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

Hydride-free Hydrogenation: Unraveling the Mechanism of Electrocatalytic Alkyne Semihydrogenation by Nickel-Bipyridine Complexes

Hydrogenation reactions of carbon-carbon unsaturated bonds are central in synthetic chemistry. Efficient catalysis of these reactions classically recourses to heterogeneous or homogeneous transition-metal species. Whether thermal or electrochemical, C–C multiple bond catalytic hydrogenation commonly involves metal hydrides as key intermediates. Here, we report that the electrocatalytic semihydrogenation of alkynes by molecular Ni complexes proceeds without the mediation of a hydride intermediate. Through a combined experimental and theoretical investigation, we disclose a mechanism that primarily involves a nickelacyclopropene resting state upon alkyne binding to a low-valent Ni(0) species. A following sequence of protonation and electron transfer steps via Ni(II) and Ni(I)-vinyl intermediates then leads to olefin release in an overall ECEC pattern as the most favored pathway. Our results also evidence that pathways involving hydride intermediates are strongly disfavored, which in turns promotes high semihydrogenation selectivity by avoiding competing hydrogen evolution. While bypassing catalytically competent hydrides, this type of mechanism still retains inner-metal-sphere characteristics with the formation of organometallic intermediates, often essential to control regio- or stereo-selectivity. We think that this approach to electrocatalytic reductions of unsaturated organic groups can open new paradigms for hydrogenation or hydroelementation reactions.</jats:p

Hydride-free Hydrogenation: Unraveling the Mechanism of Electrocatalytic Alkyne Semihydrogenation by Nickel-Bipyridine Complexes

Hydrogenation reactions of carbon-carbon unsaturated bonds are central in synthetic chemistry. Efficient catalysis of these reactions classically recourses to heterogeneous or homogeneous transition-metal species. Whether thermal or electrochemical, C–C multiple bond catalytic hydrogenation commonly involves metal hydrides as key intermediates. Here, we report that the electrocatalytic semihydrogenation of alkynes by molecular Ni complexes proceeds without the mediation of a hydride intermediate. Through a combined experimental and theoretical investigation, we disclose a mechanism that primarily involves a nickelacyclopropene resting state upon alkyne binding to a low-valent Ni(0) species. A following sequence of protonation and electron transfer steps via Ni(II) and Ni(I)-vinyl intermediates then leads to olefin release in an overall ECEC pattern as the most favored pathway. Our results also evidence that pathways involving hydride intermediates are strongly disfavored, which in turns promotes high semihydrogenation selectivity by avoiding competing hydrogen evolution. While bypassing catalytically competent hydrides, this type of mechanism still retains inner-metal-sphere characteristics with the formation of organometallic intermediates, often essential to control regio- or stereo-selectivity. We think that this approach to electrocatalytic reductions of unsaturated organic groups can open new paradigms for hydrogenation or hydroelementation reactions

Electrocatalytic Semihydrogenation of Alkynes with [Ni(bpy)₃]²⁺

Electrifying the production of base and fine chemicals calls for the development of electrocatalytic methodologies for these transformations. We show here that the semihydrogenation of alkynes, an important transformation in organic synthesis, is electrocatalyzed at room temperature by a simple complex of earth-abundant nickel, [Ni­(bpy)3]2+. The approach operates under mild conditions and is selective toward the semihydrogenated olefins with good to very good Z isomer stereoselectivity. (Spectro)­electrochemistry supports that the electrocatalytic cycle is initiated in an atypical manner with a nickelacyclopropene complex, which upon further protonation is converted into a putative cationic Ni­(II)–vinyl intermediate that produces the olefin after electron–proton uptake. This work establishes a proof of concept for homogeneous electrocatalysis applied to alkyne semihydrogenation, with opportunities to improve the yields and stereoselectivity

Hydride-Free Hydrogenation: Unraveling the Mechanism of Electrocatalytic Alkyne Semihydrogenation by Nickel–Bipyridine Complexes

Hydrogenation reactions of carbon–carbon unsaturated bonds are central in synthetic chemistry. Efficient catalysis of these reactions classically recourses to heterogeneous or homogeneous transition-metal species. Whether thermal or electrochemical, C–C multiple bond catalytic hydrogenations commonly involve metal hydrides as key intermediates. Here, we report that the electrocatalytic alkyne semihydrogenation by molecular Ni bipyridine complexes proceeds without the mediation of a hydride intermediate. Through a combined experimental and theoretical investigation, we disclose a mechanism that primarily involves a nickelacyclopropene resting state upon alkyne binding to a low-valent Ni(0) species. A following sequence of protonation and electron transfer steps via Ni(II) and Ni(I) vinyl intermediates then leads to olefin release in an overall ECEC-type pattern as the most favored pathway. Our results also evidence that pathways involving hydride intermediates are strongly disfavored, which in turn promotes high semihydrogenation selectivity by avoiding competing hydrogen evolution. While bypassing catalytically competent hydrides, this type of mechanism still retains inner-metal-sphere characteristics with the formation of organometallic intermediates, often essential to control regio- or stereoselectivity. We think that this approach to electrocatalytic reductions of unsaturated organic groups can open new paradigms for hydrogenation or hydroelementation reactions

Bioactive compounds in seaweed; functional food applications and legislation

Seaweed is more than the wrap that keeps rice together in sushi. Seaweed biomass is already used for a wide range of other products in food, including stabilising agents. Biorefineries with seaweed as feedstock are attracting worldwide interest and include low-volume, high value-added products and vice versa. Scientific research on bioactive compounds in seaweed usually takes place on just a few species and compounds. This paper reviews worldwide research on bioactive compounds, mainly of nine genera or species of seaweed, which are also available in European temperate Atlantic waters, i.e. Laminaria sp., Fucus sp., Ascophyllum nodosum, Chondrus crispus, Porphyra sp., Ulva sp., Sargassum sp., Gracilaria sp. and Palmaria palmata. In addition, Undaria pinnatifida is included in this review as this is globally one of the most commonly produced, investigated and available species. Fewer examples of other species abundant worldwide have also been included. This review will supply fundamental information for biorefineries in Atlantic Europe using seaweed as feedstock. Preliminary selection of one or several candidate seaweed species will be possible based on the summary tables and previous research described in this review. This applies either to the choice of high value-added bioactive products to be exploited in an available species or to the choice of seaweed species when a bioactive compound is desired. Data are presented in tables with species, effect and test organism (if present) with examples of uses to enhance comparisons. In addition, scientific experiments performed on seaweed used as animal feed are presented, and EU, US and Japanese legislation on functional foods is reviewed