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

Multi-physics coupling for hydrogen combustion within porous media

L’ouverture du marché du gaz naturel aux énergies renouvelables et les ambitions affichées par la France et l’Europe de réduction des émissions de gaz à effet de serre encouragent les constructeurs de chaudières à développer de nouvelles technologies. Le nombre de projets et de prototypes basés sur la combustion de mélange de gaz naturel enrichi avec de l’hydrogène est en augmentation croissante. Cette combustion pose cependant de nombreux problèmes techniques, mais également plusieurs défis scientifiques. Stabiliser ces flammes, garantir des conditions de fonctionnement stables éloignées des conditions critiques, réduire les émissions d’oxydes d’azote nécessite de développer de nouveaux outils de prévision de ces écoulements réactifs fortement couplés avec l’ensemble des modes de transfert de chaleur. L’objectif de ce travail est de développer des outils de simulation adaptés à la combustion de mélanges enrichis en hydrogène avec de l’air sur des brûleurs poreux. Bien que les propriétés fondamentales de la combustion H2/air soient largement étudiées, les mécanismes de stabilisation des flammes à la surface de brûleurs poreux ou à l’intérieur de ceux-ci, les échanges thermiques notamment par le biais du rayonnement, et la structure des flammes dans ces systèmes restent largement méconnus. Ces points seront abordés par le biais de la mise en évidence des mécanismes physiques grâce à l’expérimentation dans une configuration générique, la modélisation mathématique des phénomènes et le développement d’outils de simulation adaptés à ces systèmes.Adaption of the natural gas market to renewable energies and the ambitions declared by France and Europe to reduce greenhouse gas emissions encourage boilers to develop new technologies. The number of projects and prototypes based on the combustion of natural gas mixtures enriched with hydrogen is increasing. This combustion mode, however, raises many technical issues, but also several scientific challenges. Stabilizing these flames, ensuring stable operating conditions far from critical conditions, reducing nitrogen oxide emissions requires the development of new tools for predicting these reactive flows strongly coupled with all the modes of heat transfer. The objective of this work is to develop simulation tools adapted to the combustion of mixtures with an increasing amount of hydrogen burnt with air on porous burners. Although the fundamental properties of H2/air combustion are well known, the stabilization mechanisms of these flames above the surface of porous burners or inside the porous structure, the thermal exchanges notably by means of radiation, and the structure of the reactive layer in these systems remain largely unknown. These problems will be addressed by highlighting the main physical mechanisms through experimentation in a generic configuration, mathematical modelling of phenomena and the development of simulation tools adapted to these systems

Alteration of the Copper-Binding Capacity of Iron-Rich Humic Colloids during Transport from Peatland to Marine Waters

Blanket bogs contain vast amounts of <i>Sphagnum</i>-derived organic substances which can act as powerful chelators for dissolved iron and thus enhance its export to the coastal ocean. To investigate the variations in quantity and quality of these exports, adsorptive cathodic stripping voltammetry (CSV) was used to characterize the metal binding properties of molecular weight-fractionated dissolved organic matter (MW-fractionated DOM) in the catchment and coastal plume of a small peat-draining river over a seasonal cycle. Within the plume, both iron- and copper-binding organic ligands showed a linear, conservative distribution with increasing salinity, illustrating the high stability of peatland-derived humic substances (HS). Within the catchment, humic colloids lost up to 50% of their copper-binding capacity, expressed as a molar ratio to organic carbon, after residing for 1 week or more in the main reservoir of the catchment. Immediately downstream of the reservoir, the molar ratio [L₂]/[C_org], where L₂ was the second strongest copper-binding ligand, was 0.75 × 10^–4 when the reservoir residence time was 5 h but 0.34 × 10^–4 when it was 25 days. Residence time did not affect the carbon specific iron-binding capacity of the humic substances which was [L]/[C_org] = (0.80 ± 0.20) × 10^–2. Our results suggest that the loss of copper-binding capacity with increasing residence time is caused by intracolloidal interactions between iron and HS during transit from peat soil to river mouth

Structural Defects Play a Major Role in the Acute Lung Toxicity of Multiwall Carbon Nanotubes: Physicochemical Aspects.

Carbon nanotubes (CNT) have been reported to elicit toxic responses in vitro and in vivo, ascribed so far to metal contamination, CNT length, degree of oxidation, or extent of hydrophilicity. To examine how structural properties may modulate the toxicity of CNT, one preparation of multiwall CNT has been modified (i) by grinding (introducing structural defects) and subsequently heating either in a vacuum at 600 degrees C (causing reduction of oxygenated carbon functionalities and reduction of metallic oxides) or in an inert atmosphere at 2400 degrees C (causing elimination of metals and annealing of defects) and (ii) by heating at 2400 degrees C in an inert atmosphere and subsequently grinding the thermally treated CNT (introducing defects in a metal-deprived carbon framework). The presence of framework and surface defects, metals, and oxygenated functionalities was monitored by means of a set of techniques, including micro-Raman spectroscopy, adsorption calorimetry, X-ray photoelectron spectroscopy, inductively coupled plasma mass spectrometry, and atomic emission spectroscopy. Contrary to traditional toxicants, such as asbestos, CNT may quench rather than generate oxygenated free radicals. The potential of the modified CNT to scavenge hydroxyl radicals was thus evaluated by means of electron spin resonance spectroscopy (spin trapping). The original ground material exhibited a scavenging activity toward hydroxyl radicals, which was eliminated by heating at 2400 degrees C but restored upon grinding. This scavenging activity, related to the presence of defects, appears to go paired with the genotoxic and inflammatory potential of CNT reported in the companion paper. Thus, defects may be one of the major factors governing the toxic potential of CNT

Annotation of the <i>Staphylococcus aureus</i> Metabolome Using Liquid Chromatography Coupled to High-Resolution Mass Spectrometry and Application to the Study of Methicillin Resistance

<i>Staphylococcus aureus</i> can cause a variety of severe disease patterns and can readily acquire antibiotic resistance; however, the mechanisms by which this commensal becomes a pathogen or develops antibiotic resistance are still poorly understood. Here we asked whether metabolomics can be used to distinguish bacterial strains with different antibiotic susceptibilities. Thus, an efficient and robust method was first thoroughly implemented to measure the intracellular metabolites of <i>S. aureus</i> in an unbiased and reproducible manner. We also placed special emphasis on metabolome coverage and annotation and used both hydrophilic interaction liquid chromatography and pentafluorophenyl-propyl columns coupled to high-resolution mass spectrometry in conjunction with our spectral database developed in-house to identify with high confidence as many meaningful<i> S. aureus</i> metabolites as possible. Overall, we were able to characterize up to 210 metabolites in <i>S. aureus</i>, which represents a substantial ∼50% improvement over previously published data. We then preliminarily compared the metabolic profiles of 10 clinically relevant methicillin-resistant and susceptible strains harvested at different time points during the exponential growth phase (without any antibiotic exposure). Interestingly, the resulting data revealed a distinct behavior of “slow-growing” resistant strains, which show modified levels of several precursors of peptidoglycan and capsular polysaccharide biosynthesis

Unraveling the Mechanism of Cysteine Persulfide Formation Catalyzed by 3‑Mercaptopyruvate Sulfurtransferases

Sulfhydration of reactive cysteines in target proteins is now recognized as a major route by which H₂S mediates signal transduction and regulates various cellular processes. Among the enzymatic systems permitting the formation of cysteine persulfide from nonactivated sulfur compounds, 3-mercaptopyruvate sulfurtransferases can be considered as a model of thiolate-based chemistry for carbon–sulfur bond breaking. These ubiquitous enzymes transfer a sulfur atom from 3-mercaptopyruvate (3-MP) to a thiol acceptor via a cysteine-persulfide intermediate, but the mechanistic basis for its formation is still unclear. To address this question, kinetic approaches were developed for studying the reaction catalyzed by the human and Escherichia coli enzymes and the role of several conserved residues was also investigated. We showed that the first step of sulfur transfer that leads to pyruvate release and formation of the persulfide intermediate is very efficient for both enzymes. It critically depends on the electrostatic contribution provided by the CGSGVT catalytic loop, while any role of the so-called Ser/His/Asp triad can be excluded. Furthermore, solvent kinetic isotopic effect and proton inventory studies revealed a concerted mechanism in which the water-mediated protonation of the pyruvate enolate and S⁰ transfer from the deprotonated 3-MP to the thiolate form of the catalytic cysteine occur concomitantly

Bacterial Detection Using Unlabeled Phage Amplification and Mass Spectrometry through Structural and Nonstructural Phage Markers

According to the World Health Organization, food safety is an essential public health priority. In this context, we report a relevant proof of feasibility for the indirect specific detection of bacteria in food samples using unlabeled phage amplification coupled to ESI mass spectrometry analysis and illustrated with the model phage systems T4 and SPP1. High-resolving power mass spectrometry analysis (including bottom-up and top-down protein analysis) was used for the discovery of specific markers of phage infection. Structural components of the viral particle and nonstructural proteins encoded by the phage genome were identified. Then, targeted detection of these markers was performed on a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. <i>E. coli</i> at 1 × 10⁵, 5 × 10⁵, and 1 × 10⁶ CFU/mL concentrations was successfully detected after only a 2 h infection time by monitoring phage T4 structural markers in Luria–Bertani broth, orange juice, and French bean stew (“cassoulet”) matrices. Reproducible detection of nonstructural markers was also demonstrated, particularly when a high titer of input phages was required to achieve successful amplification. This strategy provides a highly time-effective and sensitive assay for bacterial detection

Phage Amplification and Immunomagnetic Separation Combined with Targeted Mass Spectrometry for Sensitive Detection of Viable Bacteria in Complex Food Matrices

We have developed and describe here for the first time a highly sensitive method for the fast and unambiguous detection of viable <i>Escherichia coli</i> in food matrices. The new approach is based on using label-free phages (T4), obligate parasites of bacteria, which are attractive for pathogen detection because of their inherent natural specificity and ease of use. A specific immunomagnetic separation was used to capture the progeny phages produced. Subsequently, T4 phage markers were detected by liquid chromatography coupled to targeted mass spectrometry. Combining the specificity of these three methodologies is of great interest in developing an alternative to conventional time-consuming culture-based technologies for the detection of viable bacteria for industrial applications. First, optimization experiments with phage T4 spiked in complex matrices (without a phage amplification event) were performed and demonstrated specific, sensitive, and reproducible phage capture and detection in complex matrices including Luria–Bertani broth, orange juice, and skimmed milk. The method developed was then applied to the detection of <i>E. coli</i> spiked in foodstuffs (with a phage amplification event). After having evaluated the impact of infection duration on assay sensitivity, we showed that our assay specifically detects viable <i>E. coli</i> in milk at an initial count of ≥1 colony-forming unit (cfu)/mL after an 8-h infection. This excellent detection limit makes our new approach an alternative to PCR-based assays for rapid bacterial detection

Functionalization of Small Rigid Platforms with Cyclic RGD Peptides for Targeting Tumors Overexpressing α_vβ₃‑Integrins

Gadolinium based Small Rigid Plaforms (SRPs) have previously demonstrated their efficiency for multimodal imaging and radiosensitization. Since the RGD sequence is well-known to be highly selective for α_vβ₃ integrins, a cyclic pentapeptide containing the RGD motif (cRGDfK) has been grafted onto the SRP surface. An appropriate protocol led to the grafting of two targeting ligands per nano-object. The resulting nanoparticles have demonstrated a strong association with α_vβ₃ integrins in comparison with cRADfK grafted SRPs as negative control. Flow cytometry and fluorescence microscopy have also been used to highlight the ability of the nanoparticles to target efficiently HEK293­(β3) and U87MG cells. Finally the grafted radiosensitizing nanoparticles were intravenously injected into <i>Nude</i> mice bearing subcutaneous U87MG tumors and the signal observed by optical imaging was twice as high for SRP-cRGDfK compared to their negative analogue

Discovery of JNJ-1802, a First-in-Class Pan-Serotype Dengue Virus NS4B Inhibitor

Dengue is a global public health threat, with about half of the world’s population at risk of contracting this mosquito-borne viral disease. Climate change, urbanization, and global travel accelerate the spread of dengue virus (DENV) to new areas, including southern parts of Europe and the US. Currently, no dengue-specific small-molecule antiviral for prophylaxis or treatment is available. Here, we report the discovery of JNJ-1802 as a potent, pan-serotype DENV inhibitor (EC50’s ranging from 0.057 to 11 nM against the four DENV serotypes). The observed oral bioavailability of JNJ-1802 across preclinical species, its low clearance in human hepatocytes, the absence of major in vitro pharmacology safety alerts, and a dose-proportional increase in efficacy against DENV-2 infection in mice were all supportive of its selection as a development candidate against dengue. JNJ-1802 is being progressed in clinical studies for the prevention or treatment of dengue