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

Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater, and sediment of Moreton Bay, Australia.

Planctomycetes are ubiquitous in marine environment and were reported to occur in association with multicellular eukaryotic organisms such as marine macroalgae and invertebrates. Here, we investigate planctomycetes associated with the marine sponge Niphates sp. from the sub-tropical Australian coast by assessing their diversity using culture-dependent and -independent approaches based on the 16S rRNA gene. The culture-dependent approach resulted in the isolation of a large collection of diverse planctomycetes including some novel lineages of Planctomycetes from the sponge as well as sediment and seawater of Moreton Bay where this sponge occurs. The characterization of these novel planctomycetes revealed that cells of one unique strain do not possess condensed nucleoids, a phenotype distinct from other planctomycetes. In addition, a culture-independent clone library approach identified unique planctomycete 16S rRNA gene sequences closely related to other sponge-derived sequences. The analysis of tissue of the sponge Niphates sp. showed that the mesohyl of the sponge is almost devoid of microbial cells, indicating this species is in the group of 'low microbial abundant' (LMA) sponges. The unique planctomycete 16S rRNA gene sequences identified in this study were phylogenetically closely related to sequences from LMA sponges in other published studies. This study has revealed new insights into the diversity of planctomycetes in the marine environment and the association of planctomycetes with marine sponges

Sugar uptake by the solventogenic clostridia

The acetone–butanol–ethanol fermentation of solventogenic clostridia was operated as a successful, worldwide industrial process during the first half of the twentieth century, but went into decline for economic reasons. The recent resurgence in interest in the fermentation has been due principally to the recognised potential of butanol as a biofuel, and development of reliable molecular tools has encouraged realistic prospects of bacterial strains being engineered to optimise fermentation performance. In order to minimise costs, emphasis is being placed on waste feedstock streams containing a range of fermentable carbohydrates. It is therefore important to develop a detailed understanding of the mechanisms of carbohydrate uptake so that effective engineering strategies can be identified. This review surveys present knowledge of sugar uptake and its control in solventogenic clostridia. The major mechanism of sugar uptake is the PEP-dependent phosphotransferase system (PTS), which both transports and phosphorylates its sugar substrates and plays a central role in metabolic regulation. Clostridial genome sequences have indicated the presence of numerous phosphotransferase systems for uptake of hexose sugars, hexose derivatives and disaccharides. On the other hand, uptake of sugars such as pentoses occurs via non-PTS mechanisms. Progress in characterization of clostridial sugar transporters and manipulation of control mechanisms to optimise sugar fermentation is described