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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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

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Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

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Melatonin is remarkably functionally diverse with actions as a free radical scavenger and antioxidant, circadian rhythm regulator, antiinflammatory and immunoregulating molecule, and as an oncostatic agent. We hypothesize that the initial and primary function of melatonin in photosynthetic cyanobacteria, which appeared on Earth 3.5-3.2 billion years ago, was as an antioxidant. The evolution of melatonin as an antioxidant by this organism was necessary as photosynthesis is associated with the generation of toxic-free radicals. The other secondary functions of melatonin came about much later in evolution. We also surmise that mitochondria and chloroplasts may be primary sites of melatonin synthesis in all eukaryotic cells that possess these organelles. This prediction is made on the basis that mitochondria and chloroplasts of eukaryotes developed from purple nonsulfur bacteria (which also produce melatonin) and cyanobacteria when they were engulfed by early eukaryotes. Thus, we speculate that the melatoninsynthesizing actions of the engulfed bacteria were retained when these organelles became mitochondria and chloroplasts, respectively. That mitochondria are likely sites of melatonin formation is supported by the observation that this organelle contains high levels of melatonin that are not impacted by blood melatonin concentrations. Melatonin has a remarkable array of means by which it thwarts oxidative damage. It, as well as its metabolites, is differentially effective in scavenging a variety of reactive oxygen and reactive nitrogen species. Moreover, melatonin and its metabolites modulate a large number of antioxidative and pro-oxidative enzymes, leading to a reduction in oxidative damage. The actions of melatonin on radical metabolizing/producing enzymes may be mediated by the Keap1-Nrf2-ARE pathway. Beyond its direct free radical scavenging and indirect antioxidant effects, melatonin has a variety of physiological and metabolic advantages that may enhance its ability to limit oxidative stress.