Methods to Monitor and Quantify Autophagy in the Social Amoeba Dictyostelium discoideum

Autophagy is a eukaryotic catabolic pathway that degrades and recycles cellular components to maintain homeostasis. It can target protein aggregates, superfluous biomolecular complexes, dysfunctional and damaged organelles, as well as pathogenic intracellular microbes. Autophagy is a dynamic process in which the different stages from initiation to final degradation of cargo are finely regulated. Therefore, the study of this process requires the use of a palette of techniques, which are continuously evolving and whose interpretation is not trivial. Here, we present the social amoeba Dictyostelium discoideum as a relevant model to study autophagy. Several methods have been developed based on the tracking and observation of autophagosomes by microscopy, analysis of changes in expression of autophagy genes and proteins, and examination of the autophagic flux with various techniques. In this review, we discuss the pros and cons of the currently available techniques to assess autophagy in this organism

Guidance on Dravet Syndrome from Infant to Adult Care: Road Map for Treatment Planning in Europe

Dravet syndrome (DS) is a severe, rare and complex developmental and epileptic encephalopathy affecting 1 in 16'000 live births and characterized by a drug resistant epilepsy, cognitive, psychomotor and language impairment, as well as behavioral disorders. Evidence suggests that optimal treatment of seizures in DS may improve outcomes, even though neurodevelopmental impairments are the likely result of both the underlying genetic variant and the epilepsy. We present an updated guideline for DS diagnosis and treatment, taking into consideration care of the adult patient and non-pharmaceutical therapeutic options for this disease. This up-to-date guideline, which is based on an extensive review of the literature and culminates with a new treatment algorithm for DS, is a European consensus developed through a survey involving 29 European clinical experts in DS. Theis guideline will serve professionals in their clinical practice and, as a consequence, will benefit DS patients and their families

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

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Decision Making in Phagocytosis.

Dictyostelium cells are professional phagocytes that are capable of handling particles of variable shapes and sizes. Here we offer long bacteria that challenge the uptake mechanism to its limits and report on the responses of the phagocytes if they are unable to engulf the particle by closing the phagocytic cup. Reasons for failure may be a length of the particle much larger than the phagocyte's diameter, or competition with another phagocyte. A cell may simultaneously release a particle and engulf another one. The final phase of release can be fast, causing the phagosome membrane to turn inside-out and to form a bleb. Myosin-II may be involved in the release by generating tension at the plasma membrane, it does however not accumulate on the phagosome to act there directly in expelling the particle. Labeling with GFP-2FYVE indicates that processing of the phagosome with phosphatidylinositol 3-phosphate begins at the base of a long phagosome already before closure of the cup. The decision of releasing the particle can be made even at the stage of the processed phagosome