Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Comparative Effects of Volutrauma and Atelectrauma on Lung Inflammation in Experimental Acute Respiratory Distress Syndrome.

OBJECTIVE: Volutrauma and atelectrauma promote ventilator-induced lung injury, but their relative contribution to inflammation in ventilator-induced lung injury is not well established. The aim of this study was to determine the impact of volutrauma and atelectrauma on the distribution of lung inflammation in experimental acute respiratory distress syndrome. DESIGN: Laboratory investigation. SETTING: University-hospital research facility. SUBJECTS: Ten pigs (five per group; 34.7-49.9 kg) INTERVENTIONS:: Animals were anesthetized and intubated, and saline lung lavage was performed. Lungs were separated with a double-lumen tube. Following lung recruitment and decremental positive end-expiratory pressure trial, animals were randomly assigned to 4 hours of ventilation of the left (ventilator-induced lung injury) lung with tidal volume of approximately 3 mL/kg and 1) high positive end-expiratory pressure set above the level where dynamic compliance increased more than 5% during positive end-expiratory pressure trial (volutrauma); or 2) low positive end-expiratory pressure to achieve driving pressure comparable with volutrauma (atelectrauma). The right (control) lung was kept on continuous positive airway pressure of 20 cm H2O, and CO2 was partially removed extracorporeally. MEASUREMENTS AND MAIN RESULTS: Regional lung aeration, specific [F]fluorodeoxyglucose uptake rate, and perfusion were assessed using computed and positron emission tomography. Volutrauma yielded higher [F]fluorodeoxyglucose uptake rate in the ventilated lung compared with atelectrauma (median [interquartile range], 0.017 [0.014-0.025] vs 0.013 min [0.010-0.014 min]; p < 0.01), mainly in central lung regions. Volutrauma yielded higher [F]fluorodeoxyglucose uptake rate in ventilator-induced lung injury versus control lung (0.017 [0.014-0.025] vs 0.011 min [0.010-0.016 min]; p < 0.05), whereas atelectrauma did not. Volutrauma decreased blood fraction at similar perfusion and increased normally as well as hyperaerated lung compartments and tidal hyperaeration. Atelectrauma yielded higher poorly and nonaerated lung compartments, and tidal recruitment. Driving pressure increased in atelectrauma. CONCLUSIONS: In this model of acute respiratory distress syndrome, volutrauma promoted higher lung inflammation than atelectrauma at comparable low tidal volume and lower driving pressure, suggesting that static stress and strain are major determinants of ventilator-induced lung injury