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

Development of a brood stock of the jumbo tiger prawn, Penaeus monodon Fabricius

The full-scale cultivation of sugpo, P. monodon Fabricius, could only be realized if there is an assurance of continuous supply of fry. Obviously, a steady supply will depend largely on the availability of spawners. In December 1975, roughly after 2 1/2 years of intensive study, for the first time in the world, SEAFDEC Aquaculture Department has succeeded in inducing P. monodon to mature and produce normally the first generation of postlarval fry, thereby successfully effecting the completion of P. monodon's life cycle while in captivity. Another significant study the Department has initially carried out which could help augment and stabilize the supply of spawners and eventually stimulate the establishment of more prawn hatcheries and the development of ponds for prawn culture as a major export-oriented, dollar-earning industry, is the possible development of ovarian rematuration of spent spawners

Patterns of skills acquisition in anesthesiologists during simulated interscalene block training on the soft embalmed Thiel cadaver

BACKGROUND: The demand for regional anesthesia for major surgery has increased considerably, but only a small number of anesthesiologists can provide such care. Simulations may improve clinical performance. However, opportunities to rehearse procedures are limited, and the clinical educational outcomes prescribed by the Royal College of Anesthesiologists training curriculum 2021 are difficult to attain. Educational paradigms, such as mastery learning and dedicated practice, are increasingly being used to teach technical skills to enhance skills acquisition. Moreover, high-fidelity, resilient cadaver simulators are now available: the soft embalmed Thiel cadaver shows physical characteristics and functional alignment similar to those of patients. Tissue elasticity allows tissues to expand and relax, fluid to drain away, and hundreds of repeated injections to be tolerated without causing damage. Learning curves and their intra- and interindividual dynamics have not hitherto been measured on the Thiel cadaver simulator using the mastery learning and dedicated practice educational paradigm coupled with validated, quantitative metrics, such as checklists, eye tracking metrics, and self-rating scores. OBJECTIVE: Our primary objective was to measure the learning slopes of the scanning and needling phases of an interscalene block conducted repeatedly on a soft embalmed Thiel cadaver over a 3-hour period of training. METHODS: A total of 30 anesthesiologists, with a wide range of experience, conducted up to 60 ultrasound-guided interscalene blocks over 3 hours on the left side of 2 soft embalmed Thiel cadavers. The duration of the scanning and needling phases was defined as the time taken to perform all the steps correctly. The primary outcome was the best-fit linear slope of the log-log transformed time to complete each phase. Our secondary objectives were to measure preprocedural psychometrics, describe deviations from the learning slope, correlate scanning and needling phase data, characterize skills according to clinical grade, measure learning curves using objective eye gaze tracking and subjective self-rating measures, and use cluster analysis to categorize performance irrespective of grade. RESULTS: The median (IQR; range) log-log learning slopes were −0.47 (−0.62 to −0.32; −0.96 to 0.30) and −0.23 (−0.34 to −0.19; −0.71 to 0.27) during the scanning and needling phases, respectively. Locally Weighted Scatterplot Smoother curves showed wide variability in within-participant performance. The learning slopes of the scanning and needling phases correlated: ρ=0.55 (0.23-0.76), P<.001, and ρ=−0.72 (−0.46 to −0.87), P<.001, respectively. Eye gaze fixation count and glance count during the scanning and needling phases best reflected block duration. Using clustering techniques, fixation count and glance were used to identify 4 distinct patterns of learning behavior. CONCLUSIONS: We quantified learning slopes by log-log transformation of the time taken to complete the scanning and needling phases of interscalene blocks and identified intraindividual and interindividual patterns of variability

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

non present

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