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

The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

Invasive bacteria actively induce their own uptake by phagocytosis in normally nonphagocytic cells and then either establish a protected niche within which they survive and replicate, or disseminate from cell to cell by means of an actin-based motility process. The mechanisms underlying bacterial entry, phagosome maturation, and dissemination reveal common strategies as well as unique tactics evolved by individual species to establish infection.