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

Validation of death prediction after breast cancer relapses using joint models.

BACKGROUND: Cancer relapses may be useful to predict the risk of death. To take into account relapse information, the Landmark approach is popular. As an alternative, we propose the joint frailty model for a recurrent event and a terminal event to derive dynamic predictions of the risk of death. METHODS: The proposed prediction settings can account for relapse history or not. In this work, predictions developed on a French hospital series of patients with breast cancer are externally validated on UK and Netherlands registry data. The performances in terms of prediction error and calibration are compared to those from a Landmark Cox model. RESULTS: The error of prediction was reduced when relapse information was taken into account. The prediction was well-calibrated, although it was developed and validated on very different populations. Joint modelling and Landmark approaches had similar performances. CONCLUSIONS: When predicting the risk of death, accounting for relapses led to better prediction performance. Joint modelling appeared to be suitable for such prediction. Performance was similar to the landmark Cox model, while directly quantifying the correlation between relapses and death

Truncating plakophilin-2 mutations in arrhythmogenic cardiomyopathy are associated with protein haploinsufficiency in both myocardium and epidermis.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is a hereditary cardiac condition associated with ventricular arrhythmias, heart failure, and sudden death. The disease is most often caused by mutations in the desmosomal gene for plakophilin-2 (PKP2), which is expressed in both myocardial and epidermal tissue. This study aimed to investigate protein expression in myocardial tissue of patients with AC carrying PKP2 mutations and elucidate whether keratinocytes of the same individuals exhibited a similar pattern of protein expression. METHODS AND RESULTS: Direct sequencing of 5 AC genes in 71 unrelated patients with AC identified 10 different PKP2 mutations in 12 index patients. One patient, heterozygous for a PKP2 nonsense mutation, developed severe heart failure and underwent cardiac transplantation. Western blotting and immunohistochemistry of the explanted heart showed a significant decrease in PKP2 protein expression without detectable amounts of truncated PKP2 protein. Cultured keratinocytes of the patient showed a similar reduction in PKP2 protein expression. Nine additional PKP2 mutations were investigated in both cultured keratinocytes and endomyocardial biopsies from affected individuals. It was evident that PKP2 mutations introducing a premature termination codon in the reading frame were associated with PKP2 transcript and protein levels reduced to ≈50%, whereas a missense variant did not seem to affect the amount of PKP2 protein. CONCLUSIONS: The results of this study showed that truncating PKP2 mutations in AC are associated with low expression of the mutant allele and that the myocardial protein expression of PKP2 is mirrored in keratinocytes. These findings indicate that PKP2 haploinsufficiency contributes to pathogenesis in AC