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 flux 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, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Rather than enjoying a fine ebook as soon as a mug of coffee in the afternoon, instead they juggled when some harmful virus inside their computer. Methods Of Enzymatic Analysis is clear in our digital library an online admission to it is set as public appropriately you can download it instantly. Our digital library saves in complex countries, allowing you to get the most less latency period to download any of our books considering this one. Merely said, the Methods Of Enzymatic Analysis is universally compatible behind any devices to read.

Methods Of Enzymatic Analysis

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.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

A great challenge for ecology in the coming decades is to understand the role humans play in eco-evolutionary dynamics. If, as emerging evidence shows, rapid evolutionary change affects ecosystem functioning and stability, current rapid environmental change and its evolutionary effects might have significant implications for ecological and human wellbeing on a relatively short time scale. Humans are major selective agents with potential for unprecedented evolutionary consequences for Earth's ecosystems, especially as cities expand rapidly. In this review, I identify emerging hypotheses on how urbanization drives eco-evolutionary dynamics. Studying how human-driven micro-evolutionary changes interact with ecological processes offers us the chance to advance our understanding of eco-evolutionary feedbacks and will provide new insights for maintaining biodiversity and ecosystem function over the long term.

https://www.cell.com/trends/ecology-evolution/pdf/S0169-5347(14)00249-3.pdf

Eco-evolutionary dynamics in an urbanizing planet.

Stress in teleosts is an increasingly studied topic because of its interaction with growth, reproduction, immune system and ultimately fitness of the animal. Whether it is for evaluating welfare in aquaculture, adaptive capacities in fish ecology, or to investigate effects of human-induced rapid environmental change, new experimental methods to describe stress physiology in captive or wild fish have flourished. Cortisol has proven to be a reliable indicator of stress and is considered the major stress hormone. Initially principally measured in blood, cortisol measurement methods are now evolving towards lower invasiveness and to allow repeated measurements over time. We present an overview of recent achievements in the field of cortisol measurement in fishes, discussing new alternatives to blood, whole body and eggs as matrices for cortisol measurement, notably mucus, faeces, water, scales and fins. In parallel, new analytical tools are being developed to increase specificity, sensitivity and automation of the measure. The review provides the founding principles of these techniques and introduces their potential as continuous monitoring tools. Finally, we consider promising avenues of research that could be prioritised in the field of stress physiology of fishes.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/jfb.13904

Measuring cortisol, the major stress hormone in fishes.

The capacity of fishes to cope with environmental variation is considered to be a main determinant of their fitness and is partly determined by their stress physiology. By 2100, global ocean temperature is expected to rise by 1-4°C, with potential consequences for stress physiology. Global warming is affecting animal populations worldwide through chronic temperature increases and an increase in the frequency of extreme heatwave events. As ectotherms, fishes are expected to be particularly vulnerable to global warming. Although little information is available about the effects of global warming on stress physiology in nature, multiple studies describe the consequences of temperature increases on stress physiology in controlled laboratory conditions, providing insight into what can be expected in the wild. Chronic temperature increase constitutes a physiological load that can alter the ability of fishes to cope with additional stressors, which might compromise their fitness. In addition, rapid temperature increases are known to induce acute stress responses in fishes and might be of ecological relevance in particular situations. This review summarizes knowledge about effects of temperature increases on the stress physiology of fishes and discusses these in the context of global warming.

/pdf/temperature-increase-and-its-effects-on-fish-stress-1nu0bg6r4e.pdf

Temperature increase and its effects on fish stress physiology in the context of global warming

1. Introduction 2. A Conceptual Framework for Growth 2.1. The Dynamic Energy Budget (DEB) Model for Growth 2.2. Myocyte Growth 3. Stress Effect on Energy Available for Growth 3.1. Food Intake 3.2. Energy Substrate Absorption at the Gut 3.3. Energy Demand for Maintenance 4. Stress Effects on Promoters of Muscle Formation 4.1. Stress Effects on Myogenesis 4.2. Stress Regulation of the GH–IGF Axis 5. Conclusion and Knowledge Gaps In fish, muscle contributes over half the body mass and thus changes in the size of this organ system is considered to be of primary importance to growth. Muscle growth is the final consequence of a complex set of processes starting with the absorption of nutrients from the environment to their allocation for increases in myocyte number and size. Stress affects these processes, including energy utilization, absorption, and allocation, resulting in reduced muscle growth. Situations encountered in the wild such as predator avoidance or deteriorated water quality, or in captive situations such as handling, crowding, sorting, and grading may be stressful to the animal, entailing reallocation of energy away from growth to cope with stress. Here we highlight the potential role of cortisol, the principal glucocorticoid in teleosts, in regulating processes leading to muscle growth suppression. Stress-mediated elevation in plasma cortisol level affects energy intake, absorption, and utilization, including protein turnover and modulation of protective proteins expression, all leading to a decrease in energy available for muscle growth. Cortisol also modulates muscle growth regulators, including growth factors and transcription factors, causing growth suppression. Overall, there is a paucity of information on the mechanisms leading to stress effects on growth, but the available literature suggests a key role for stressor-mediated elevation in circulating cortisol levels and modulating muscle protein accretion in fish.

Stress and Growth

A new method for measuring group behaviours of fish shoals from recorded videos taken in near aquaculture conditions

Many fish species experience long periods of fasting often associated with seasonal reductions in water temperature and prey availability or spawning migrations. During periods of nutrient restriction, changes in metabolism occur to provide cellular energy via catabolic processes. Muscle is particularly affected by prolonged fasting as proteins of this tissue act as a major energy source. However, the molecular components involved in muscle protein degradation as well as the regulatory networks that control their function are still incompletely defined in fish. The present work aimed to characterize the response of the autophagy-lysosomal degradative pathway to nutrient and serum availability in primary culture of rainbow trout myoblasts. In this aim, 4-day-old cells were incubated in a serum and amino acid-rich medium (complete medium), a serum and amino acid-deprived medium (minimal medium) or a minimal medium plus amino acids, and both the transcription-independent short-term response and the transcription-dependent long-term response of the autophagy-lysosomal degradative pathway were analyzed. We report that serum and amino acids withdrawal is accompanied by a rapid increase of autophagosome formation but also by a slower induction of the expression of several autophagy-related genes (LC3B, gabarapl1, atg4b). We also showed that this latter response is controlled by amino acid (AA) availability and that both TOR-dependent and TOR-independent pathways are involved in this effect. Together these results suggest an important role for AA released by muscle proteolysis during the fasting period in regulating the subtle balance between using proteins as disposable furniture to provide energy, and conserving muscle through protein sparing.

https://www.tandfonline.com/doi/pdf/10.4161/auto.18863

Bastien Sadoul

Papers

Measuring cortisol, the major stress hormone in fishes.

Temperature increase and its effects on fish stress physiology in the context of global warming

Stress and Growth

A new method for measuring group behaviours of fish shoals from recorded videos taken in near aquaculture conditions

Amino acids downregulate the expression of several autophagy-related genes in rainbow trout myoblasts.