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

Complementary and alternative medicine in the (symptomatic) treatment of acute tonsillitis in children: A systematic review

Background: Acute tonsillitis (including tonsillopharyngitis) in childhood is a common disease with a peak of illness in school-age. Most of these cases have a viral origin and antibiotic therapy is not indicated, therefore, effective symptomatic therapy is required. For this reason, complementary, alternative and integrative medicine therapies might be a solution. Objective: The aim of this review is to demonstrate study status of such therapies. Methods: The databases PubMed, Cochrane Library, OVID, CAMbase, CAM-QUEST® and Anthromedics were systematically screened for studies investigating complementary, alternative and integrative therapy approaches in paediatric cohorts. Studies were analyzed by therapy approach, study design, cohort and outcome, using the PRISMA 2020 checklist. Results: The systematic literature search resulted in 321 articles. Five publications corresponded to the search criteria and were assigned to the following specific therapeutic categories: herbal medicine (3), homeopathy (1) and ayurvedic medicine (1). Clinical trials were found for the herbal compounds BNO 1030 (Impupret®) and EPs® 7630 (Umckaloabo), the homeopathic complex Tonzolyt® and the ayurvedic medicine Kanchnara-Guggulu and Pratisarana of Tankana‑Madhu. Antimicrobial effects of essential oils and carvacrol as single agents as well as in combination with erythromycin were analysed in an in vitro study. Conclusion: Clinical studies indicate an improvement of symptoms and a good tolerability of all investigated remedies of complementary, alternative and integrative medicine in the treatment of tonsillitis in childhood. Nevertheless, quality and quantity of the studies were insufficient to make a reliable conclusion regarding effectiveness. Therefore, more clinical trials are urgently needed to achieve a meaningful result

Fragmentation experiments with Havre melt: dry and induced fuel-coolant interaction runs

Here we present the data records (raw data) from 19 fragmentation experiments. In these runs silicic HVR254 dome rock (retrieved from the submarine Havre volcano) was crushed, remelted and fragmented using two different experimental settings: 1. dry runs (records labelled "D"): melt was fragmented by injection of pressurized Ar gas. 2. induced fuel-coolant interaction runs (records labelled "IFCI"): a water layer was established on top of the melt, before gas was injected from below. This caused fragmentation of the melt plug under IFCI conditions. Note that the runs D07, D08, D09, IFCI08 and IFCI09 used a reduced melt mass (100g instead of 250g). Files contain (separated by column) records of: time, trigger signal, force, pressure, microphone, electric field, seismic data. The units and amplification settings used are provided in the file headers. In addition, the results of morphometry analysis (t-tests) are provided in a pdf file. The morphometric analyses of natural ash focused exclusively on the curvi-planar grains dominant in Havre ash samples, labelled "Nat1" - "Nat6". Four types of experimental grains were compared with them: • “DG”: particles from dry runs, from the lab floor • “IG”: grains from open IFCI runs, from the lab floor • “IW”: very small particles from open IFCI runs deposited in water droplets on the walls and ceiling around the experimental area • “IU”: particles from IFCI runs with U-tube, from the water bow

Null Mutation in PGAP1 Impairing Gpi-Anchor Maturation in Patients with Intellectual Disability and Encephalopathy

<div><p>Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene <i>PGAP1</i>. <i>PGAP1</i> was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type <i>PGAP1</i> cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene <i>PGAP1</i>. Our results add <i>PGAP1</i> to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.</p></div

FACS analysis of GPI-APs on LCLs and their PI-PLC sensitivity.

<p>(A, B) Cells from one of the affected siblings (III-3) and the parents were transfected with empty pMEoriP vector (A) and pMEoriP-FLAG-humanPGAP1 (B). Cells from the healthy sister were used without transfection. Four days after transfection, cells were treated with (solid lines) or without (dotted lines) 10 unit/ml of PI-PLC for 1.5 h at 37°C, and the surface expression of CD59, DAF and CD48 were assessed by flow cytometry.</p

Pedigree of family MR079 and a PGAP1 mutation.

<p>Pedigree of family MR079 and a PGAP1 mutation.</p

Molecular modeling of PGAP1.

<p>(A) Model of PGAP1 highlighting the position of Leu197. The two views differ by a rotation of 90° around the horizontal axis. (B) Interactions of Leu197 (green) with residues Leu184 and Ile194 of the hydrophobic core. (C) Interactions of Ile198 (green) in the Leu197del mutant. Clashes with the adjacent amino acids Leu184 and Ile194 are indicated by cyan arrows. Residues 203–316 are not shown in (B) and (C) for reasons of clarity.</p

COVID-19-related mortality in kidney transplant and dialysis patients: results of the ERACODA collaboration.

BACKGROUND: Patients on kidney replacement therapy comprise a vulnerable population and may be at increased risk of death from coronavirus disease 2019 (COVID-19). Currently, only limited data are available on outcomes in this patient population. METHODS: We set up the ERACODA (European Renal Association COVID-19 Database) database, which is specifically designed to prospectively collect detailed data on kidney transplant and dialysis patients with COVID-19. For this analysis, patients were included who presented between 1 February and 1 May 2020 and had complete information available on the primary outcome parameter, 28-day mortality. RESULTS: Of the 1073 patients enrolled, 305 (28%) were kidney transplant and 768 (72%) dialysis patients with a mean age of 60 ± 13 and 67 ± 14 years, respectively. The 28-day probability of death was 21.3% [95% confidence interval (95% CI) 14.3-30.2%] in kidney transplant and 25.0% (95% CI 20.2-30.0%) in dialysis patients. Mortality was primarily associated with advanced age in kidney transplant patients, and with age and frailty in dialysis patients. After adjusting for sex, age and frailty, in-hospital mortality did not significantly differ between transplant and dialysis patients [hazard ratio (HR) 0.81, 95% CI 0.59-1.10, P = 0.18]. In the subset of dialysis patients who were a candidate for transplantation (n = 148), 8 patients died within 28 days, as compared with 7 deaths in 23 patients who underwent a kidney transplantation <1 year before presentation (HR adjusted for sex, age and frailty 0.20, 95% CI 0.07-0.56, P < 0.01). CONCLUSIONS: The 28-day case-fatality rate is high in patients on kidney replacement therapy with COVID-19 and is primarily driven by the risk factors age and frailty. Furthermore, in the first year after kidney transplantation, patients may be at increased risk of COVID-19-related mortality as compared with dialysis patients on the waiting list for transplantation. This information is important in guiding clinical decision-making, and for informing the public and healthcare authorities on the COVID-19-related mortality risk in kidney transplant and dialysis patients

Genetic Susceptibility for Atrial Fibrillation in Patients Undergoing Atrial Fibrillation Ablation

Background: Ablation is a widely used therapy for atrial fibrillation (AF); however, arrhythmia recurrence and repeat procedures are common. Studies examining surrogate markers of genetic susceptibility to AF, such as family history and individual AF susceptibility alleles, suggest these may be associated with recurrence outcomes. Accordingly, the aim of this study was to test the association between AF genetic susceptibility and recurrence after ablation using a comprehensive polygenic risk score for AF. Methods: Ten centers from the AF Genetics Consortium identified patients who had undergone de novo AF ablation. AF genetic susceptibility was measured using a previously described polygenic risk score (N=929 single-nucleotide polymorphisms) and tested for an association with clinical characteristics and time-to-recurrence with a 3 month blanking period. Recurrence was defined as >30 seconds of AF, atrial flutter, or atrial tachycardia. Multivariable analysis adjusted for age, sex, height, body mass index, persistent AF, hypertension, coronary disease, left atrial size, left ventricular ejection fraction, and year of ablation. Results: Four thousand two hundred seventy-six patients were eligible for analysis of baseline characteristics and 3259 for recurrence outcomes. The overall arrhythmia recurrence rate between 3 and 12 months was 44% (1443/3259). Patients with higher AF genetic susceptibility were younger (P<0.001) and had fewer clinical risk factors for AF (P=0.001). Persistent AF (hazard ratio [HR], 1.39 [95% CI, 1.22-1.58]; P<0.001), left atrial size (per cm: HR, 1.32 [95% CI, 1.19-1.46]; P<0.001), and left ventricular ejection fraction (per 10%: HR, 0.88 [95% CI, 0.80-0.97]; P=0.008) were associated with increased risk of recurrence. In univariate analysis, higher AF genetic susceptibility trended towards a higher risk of recurrence (HR, 1.08 [95% CI, 0.99-1.18]; P=0.07), which became less significant in multivariable analysis (HR, 1.06 [95% CI, 0.98-1.15]; P=0.13). Conclusions: Higher AF genetic susceptibility was associated with younger age and fewer clinical risk factors but not recurrence. Arrhythmia recurrence after AF ablation may represent a genetically different phenotype compared to AF susceptibility