Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Access Safety Systems – New Concepts from the LHC Experience

The LHC Access Safety System has introduced a number of new concepts into the domain of personnel protection at CERN. These can be grouped into several categories: organisational, architectural and concerning the end-user experience. By anchoring the project on the solid foundations of the IEC 61508/61511 methodology, the CERN team and its contractors managed to design, develop, test and commission on time a SIL3 safety system. The system uses a successful combination of the latest Siemens redundant safety programmable logic controllers with a traditional relay logic hardwired loop. The external envelope barriers used in the LHC include personnel and material access devices, which are interlocked door-booths introducing increased automation of individual access control, thus removing the strain from the operators. These devices ensure the inviolability of the controlled zones by users not holding the required credentials. To this end they are equipped with personnel presence detectors and the access control includes a state of the art biometry check. Building on the LHC experience, new projects targeting the refurbishment of the existing access safety infrastructure in the injector chain have started. This paper summarises the new concepts introduced in the LHC access control and safety systems, discusses the return of experience and outlines the main guiding principles for the renewal stage of the personnel protection systems in the LHC injector chain in a homogeneous manner

Data Management and Tools for the Access to the Radiological Areas at CERN

As part of the refurbishment of the PS accelerator complex Personnel Protection System, the Radiation Protection (RP) checkpoints and buffer zones, for the radiological controls of equipment removed from the beam areas, have been incorporated into the design of the new access points

Refurbishing of the CERN PS Complex Personnel Protection System

In 2010, the refurbishment of the Personnel Protection System of the CERN Proton Synchrotron (PS) primary beam areas started

CERN Proton Synchrotron East Area Facility: Upgrades and renovation during Long Shutdown 2

In this document, we present the upgrade of the East Experimental Area facility which took place during the Long Shutdown 2 (2019–2021). This document covers the renovation of the East Hall beamlines and infrastructure according to a new layout with the goal of improving the magnet and radiation situation in general. The performance of the new beamlines will be optimized in terms of maximum momentum and choice of particle type. Thanks to a cycled powering mode of the magnets instead of a steady state one, considerable energy savings will be possible. This report summarizes the various detailed studies completed from 2016 to 2019

Development and validation of a score to predict postoperative respiratory failure in a multicentre European cohort : A prospective, observational study

BACKGROUND Postoperative respiratory failure (PRF) is the most frequent respiratory complication following surgery. OBJECTIVE The objective of this study was to build a clinically useful predictive model for the development of PRF. DESIGN A prospective observational study of a multicentre cohort. SETTING Sixty-three hospitals across Europe. PATIENTS Patients undergoing any surgical procedure under general or regional anaesthesia during 7-day recruitment periods. MAIN OUTCOME MEASURES Development of PRF within 5 days of surgery. PRF was defined by a partial pressure of oxygen in arterial blood (PaO2) less than 8 kPa or new onset oxyhaemoglobin saturation measured by pulse oximetry (SpO(2)) less than 90% whilst breathing room air that required conventional oxygen therapy, noninvasive or invasive mechanical ventilation. RESULTS PRF developed in 224 patients (4.2% of the 5384 patients studied). In-hospital mortality [95% confidence interval (95% CI)] was higher in patients who developed PRF [10.3% (6.3 to 14.3) vs. 0.4% (0.2 to 0.6)]. Regression modelling identified a predictive PRF score that includes seven independent risk factors: low preoperative SpO(2); at least one preoperative respiratory symptom; preoperative chronic liver disease; history of congestive heart failure; open intrathoracic or upper abdominal surgery; surgical procedure lasting at least 2 h; and emergency surgery. The area under the receiver operating characteristic curve (c-statistic) was 0.82 (95% CI 0.79 to 0.85) and the Hosmer-Lemeshow goodness-of-fit statistic was 7.08 (P = 0.253). CONCLUSION A risk score based on seven objective, easily assessed factors was able to predict which patients would develop PRF. The score could potentially facilitate preoperative risk assessment and management and provide a basis for testing interventions to improve outcomes. The study was registered at ClinicalTrials.gov (identifier NCT01346709)