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

A transferability study of the kernel-based reclassification algorithm for habitat delineation

Wetland mapping using Earth observation (EO) data has proved to be a challenging task for practitioners due to the complexity in the spatial structure and composition, the wide within-class spectral variability and the absence of easily distinguishable boundaries between habitat types. Furthermore, the inherent temporal water instability of these landscapes poses an obstacle to the integration of field data with remote sensing data, which also are not acquired simultaneously at all times.To cope with these limitations we tested the applicability of the Kernel-based reclassification (KRC) algorithm on very high spatial resolution (VHR) satellite imagery over a wetland. A composite multi-temporal (i.e. dual-date) VHR WorldView-2 image consisting of spectral bands and indices derived from two images acquired during flooded and dry water conditions were employed. This dataset stresses the seasonal variations of the habitat in response to environmental changes (i.e. flooding) occurring between the two acquisition dates. Multi-temporal imagery is an important information source for fine mapping of wetlands such are river deltas. A multi-temporal approach could reveal even more specific information during the phenology of these habitats.The methodology was applied firstly to Axios and then to Aliakmonas river deltas in Northern Greece. The results revealed an overall accuracy of 53% in the first and more complex site, and 86% in the second sit

Evaluating sentinel-2 for lakeshore habitat mapping based on airborne hyperspectral data

Monitoring of lakeshore ecosystems requires fine-scale information to account for the high biodiversity typically encountered in the land-water ecotone. Sentinel-2 is a satellite with high spatial and spectral resolution and improved revisiting frequency and is expected to have significant potential for habitat mapping and classification of complex lakeshore ecosystems. In this context, investigations of the capabilities of Sentinel-2 in regard to the spatial and spectral dimensions are needed to assess its potential and the quality of the expected output. This study presents the first simulation of the high spatial resolution (i.e., 10 m and 20 m) bands of Sentinel-2 for lakeshore mapping, based on the satellite’s Spectral Response Function and hyperspectral airborne data collected over Lake Balaton, Hungary in August 2010. A comparison of supervised classifications of the simulated products is presented and the information loss from spectral aggregation and spatial upscaling in the context of lakeshore vegetation classification is discussed. We conclude that Sentinel-2 imagery has a strong potential for monitoring fine-scale habitats, such as reed beds