Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Upstream of the SOS response: figure out the trigger

The bacterial SOS regulon encodes a response to DNA damage that not only functions to relieve the incurred damage but also enhances adaptation through mutagenesis and the lateral spread of virulence factors. Recent papers have demonstrated that certain stimuli can indirectly generate the SOS-inducing signal by activation of endogenous DNA damage mechanisms rather than by direct DNA damage. We suggest that these endogenous triggers have been recruited by bacteria to enable adaptation to various types of stresses.status: publishe

Mrr instigates the SOS response after high pressure stress in Escherichia coli

The bacterial SOS response is not only a vital reply to DNA damage but also constitutes an essential mechanism for the generation of genetic variability that in turn fuels adaptation and resistance development in bacterial populations. Despite the extensive depiction of the SOS regulon itself, its activation by stresses different from typical DNA damaging treatments remains poorly characterized. Recently, we reported the RecA- and LexA-dependent induction of the SOS response in Escherichia coli MG1655 after exposure to high hydrostatic pressure (HP, similar to 100 MPa), a physical stress of which the cellular effects are not well known. We now found this HP mediated SOS response to depend on RecB and not on RecF, which is a strong indication for the involvement of double strand breaks. As the pressures used in this work are thermodynamically unable to break covalent bonds in DNA, we hypothesized the involvement of a cellular function or pathway in the formation of this lesion. A specialized screening allowed us to identify the cryptic type IV restriction endonuclease Mrr as the final effector of this pathway. The HP SOS response and its corresponding phenotypes could be entirely attributed to the HP triggered activation of Mrr restriction activity. Several spontaneously occurring alleles of mrr, incapable of triggering the HP-induced SOS response, were isolated and characterized. These results provide evidence for a specific pathway that transmits the perception of HP stress to induction of the SOS response and support a role for Mrr in bacterial stress physiology.status: publishe

From field barley to malt: Detection and specification of microbial activity for quality aspects

Barley grain carries a numerous, variable, and complex microbial population that mainly consists of bacteria, yeasts, and filamentous fungi and that can partly be detected and quantified using plating methods and microscopic and molecular techniques. The extent and the activity of this microflora are determined by the altering state of the grain and the environmental conditions in the malt production chain. Three ecological systems can be distinguished: the growing cereal in the field, the dry barley grain under storage, and the germinating barley kernel during actual malting. Microorganisms interact with the malting process both by their presence and by their metabolic activity. In this respect, interference with the oxygen uptake by the barley grain and secretion of enzymes, hormones, toxins, and acids that may affect the plant physiological processes have been studied. As a result of the interaction, microorganisms can cause important losses and influence malt quality as measured by brewhouse performance and beer quality. Of particular concern is the occurrence of mycotoxins that may affect the safety of malt. The development of the microflora during malt production can to a certain extent be controlled by the selection of appropriate process conditions. Physical and chemical treatments to inactivate the microbial population on the barley grain are suggested. Recent developments, however, aim to control the microbial activity during malt production by promoting the growth of desirable microbial cultures, selected either as biocontrol agents inhibiting mycotoxin-producing molds or as starter cultures actively contributing to malt modification. Such techniques may offer natural opportunities to improve the quality and safety of malt.status: publishe

Food applications of bacterial cell wall hydrolases

Bacterial cell wall hydrolases (BCWHs) display a remarkable structural and functional diversity that offers perspectives for novel food applications, reaching beyond those of the archetype BCWH and established biopreservative hen egg white lysozyme. Insights in BCWHs from bacteriophages to animals have provided concepts for tailoring BCWHs to target specific pathogens or spoilage bacteria, or, conversely, to expand their working range to Gram-negative bacteria. Genetically modified foods expressing BCWHs in situ showed successful, but face regulatory and ethical concerns. An interesting spin-off development is the use of cell wall binding domains of bacteriophage BCWHs for detection and removal of foodborne pathogens. Besides for improving food safety or stability, BCWHs may also find use as functional food ingredients with specific health effects.status: publishe

Fundamental research enables process optimization in the sugar Industry

Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. However, this thick juice storage commonly faces problems due to juice degradation. The precise cause for this problem was ill defined but believed to be of microbial origin. In this research, the microbial population dynamics during thick juice storage was described and we identified the causative degradation flora. Finally, optimal good storage practices were defined with the ultimate goal of preventing thick juice degradation. The thick juice microflora has been thoroughly studied with both culture-based and culture-independent techniques, encompassing the application of 16S rRNA gene clone libraries and T-RFLP analysis, providing a more comprehensive representation of the thick juice microflora than the previous studies. The initial, heterogeneous microflora in freshly produced thick juice evolved to the dominance (>99%) of Tetragenococcus halophilus during storage. Based on its high population density (106–107 cfu/ml), the ability to consume sucrose and the similar acidification pattern of experimentally infested thick juice, T. halophilus is proposed to be the key player in thick juice degradation. Remarkably, T. halophilus has thus far been associated only with high salt food products and our work is the first to associate it with high sugar matrices. In addition to T. halophilus, other bacteria such as Staphylococcus and Bacillus species were consistently present, though in lower steady concentrations of 103 cfu/ml. In order to be able to detect the different bacteria that may occur in thick juice, a DNA array was developed containing detector oligonucleotides for the genera Bacillus, Kocuria, Staphylococccus and Tetragenococcus, and the species Aerococcus viridans, Leuconostoc mesenteroides and T. halophilus. The developed macroarray was shown reliable and sensitive (up to 102 cfu/ml) and has potential for monitoring the thick juice microflora during storage as an early warning system. Finally, best available storage practices were defined based on laboratory and pilot scale storage experiments using the independent variables solids content, pH, storage temperature and biocide concentration. In conclusion, this work has contributed to a better description of the microbial population dynamics during thick juice storage and degradation, and to the definition of improved storage practices that will be useful for the sugar industry.status: publishe