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

APEX simulation of best irrigation and N management strategies for off-site N pollution control in three Mediterranean irrigated watersheds

42 Pags., 9 Tabls., 5 Figs. The definitive version is available at: http://www.sciencedirect.com/science/journal/03783774One of the main constraints of irrigated agriculture is off-siteNpollution due to export of nitrate in irrigation return flows (IRF). Models capable of simulating the growth of crops and the N loads in IRF as affected by irrigation and N fertilization may be valuable tools in watershed studies. The Agricultural Policy Environmental eXtender (APEX) model was used to assess bestmanagement practices for reducing off-siteN loads in the IRF of three Mediterranean irrigated watersheds (Akarsu in Turkey, La Violada in Spain and Sidi Rached in Algeria). The watersheds (ranging from 4013 to 10,971 ha) were monitored along three hydrological years to determine the volume of IRF and the NO3-N concentrations and loads in IRF. APEX was calibrated with the data of the first two years and validated with the last year's data. APEX adequately simulated crop evapotranspiration and the volume of IRF and N loads in the IRF (errors < 20%). Simulated annual values were in general more accurate than simulated monthly values. APEX predicted that improving irrigationmanagement (change of irrigation system and/or scheduling) will decrease N loads in IRF over current values by 45% (Akarsu), 40% (La Violada), and 8% (Sidi Rached). However, improved N fertilization only will reduce N loads in IRF by 17% (Akarsu) or below 5% (La Violada and Sidi Rached). Improving irrigationmanagement will increase IRF NO3-N concentrations by 19% in La Violada and will decrease or will remain the same in the other two watersheds. APEXsimulations identified the main soils (shallow and low water holding capacity soils) and crops (heavily fertilized or shallow-root crops) N polluters within the studied watersheds. Overall, APEX simulated that the improvement of irrigation performance was the bestmanagementstrategy to decrease off-siteNpollution while maintaining or increasing crop yields in the three studied Mediterranean watersheds.This study was supported by the European Commission research project (INCO-CT-2005-015031).Peer reviewe

Accelerating real-time shading with reverse reprojection caching

Evaluating pixel shaders consumes a growing share of the computational budget for real-time applications. However, the significant temporal coherence in visible surface regions, lighting conditions, and camera location allows reusing computationally-intensive shading calculations between frames to achieve significant performance improvements at little degradation in visual quality. This paper investigates a caching scheme based on reverse reprojection which allows pixel shaders to store and reuse calculations performed at visible surface points. We provide guidelines to help programmers select appropriate values to cache and present several policies for keeping cached entries up-to-date. Our results confirm this approach offers substantial performance gains for many common real-time effects, including precomputed global lighting effects, stereoscopic rendering, motion blur, depth of field, and shadow mapping. Copyright © 2007 by the Association for Computing Machinery, Inc

Adsorption of human serum albumin on the chrysotile surface: a molecular dynamics and spectroscopic investigation

The human serum albumin (HSA) secondary structure modifications induced by the chrysotile surface have been investigated via computational molecular dynamics (MD) and experimental infrared spectroscopy (FTIR) on synthetic chrysotile nanocrystals coated with different amount of HSA. MD simulations, conducted by placing various albumin subdomains close to the fixed chrysotile surface, show an initial adsorption phase, accompanied by local rearrangements of the albumin motifs in contact with the chrysotile layer. Next, large-scale rearrangements follow with consequent secondary structure modifications

Polymorphisms in CEP68 gene associated with risk of immediate selective reactions to non-steroidal anti-inflammatory drugs

Non-steroidal anti-inflammatory drugs (NSAIDs) are the main triggers of drug hypersensitivity reactions. Such reactions can be pharmacologically or immunologically mediated, but in both cases individual susceptibility can be influenced by genetic factors. Polymorphisms in centrosomal protein of 68 kDa (CEP68) have been associated with pharmacologically mediated NSAIDs reactions. Here, we evaluated this gene in immunologically mediated single-NSAID-induced urticaria/angioedema or anaphylaxis (SNIUAA) by analyzing 52 single nucleotide polymorphisms in CEP68 in 176 patients and 363 NSAIDs-tolerant controls. Two intronic variants (rs2241160 and rs2241161) were significantly associated with an increased risk of SNIUAA, suggesting CEP68 to be a key player in both types of NSAIDs hypersensitivity. However, we found no overlap with genetic variants previously associated with pharmacologically mediated hypersensitivity, pointing to a complex role for this gene and its potential use in the development of biomarkers of clinical utility to diagnose patients at risk of these reactions and to differentiate entities

At-admission prediction of mortality and pulmonary embolism in an international cohort of hospitalised patients with COVID-19 using statistical and machine learning methods

By September 2022, more than 600 million cases of SARS-CoV-2 infection have been reported globally, resulting in over 6.5 million deaths. COVID-19 mortality risk estimators are often, however, developed with small unrepresentative samples and with methodological limitations. It is highly important to develop predictive tools for pulmonary embolism (PE) in COVID-19 patients as one of the most severe preventable complications of COVID-19. Early recognition can help provide life-saving targeted anti-coagulation therapy right at admission. Using a dataset of more than 800,000 COVID-19 patients from an international cohort, we propose a cost-sensitive gradient-boosted machine learning model that predicts occurrence of PE and death at admission. Logistic regression, Cox proportional hazards models, and Shapley values were used to identify key predictors for PE and death. Our prediction model had a test AUROC of 75.9% and 74.2%, and sensitivities of 67.5% and 72.7% for PE and all-cause mortality respectively on a highly diverse and held-out test set. The PE prediction model was also evaluated on patients in UK and Spain separately with test results of 74.5% AUROC, 63.5% sensitivity and 78.9% AUROC, 95.7% sensitivity. Age, sex, region of admission, comorbidities (chronic cardiac and pulmonary disease, dementia, diabetes, hypertension, cancer, obesity, smoking), and symptoms (any, confusion, chest pain, fatigue, headache, fever, muscle or joint pain, shortness of breath) were the most important clinical predictors at admission. Age, overall presence of symptoms, shortness of breath, and hypertension were found to be key predictors for PE using our extreme gradient boosted model. This analysis based on the, until now, largest global dataset for this set of problems can inform hospital prioritisation policy and guide long term clinical research and decision-making for COVID-19 patients globally. Our machine learning model developed from an international cohort can serve to better regulate hospital risk prioritisation of at-risk patients. © The Author(s) 2024

Search for intermediate-mass black hole binaries in the third observing run of Advanced LIGO and Advanced Virgo

International audienceIntermediate-mass black holes (IMBHs) span the approximate mass range 100−105 M⊙, between black holes (BHs) that formed by stellar collapse and the supermassive BHs at the centers of galaxies. Mergers of IMBH binaries are the most energetic gravitational-wave sources accessible by the terrestrial detector network. Searches of the first two observing runs of Advanced LIGO and Advanced Virgo did not yield any significant IMBH binary signals. In the third observing run (O3), the increased network sensitivity enabled the detection of GW190521, a signal consistent with a binary merger of mass ∼150 M⊙ providing direct evidence of IMBH formation. Here, we report on a dedicated search of O3 data for further IMBH binary mergers, combining both modeled (matched filter) and model-independent search methods. We find some marginal candidates, but none are sufficiently significant to indicate detection of further IMBH mergers. We quantify the sensitivity of the individual search methods and of the combined search using a suite of IMBH binary signals obtained via numerical relativity, including the effects of spins misaligned with the binary orbital axis, and present the resulting upper limits on astrophysical merger rates. Our most stringent limit is for equal mass and aligned spin BH binary of total mass 200 M⊙ and effective aligned spin 0.8 at 0.056 Gpc−3 yr−1 (90% confidence), a factor of 3.5 more constraining than previous LIGO-Virgo limits. We also update the estimated rate of mergers similar to GW190521 to 0.08 Gpc−3 yr−1.Key words: gravitational waves / stars: black holes / black hole physicsCorresponding author: W. Del Pozzo, e-mail: [email protected]† Deceased, August 2020