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

Temperature alters dicyandiamide (DCD) efficacy for multiple reactive nitrogen species in urea-amended soils: Experiments and modeling

Dicyandiamide (DCD) is a nitrification inhibitor (NI) used to reduce reactive nitrogen (N) losses from soils. While commonly used, its effectiveness varies widely. Few studies have measured DCD and temperature effects on a complete set of soil N variables, including nitrite (NO₂¯) measured separately from nitrate (NO₃‾). Here the DCD reduction efficiencies (RE) for nine N availability metrics were quantified in two soils (a loam and silt loam) using aerobic laboratory microcosms at 5–30 °C. Both regression analysis and process modeling were used to characterize the responses. Four metrics accounted for NO₃‾ production and included total mobilized N, net nitrification, maximum nitrification rate, and cumulative NO₃‾ (cNO₃‾). The REs for these NO₃‾ -associated production variables decreased linearly with temperature, and in all cases were below 60% at temperatures ≥22 °C, except for cNO₃‾ in one soil. In contrast, REs for NO₂‾ and nitric oxide (NO) gas production were less sensitive to temperature, ranging from 80 to 99% at 22 °C and 50–95% at 30 °C. Addition of DCD suppressed nitrous oxide (N₂O) production in both soils by 20–80%, but increased ammonia volatilization by 36–210%. The time at which the maximum reduction efficiency occurred decreased exponentially with increasing temperature for most variables. The two-step nitrification process model (2SN) was modified to include competitive inhibition coupled to first-order DCD decomposition. Model versus data comparisons suggested that DCD had indirect effects on NO₂‾ kinetics that contributed to the greater suppression of NO₂‾ and NO relative to NO₃‾. This study also points to the need for NIs that are more stable under increased temperature. The methods used here could help to assess the efficacy and temperature sensitivity of other NIs as well as new microbial inhibitors that may be develope