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

Segmentation of the Carotid Lumen and Vessel Wall using Deep Learning and Location Priors

In this report we want to present our method and results for the Carotid Artery Vessel Wall Segmentation Challenge. We propose an image-based pipeline utilizing the U-Net architecture and location priors to solve the segmentation problem at hand.Comment: Challenge Report - Preprin

Computer-assisted contralateral side comparison of the ankle joint using flat panel technology

Purpose!#!Reduction and osteosynthesis of ankle fractures is a challenging surgical procedure when it comes to the verification of the reduction result. Evaluation is conducted using intra-operative imaging of the injured ankle and depends on the expertise of the surgeon. Studies suggest that intra-individual variance of the ankle bone shape and pose is considerably lower than the inter-individual variance. It stands to reason that the information gain from the healthy contralateral side can help to improve the evaluation.!##!Method!#!In this paper, an assistance system is proposed that provides a side-to-side view of the two ankle joints for visual comparison and instant evaluation using only one 3D C-arm image. Two convolutional neural networks (CNN) are employed to extract the relevant image regions and pose information of each ankle so that they can be aligned with each other. A first U-Net uses a sliding window to predict the location of each ankle. The standard plane estimation is formulated as segmentation problem so that a second U-Net predicts the three viewing planes for alignment.!##!Results!#!Experiments were conducted to assess the accuracy of the individual steps on 218 unilateral ankle datasets as well as the overall performance on 7 bilateral ankle datasets. The experiments on unilateral ankles yield a median position-to-plane error of [Formula: see text] mm and a median angular error between 2.98[Formula: see text] and 3.71[Formula: see text] for the plane normals.!##!Conclusion!#!Standard plane estimation via segmentation outperforms direct pose regression. Furthermore, the complete pipeline was evaluated including ankle detection and subsequent plane estimation on bilateral datasets. The proposed pipeline enables a direct contralateral side comparison without additional radiation. This has the potential to ease and improve the intra-operative evaluation for the surgeons in the future and reduce the need for revision surgery

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