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

Nouvel ergomètre de terrain pour mesurer de manière reproductible la force maximale et le taux de développement de la force des ischio-jambiers

International audienceObjectives. — Hamstring strain injuries are common in sprint-based activities and multifacto- rial. One of the main risk factors is the deficit of hamstring eccentric strength, which is not easily assessed in field conditions. Therefore, a new ergometer named ‘‘Hamtech’’ has been developed to accurately and practically assess hamstring force output. The aim of this study was to test the reproducibility of this novel ergometer during maximal isometric and eccentric contractions.Materials and methods. — Thirteen soccer players (age: 20.7 ± 1.6 years; stature: 178.1±4.9cm; body mass: 72.1±6.3kg) were recruited. After two familiarization ses- sions, hamstring force production was recorded during maximal isometric and eccentric contractions using the Hamtech. The coefficients of variations and intra-class correlations coefficients were used to quantify reproducibility.Results. — This ergometer allows reproducible measurements of unilateral (isomet- ric+eccentric) and bilateral (eccentric) peak force. However, the reproducibility was moderate to good (unilateral) and poor (bilateral) for the knee angle at the eccentric peak force. A good reproducibility was observed for the rate of force development at 200-ms during maximal isometric contractions. The Hamtech offers a simple and reproducible solution to measure hamstring force production during isometric and eccentric contractions. The use of unilateral hamstring muscles at long muscular length might provide a valuable alternative to the classical bilateral condition to assess the hamstring force production.Objectifs : Les blessures aux ischio-jambiers sont fréquentes dans les activités basées sur le sprint et sont multifactorielles. L'un des principaux facteurs de risque est le déficit de force excentrique des ischio-jambiers, qui n'est pas facile à évaluer sur le terrain. Par conséquent, un nouvel ergomètre appelé "Hamtech" a été développé pour évaluer de manière précise et pratique, la force des ischio-jambiers. L'objectif de cette étude était de tester la reproductibilité de ce nouvel ergomètre lors de contractions isométriques et excentriques maximales

Characterization of two distinct liver progenitor cell subpopulations of hematopoietic and hepatic origins

Despite extensive studies, the hematopoietic versus hepatic origin of liver progenitor oval cells remains controversial. The aim of this study was to determine the origin of such cells after liver injury and to establish an oval cell line. Rat liver injury was induced by subcutaneous insertion of 2-AAF pellets for 7 days with subsequent injection of CCl(4). Livers were removed 9 to 13 days post-CCl(4) treatment. Immunohistochemistry was performed using anti-c-kit, OV6, Thy1, CK19, AFP, vWF and Rab3b. Isolated non-parenchymal cells were grown on mouse embryonic fibroblast, and their gene expression profile was characterized by RT-PCR. We identified a subpopulation of OV6/CK19/Rab3b-expressing cells that was activated in the periportal region of traumatized livers. We also characterized a second subpopulation that expressed the HSCs marker c-kit but not Thy1. Although we successfully isolated both cell types, OV6/CK19/Rab3b(+) cells fail to propagate while c-kit(+)-HSCs appeared to proliferate for up to 7 weeks. Cells formed clusters which expressed c-kit, Thy1 and albumin. Our results indicate that a bona fide oval progenitor cell population resides within the liver and is distinct from c-kit(+)-HSCs. Oval cells require the hepatic niche to proliferate, while cells mobilized from the circulation proliferate and transdifferentiate into hepatocytes without evidence of cell fusion

Deficiency in the extracellular signal-regulated kinase 1 (ERK1) protects leptin-deficient mice from insulin resistance without affecting obesity.

International audienceAIMS/HYPOTHESIS: Extracellular signal-regulated kinase (ERK) activity is increased in adipose tissue in obesity and type 2 diabetes mellitus and strong evidences suggests that it is implicated in the downregulation of insulin signalling and action in the insulin-resistant state. To determine the role of ERK1 in obesity-associated insulin resistance in vivo, we inactivated Erk1 (also known as Mapk3) in obese leptin-deficient mice (ob/ob). METHODS: Mice of genotype ob/ob-Erk1 (-/-) were obtained by crossing Erk1 (-/-) mice with ob/ob mice. Glucose tolerance and insulin sensitivity were studied in 12-week-old mice. Tissue-specific insulin sensitivity, insulin signalling, liver steatosis and adipose tissue inflammation were determined. RESULTS: While ob/ob-Erk1 (-/-) and ob/ob mice exhibited comparable body weight and adiposity, ob/ob-Erk1 (-/-) mice did not develop hyperglycaemia and their glucose tolerance was improved. Hyperinsulinaemic-euglycaemic clamp studies demonstrated an increase in whole-body insulin sensitivity in the ob/ob-Erk1 (-/-) mice associated with an increase in both insulin-stimulated glucose disposal in skeletal muscles and adipose tissue insulin sensitivity. This occurred in parallel with improved insulin signalling in both tissues. The ob/ob-Erk1 (-/-) mice were also partially protected against hepatic steatosis with a strong reduction in acetyl-CoA carboxylase level. These metabolic improvements were associated with reduced expression of mRNA encoding inflammatory cytokine and T lymphocyte markers in the adipose tissue. CONCLUSIONS/INTERPRETATION: Our results demonstrate that the targeting of ERK1 could partially protect obese mice against insulin resistance and liver steatosis by decreasing adipose tissue inflammation and by increasing muscle glucose uptake. Our results indicate that deregulation of the ERK1 pathway could be an important component in obesity-associated metabolic disorders