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

First archaeal rDNA sequences from Argentine coastal waters: Unexpected PCR characterization using eukaryotic primers

Many members of Archaea, a group of prokaryotes recognized three decades ago, colonize extreme environments; however, new research has shown that Archaeans are also abundant components of plankton in the open sea, where they play a key role in the biogeochemical cycles. Although the widespread distribution of Archaea in the marine environment is well documented there are no reports on the detection of Archaea in the Southwest Atlantic Ocean. During the search for picophytoplankton sequences using eukaryotic universal primers, we retrieved archaeal rDNA sequences from surface samples collected during the spring at a fixed monitoring station (EPEA) in the Argentine Sea. From environmental DNA and using PCR methodology, two DNA fragments of about 1700 and 1450 bp were visualized after electrophoresis in agarose gels, and separately purified, cloned, and sequenced. BLAST analysis showed that sequences of the highest size corresponded to eukaryotic organisms and, unexpectedly, those of about 1460 bp corresponded to archaeal organisms. Phylogenetic analysis showed that archaeal sequences belong to Euryarchaeota of marine group II, characterized as a methanogenic lineage. This is the first report on the presence of group II Euryarchaeota sequences in environmental water samples of the Argentine Sea. The fact that Archaea sequences were amplified with primers non-specific for this group may suggest an unexpected abundance of these organisms in the early spring in the Argentine Sea.