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

Contrasting effects of tropospheric ozone on five native herbs which coexist in calcareous grassland

The aim of this study was to examine the effects of increased tropospheric ozone concentrations on the growth and morphology of five native herbs commonly found to coexist in calcareous grassland in areas of Britain and continental Europe: Anthyllis vulneraria L., Cirsium acaule (L.) Scop., Festuca ovina L., Pilosella offtcinarum F. Shultz & Shultz-Bip and Lotus comiculatus L.In a chronic fumigation (mean O3 concentration of 71 ppb (71 nl 1?1) for 7 h d?1 AOT40 4585 ppb-h) which lasted for 21 d, the effects of ozone were assessed using classical growth analysis. Large reductions in mean relative growth rates for shoot and root weight and root length were observed for the two legumes (Fabaceae) Lotus corniculatus and Anthyllis vulneraria, although these were only statistically significant for Lotus corniculatus. Significant reductions in specific root length (length per unit dry weight) were found for Cirsium acaule and Pilosella officinarum (Asteraceae), while for Festuca ovina (Poaceae) the allometric coefficient was reduced significantly following exposure to ozone.An acute fumigation (mean O3 concentration of 196 ppb, 7 h) resulted in a range of visible injury, from no injury (Festuca ovina and Pilosella officinarum) through moderate levels of injury (Cirsium acaule and Lotus corniculatus) to extensive and widespread injury (Anthyllis vulneraria). Scoring of visible damage showed that this was only statistically significant for the two legumes, Lotus corniculatus and Anthyllis vulneraria.These results suggest that native herbs may differ in their sensitivity to tropospheric ozone. Both chronic and acute exposures revealed that members of the Fabaceae may be most sensitive to ozone pollution, but the study also suggests that subtle changes in root morphology occurred for members of the Asteraceae. These findings are discussed in relation to the critical levels of ozone set recently for plants and the implications of increasing tropospheric ozone for the conservation of native plant communities