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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Development of a Mechanically Assisted Pneumatic Metering Mechanism for High-Speed Planting of Soybean

84-91A planter for planting soybean seeds up to 1.94 m∙s−1 (7 km∙h−1) was designed. In this machine, a hybrid metering mechanism was developed in which singulation was done with the help of vacuum pressure and conveying of seeds was done by cellular ring. The metering mechanism was evaluated for seed plates with three different numbers of holes (64, 74 and 84); three vacuum pressures (2, 3 and 4 kPa) and at three forward speeds (0.83, 1.39 and 1.94 m∙s−1). The effects of forward speeds on different indices were found insignificant at 5% confidence level. The optimum value of different indices i.e., miss index, multiple index, quality of feed index and precision index were observed for 84 number of seed holes at 3 kPa vacuum pressure for 0.83, 1.39 and 1.94 m∙s−1. In field evaluation of the machine, the average width of operation, plant to plant spacing, depth of operation, field capacity, and field efficiency were 2.1 m, 9.7 × 10−2 m, 5.2 × 10−2 m and 0.9 ha∙h−1, 72% at 1.94 m∙s−1 speed of operation and 0.1 m plant to plant spacing setting. The metering mechanism was found suitable for planting soybean up to 1.94 m∙s−1 (7 km∙h−1)

Reservoir Units Optimization in Pneumatic Spray Delivery-Based Fixed Spray System for Large-Scale Commercial Adaptation

A pneumatic spray delivery (PSD)-based solid set canopy delivery system (SSCDS) consists of in-line reservoirs and micro-emitter assemblies distributed throughout perennial crop canopies. The existing PSD-based SSCDS uses a large number of reservoirs, i.e., one unit per 3 m of linear spacing, which resulted in high installation and maintenance costs. These reservoirs also produces up to 25% post-spray chemical losses. Therefore, this study aimed to optimize the volumetric capacity and functionality of the existing reservoir for an efficient spray performance and the large-scale commercial adaptation of PSD-based SSCDS. Three reservoirs with volumetric capacities of 370 (1×), 740 (2×), and 1110 mL (3×) were developed to cover a spray span of 3.0, 6.1, and 9.1 m, respectively. Five system configurations with modified reservoirs and spray outlets were evaluated in the laboratory for pressure drop and spray uniformity. The three best system configurations were then field evaluated in a high-density apple orchard. These configurations had reservoirs with 1×, 2×, and 3× volumetric capacity and micro-emitters installed in a three-tier arrangement. Each replicate configuration was installed as a 77 m loop length encompassing 50 apple trees trained in a tall spindle architecture. A pair of water-sensitive paper (WSPs) samplers (25.4 × 25.4 mm) were placed on the abaxial and adaxial leaf surfaces in the bottom, middle, and top third of the canopy to evaluate the spray coverage (%). The PSD-based SSCDS showed no significant difference at the 5% level in terms of coverage among the three reservoir treatments. Coverage was more evenly distributed among the top, middle, and bottom zones for the 2× and 3× as compared to the 1× reservoir treatment. Overall, compared to the 1× reservoirs, the 2× and 3× reservoirs could potentially reduce the system costs by USD 20,000 and USD 23,410 ha−1, respectively, for tall spindle apple orchards and potentially reduce maintenance needs as well

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

No abstract available