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

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

International audienceIn 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

International Society of Sports Nutrition Position Stand: protein and exercise

Abstract Position statement The International Society of Sports Nutrition (ISSN) provides an objective and critical review related to the intake of protein for healthy, exercising individuals. Based on the current available literature, the position of the Society is as follows: 1) An acute exercise stimulus, particularly resistance exercise, and protein ingestion both stimulate muscle protein synthesis (MPS) and are synergistic when protein consumption occurs before or after resistance exercise. 2) For building muscle mass and for maintaining muscle mass through a positive muscle protein balance, an overall daily protein intake in the range of 1.4–2.0 g protein/kg body weight/day (g/kg/d) is sufficient for most exercising individuals, a value that falls in line within the Acceptable Macronutrient Distribution Range published by the Institute of Medicine for protein. 3) There is novel evidence that suggests higher protein intakes (>3.0 g/kg/d) may have positive effects on body composition in resistance-trained individuals (i.e., promote loss of fat mass). 4) Recommendations regarding the optimal protein intake per serving for athletes to maximize MPS are mixed and are dependent upon age and recent resistance exercise stimuli. General recommendations are 0.25 g of a high-quality protein per kg of body weight, or an absolute dose of 20–40 g. 5) Acute protein doses should strive to contain 700–3000 mg of leucine and/or a higher relative leucine content, in addition to a balanced array of the essential amino acids (EAAs). 6) These protein doses should ideally be evenly distributed, every 3–4 h, across the day. 7) The optimal time period during which to ingest protein is likely a matter of individual tolerance, since benefits are derived from pre- or post-workout ingestion; however, the anabolic effect of exercise is long-lasting (at least 24 h), but likely diminishes with increasing time post-exercise. 8) While it is possible for physically active individuals to obtain their daily protein requirements through the consumption of whole foods, supplementation is a practical way of ensuring intake of adequate protein quality and quantity, while minimizing caloric intake, particularly for athletes who typically complete high volumes of training. 9) Rapidly digested proteins that contain high proportions of essential amino acids (EAAs) and adequate leucine, are most effective in stimulating MPS. 10) Different types and quality of protein can affect amino acid bioavailability following protein supplementation. 11) Athletes should consider focusing on whole food sources of protein that contain all of the EAAs (i.e., it is the EAAs that are required to stimulate MPS). 12) Endurance athletes should focus on achieving adequate carbohydrate intake to promote optimal performance; the addition of protein may help to offset muscle damage and promote recovery. 13) Pre-sleep casein protein intake (30–40 g) provides increases in overnight MPS and metabolic rate without influencing lipolysis

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

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