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

Laboratory and Field Inter-Comparisons of NO2 Diffusive Samplers

Abstract not availableJRC.H-Institute for environment and sustainability (Ispra

Laboratory and Field Comparison of Measurements Obtained Using the Available Diffusive Samplers for Ozone and Nitrogen Dioxide in Ambient Air

This study presents an evaluation of the extent of differences between measurements performed by O3 and NO2 diffusive samplers and by the reference methods for diffusive samplers commercially available. The tests were performed in an exposure chamber under extreme conditions of controlling factors and under field conditions. For NO2, the results of the laboratory experiments showed that most of the diffusive samplers were affected by extreme exposure conditions. The agreement between the samplers and the reference method was better for the field tests than for the laboratory ones. The estimate of the uptake rate for the exposure conditions using a model equation improved the agreement between the diffusive samplers and the reference methods. The agreement between O3 measured by the diffusive samplers and by the reference method was satisfactory for 1-week exposure. For 8-hour exposures, the diffusive samplers with high uptake rates quantifiyed better the O3 concentration than the samplers with low uptake rates. As for NO2, the results of the O3 field tests were in better agreement with the reference method than the ones of the laboratory tests. The field tests showed that the majority of diffusive samplers fulfils the 25% uncertainty requirement of the NO2 European Directive and the 30% uncertainty requirement of the O3 European Directive for 1-week exposure .JRC.H.4-Transport and air qualit

Feasibility Study of Preparation and Certification of Reference Materials for Nitrogen Dioxide and Sulfur Dioxide in Diffusive Samplers

Feasibility study of preparation and certification of reference materials for nitrogen dioxide and sulfur dioxide in diffusive samplers Michel Gerboles*, Daniela Buzica, Luciano Amantini, Friedrich Lagler and Theo Hafkenscheid+ European Commission - Joint Research Centre, Institute for Environment and Sustainability, Emissions and Health Unit, 21020 Ispra, Italy. Fax: +39-332-785652; Tel: +39-332-789364; E-mail: [email protected] +Dutch Measurement Institute, Delft, Netherlands This paper presents the results of a feasability study for the preparation and certification of reference materials (RMs) for nitrogen dioxide (NO2) and sulphur dioxide (SO2) in diffusive samplers. RMs for NO2 were prepared by exposure to gas mixtures in a chamber while the RMs for SO2 were prepared by liquid spiking. Certification of RMs for NO2 was found feasible with a certified uncertainty of 5.8 % and a proposed shelf life of 5 years. The uncertainty was calculated with contribution from the homogeneity of preparation, stability and transport of the CRMs and from an external verification of the certified value. To reach 5.8 % of uncertainty, the contribution of the differences between the results of analysis by ion chromatography and colorimetry must be eliminated. It is proposed to solve this by pre-extracting the samplers with water before analysis. The results of this study indicate that the samplers are stable for at least two years before and after exposure when stored in refrigerator. On the opposite, the certification of RMs for SO2 was found not feasible due to instability problems. This instability was attributed to reaction of sulfate on the walls of the samplers. Alternatively, the preparation of RMs by simultaneous exposure to SO2 and NO2 have been tested. Satisfying homogeneities has been reached both for NO2 and SO2.JRC.H.4-Transport and air qualit

Spatial Distribution Patterns of Metals in the Surface Sediments of Lake Orta (Italy)

Abstract not availableNA-NOT AVAILABL

Modelling of the Uptake Rate of the Nitrogen Dioxide Palmes Diffusive Sampler Based on the Effect of Environmental Factors

The influence of environmental parameters on the uptake rate of the nitrogen dioxide (NO2) Palmes diffusive sampler was investigated. The main factors affecting the uptake rate were found to be wind speed, the preponderant factor, followed by relative humidity and temperature. The NO2 concentration and exposure time, as well as the interactions among the factors were not found to have a significant influence on the uptake rate of the Palmes sampler. As a result, a model able to predict the uptake rate of the Palmes sampler was established. In addition, by using the model-predicted uptake rate, the agreement between chemiluminescence and the Palmes sampler during the field tests was improved. The NO2 Palmes diffusive sampler was shown to comply with the requirement on accuracy defined by the European Directive for the indicative methods of measurements.JRC.H.4-Transport and air qualit

Modification of the Palmes diffusion tube and semi-empirical modelling of the uptake rate for monitoring nitrogen dioxide

Hereafter, a modification of the traditional Palmes diffusion tube by fitting a membrane at the open end of the tube is presented. The membrane is placed to avoid the effect of turbulence due to wind speed. Further optimisations of the volume of absorbent and number of meshes retaining the absorbent are also described. Finally, the sampler is placed in an air proof container and is ready to use in order to improve the quality of measurement. In order to evaluate the performance of the membrane-closed Palmes diffusion tube to monitor nitrogen dioxide (NO2), experiments were carried out in an exposure chamber. The relative effect of NO2, temperature, humidity, averaging time and wind speed on the uptake rate of the sampler were then studied. Among the uncontrolled variables, temperature and humidity are found to be the most important ones leading to variability of the uptake rate. Two equations, used to estimate the uptake rate are established. These equations were then applied on some field data and laboratory inter-comparison tests. The agreement between the chemiluminescence method and diffusive samplers changes from 20% of mean bias with the traditional Palmes diffusion tubes to 5% of mean bias with the membraneclosed Palmes tube (MCPT) at the same monitoring site. In the laboratory inter-comparison, the MCPT has shown to be able to correctly measure NO2 under two extreme exposure conditions in contrast with other diffusive samplers. The uncertainty of the sampler is evaluated. It fulfils the European Directive data quality objective of 25% for accuracy and therefore the sampler qualifies as an indicative method for NO2 monitoring.JRC.H.4-Transport and air qualit

Modelling of The Uptake Rate of the Nitrogen Dioxide Palmes Diffusive Sampler Based on the Effect on the Environmenttal Factors.

Abstract not availableJRC.H-Institute for environment and sustainability (Ispra

Progress of Molecular Targeted Therapies for Advanced Renal Cell Carcinoma

Vascular endothelial growth factor (VEGF) plays a crucial role in tumor angiogenesis. VEGF expression in metastatic renal cell carcinoma (mRCC) is mostly regulated by hypoxia, predominantly via the hypoxia-induced factor (HIF)/Von Hippel-Lindau (VHL) pathway. Advances in our knowledge of VEGF role in tumor angiogenesis, growth, and progression have permitted development of new approaches for the treatment of mRCC, including several agents targeting VEGF and VEGF receptors: tyrosine kinase pathway, serine/threonine kinases, α5β1-integrin, deacetylase, CD70, mammalian target of rapamycin (mTOR), AKT, and phosphatidylinositol 3′-kinase (PI3K). Starting from sorafenib and sunitinib, several targeted therapies have been approved for mRCC treatment, with a long list of agents in course of evaluation, such as tivozanib, cediranib, and VEGF-Trap. Here we illustrate the main steps of tumor angiogenesis process, defining the pertinent therapeutic targets and the efficacy and toxicity profiles of these new promising agents