Quantifying autophagy using novel LC3B and p62 TR-FRET assays

<div><p>Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.</p></div

LC3B-II and p62 quantification in response to tool compounds treatment.

<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with a serially diluted autophagy inhibitor (bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment. LC3B-II and p62 were measured with TR-FRET (A-C). The response to compound treatment is reported as percentage average of three replicates with respect to vehicle (DMSO) treated samples (100%). Cell viability was evaluated by H33342 stained nuclei count for HEK293T (A) and astrocytes (C-E) and by neurite length/soma (morphometric readout) on MAP2 stained neurons (B). Astrocytes were also treated with SU11652 and NVP-TAE684 and LC3B-II, p62 and viability were measured with TR-FRET (D-E). Each data point is the mean±SEM (N = 3).</p

Co-treatment with bafilomycin A1 to distinguish autophagy enhancers versus blockers.

<p>Rat primary astrocytes were treated with 10 μM KU0063794, 1 μM SU11652 or 5 μM NVP-TAE684 (concentration selected from information in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194423#pone.0194423.g002" target="_blank">Fig 2</a>) followed by either 50 nM bafilomycin A1 (+Baf) or vehicle (DMSO, -Baf) for an additional 4 hours. Control samples were treated with vehicle (DMSO) for 2 hours followed by either bafilomycin A1 (50 nM) or vehicle (DMSO) for an additional 4 hours. LC3B-II TR-FRET signals are reported as fold increase with respect to the vehicle. Co-treatment of KU0063794 and bafilomycin A1 increased LC3B-II, compared to KU0063794 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukey’s multiple comparison test, *p<0.05); co-treatment of SU11652 and bafilomycin A1 increased LC3B-II, compared to SU11652 or bafilomycin A1 alone (N = 2, one-way ANOVA, p<0.01; Tukey’s multiple comparison test, *p<0.05); co-treatment of NVP-TAE684 and bafilomycin A1 did not alter the LC3B-II signal (N = 2, one-way ANOVA, p>0.05; (A). Western blots (B) confirm the TR-FRET data.</p

LC3B-II and p62 protein responses after tool compound.

<p>HEK293T cells (A), rat cortico-striatal neurons (B) and rat astrocytes (C) were treated with an autophagy inhibitor (5 nM bafilomycin A1) or upregulator (KU0063794) and examined at 2, 6 and 24 hours post-treatment, compared to DMSO. Western blot analysis is presented for LC3B-I/II and p62 levels, with a GAPDH loading control.</p

Effects of pre‐operative isolation on postoperative pulmonary complications after elective surgery: an international prospective cohort study

We aimed to determine the impact of pre-operative isolation on postoperative pulmonary complications after elective surgery during the global SARS-CoV-2 pandemic. We performed an international prospective cohort study including patients undergoing elective surgery in October 2020. Isolation was defined as the period before surgery during which patients did not leave their house or receive visitors from outside their household. The primary outcome was postoperative pulmonary complications, adjusted in multivariable models for measured confounders. Pre-defined sub-group analyses were performed for the primary outcome. A total of 96,454 patients from 114 countries were included and overall, 26,948 (27.9%) patients isolated before surgery. Postoperative pulmonary complications were recorded in 1947 (2.0%) patients of which 227 (11.7%) were associated with SARS-CoV-2 infection. Patients who isolated pre-operatively were older, had more respiratory comorbidities and were more commonly from areas of high SARS-CoV-2 incidence and high-income countries. Although the overall rates of postoperative pulmonary complications were similar in those that isolated and those that did not (2.1% vs 2.0%, respectively), isolation was associated with higher rates of postoperative pulmonary complications after adjustment (adjusted OR 1.20, 95%CI 1.05-1.36, p = 0.005). Sensitivity analyses revealed no further differences when patients were categorised by: pre-operative testing; use of COVID-19-free pathways; or community SARS-CoV-2 prevalence. The rate of postoperative pulmonary complications increased with periods of isolation longer than 3 days, with an OR (95%CI) at 4-7 days or &gt;= 8 days of 1.25 (1.04-1.48), p = 0.015 and 1.31 (1.11-1.55), p = 0.001, respectively. Isolation before elective surgery might be associated with a small but clinically important increased risk of postoperative pulmonary complications. Longer periods of isolation showed no reduction in the risk of postoperative pulmonary complications. These findings have significant implications for global provision of elective surgical care