activation. METHODS #ENTITYSTARTX00026; FINDINGS: We describe here a versatile system for reconstituting protease activation networks in yeast and assaying the activity of these pathways using a cleavable transcription factor substrate in conjunction with reporter gene read-outs. The utility of these versatile assay components and their application for screening strategies was validated for all ten human Caspases, a family of intracellular proteases involved in cell death and inflammation, including implementation of assays for high throughput screening (HTS) of chemical libraries and functional screening of cDNA libraries. The versatility of the technology was also demonstrated for human autophagins, cysteine proteases involved in autophagy. CONCLUSIONS: Altogether, the yeast-based systems described here for monitoring activity of ectopically expressed mammalian proteases provide a fascile platform for functional genomics and chemical library screening.

Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial.

CONTEXT: The expression and release of tissue factor is a major trigger for the activation of coagulation in patients with sepsis. Tissue factor pathway inhibitor (TFPI) forms a complex with tissue factor and blood protease factors leading to inhibition of thrombin generation and fibrin formation. OBJECTIVES: To determine if administration of tifacogin (recombinant TFPI) provides mortality benefit in patients with severe sepsis and elevated international normalized ratio (INR) and to assess tifacogin safety in severe sepsis, including patients with low INR. DESIGN AND SETTING: A randomized, double-blind, placebo-controlled, multicenter, phase 3 clinical trial conducted from March 21, 2000, through September 27, 2001, in 245 hospitals in 17 countries in North America, Europe, and Israel. PATIENTS: The primary efficacy population consisted of 1754 patients (> or =18 years) with severe sepsis and a high INR (> or =1.2) randomly assigned to intravenous infusion of either tifacogin (0.025 mg/kg per hour for 96 hours, n = 880) or placebo (arginine citrate buffer, n = 874), and 201 patients with a low INR (<1.2) randomly assigned to receive the same dose of either tifacogin or placebo. MAIN OUTCOME MEASURE: All-cause 28-day mortality. RESULTS: Overall mortality at 28 days in the tifacogin-treated group (n = 880) vs the placebo group (n = 874) for high INR was 34.2% vs 33.9%, respectively (P =.88, Pearson chi2 test; P =.75, logistic regression model). None of the protocol-specified secondary end points differed between the tifacogin vs placebo groups. An analysis on the first 722 patients demonstrated a mortality rate of 38.9% for placebo vs 29.1% for tifacogin (P =.006, Pearson chi2 test). Tifacogin significantly attenuated prothrombin fragment 1.2 and thrombin:antithrombin complex levels (P<.001, 2-sample t test) in patients with high and low INR. Overall mortality was lower in the tifacogin response in patients with low INR (12%; n = 83) vs placebo (22.9%; n = 118) (P =.051, Pearson chi2 test; P =.03, logistic regression model). There was an increase in serious adverse events with bleeding in the tifacogin group in both cohorts (6.5% tifacogin and 4.8% placebo for high INR; 6.0% tifacogin and 3.3% placebo for low INR). CONCLUSIONS: Treatment with tifacogin had no effect on all-cause mortality in patients with severe sepsis and high INR. Tifacogin administration was associated with an increase in risk of bleeding, irrespective of baseline INR.

A single preoperative oral dose of valdecoxib, a new cyclooxygenase-2 specific inhibitor, relieves post-oral surgery or bunionectomy pain.

BACKGROUND: The trend toward day-case surgery, with discharge on oral medication, has highlighted the need for effective and safe analgesics that facilitate a rapid recovery and discharge time. This study evaluated the analgesic efficacy, dose dependency, duration of action, and safety of the cyclooxygenase-2 specific inhibitor, valdecoxib, administered before oral or orthopedic surgery. METHODS: Eligible healthy adult patients were scheduled to undergo either extraction of two impacted third molar teeth (n = 284) or bunionectomy surgery (n = 223) with local anesthesia in two randomized, double-blind, placebo-controlled studies conducted at three centers in the United States. Patients received a single, preoperatively administered oral dose of placebo or 10 (oral surgery only), 20, 40, or 80 mg valdecoxib. Analgesic efficacy was assessed postoperatively, over a 24-h treatment period, or until the patient required rescue medication. Efficacy measures included time to rescue medication, proportion of patients requiring such rescue, pain intensity, and the Patient's Global Evaluation of Study Medication. RESULTS: In both studies, all doses of valdecoxib produced analgesia with a duration (time to rescue analgesia) and magnitude (Pain Intensity, Patient's Global Evaluation) significantly greater than placebo. A dose-dependent effect was observed up to 40 mg valdecoxib, with an 80-mg dose providing no additional analgesic benefit. In both models, all doses of valdecoxib were well tolerated, with no clinically significant treatment-related gastrointestinal, renal, or platelet-derived adverse events, and no evidence of a dose-related increase in adverse events. CONCLUSIONS: Preoperative orally administered valdecoxib provides well-tolerated and effective analgesia for mild to moderate postoperative pain.