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 flux 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, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース)

This critical review provides a survey illustrated by recent references of different strategies to achieve a sustainable conversion of biomass to bioproducts. Because of the huge number of chemical products that can be potentially manufactured, a selection of starting materials and targeted chemicals has been done. Also, thermochemical conversion processes such as biomass pyrolysis or gasification as well as the synthesis of biofuels were not considered. The synthesis of chemicals by conversion of platform molecules obtained by depolymerisation and fermentation of biopolymers is presently the most widely envisioned approach. Successful catalytic conversion of these building blocks into intermediates, specialties and fine chemicals will be examined. However, the platform molecule value chain is in competition with well-optimised, cost-effective synthesis routes from fossil resources to produce chemicals that have already a market. The literature covering alternative value chains whereby biopolymers are converted in one or few steps to functional materials will be analysed. This approach which does not require the use of isolated, pure chemicals is well adapted to produce high tonnage products, such as paper additives, paints, resins, foams, surfactants, lubricants, and plasticisers. Another objective of the review was to examine critically the green character of conversion processes because using renewables as raw materials does not exempt from abiding by green chemistry principles (368 references).

Conversion of biomass to selected chemical products

Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.

Autophagosome formation: core machinery and adaptations

Auxin: regulation, action, and interaction.

ω6- and ω3-polyunsaturated C20 fatty acids represent important components of the human diet. A more regular consumption and an accordingly sustainable source of these compounds are highly desirable. In contrast with the very high levels to which industrial fatty acids have to be enriched in plant oils for competitive use as chemical feedstocks, much lower percentages of very-long-chain polyunsaturated fatty acids (VLCPUFA) in edible plant oils would satisfy nutritional requirements. Seed-specific expression in transgenic tobacco (Nicotiana tabacum) and linseed (Linum usitatissimum) of cDNAs encoding fatty acyl-desaturases and elongases, absent from all agronomically important plants, resulted in the very high accumulation of Δ6-desaturated C18 fatty acids and up to 5% of C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acid. Detailed lipid analyses of developing seeds from transgenic plants were interpretated as indicating that, after desaturation on phosphatidylcholine, Δ6-desaturated products are immediately channeled to the triacylglycerols and effectively bypass the acyl-CoA pool. Thus, the lack of available Δ6-desaturated acyl-CoA substrates in the acyl-CoA pool limits the synthesis of elongated C20 fatty acids and disrupts the alternating sequence of lipid-linked desaturations and acyl-CoA dependent elongations. As well as the successful production of VLCPUFA in transgenic oilseeds and the identification of constraints on their accumulation, our results indicate alternative strategies to circumvent this bottleneck.

Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation.

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

Very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are valuable commodities that provide important human health benefits. We report the transgenic production of significant amounts of AA and EPA in Brassica juncea seeds via a stepwise metabolic engineering strategy. Using a series of transformations with increasing numbers of transgenes, we demonstrate the incremental production of VLCPUFAs, achieving AA levels of up to 25% and EPA levels of up to 15% of total seed fatty acids. Both fatty acids were almost exclusively found in triacylglycerols, with AA located preferentially at sn-2 and sn-3 positions and EPA distributed almost equally at all three positions. Moreover, we reconstituted the DHA biosynthetic pathway in plant seeds, demonstrating the practical feasibility of large-scale production of this important omega-3 fatty acid in oilseed crops.

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

When considering the evolution of desaturases, several different aspects come into focus, the most obvious ones being phylogenetic origins and differentiation of regioselectivities of these enzymes. In this general context the term desaturase includes all enzymes able to activate oxygen and to use this reagent for a subsequent modification of C–H bonds at saturated or monounsaturated carbons in substrates as diverse as alkyl groups, acyl residues in thio-, amideor oxygen-ester linkage, carotenoids, sphingolipids, aldehydes and sterols [1,2]. The presently known oxygen-dependent modifications do not only include the formation of cisand transdouble bonds, they also result in the production of acetylenic bonds, insertion of hydroxy or epoxy groups, and even the postulated decarbonylation of aldehydes or dehydrogenation of ubiquinols [3]. This wide spectrum of reactions is catalysed by proteins which all (as extrapolated from the few examples actually studied in detail) may house a di-iron complex held in place by the side chains of suitable amino acids (histidine, aspartate, glutamate and glutamine), although some similar reactions are catalysed by the heme iron of cytochrome P450 isoforms [4]. It should also be pointed out that the mitochondrial dehydrogenation of ubiquinol by the alternative oxidase [3] does not attack a C–H–, but an O–H bond. If the activity of this enzyme does in fact rely on a di-iron centre, it seems to make use of an overpowered reagent for a reaction which normally involves the completely different di-iron–sulphur cluster of the Rieske protein.

The evolution of desaturases.

Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1- 14 C]acetate were purified from leaves of both 16:3 ( Solanum nodiflorum, Chenopodium album ) and 18:3 plants ( Amaranthus lividus, Pisum sativum ). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[ 14 C]gal were similar for both types of chloroplasts. The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage. Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [ 14 C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn -1 and sn -2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants.

https://www.jstor.org/stable/pdfplus/4268018.pdf

Ernst Heinz

Papers

Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation.

Defensins from Insects and Plants Interact with Fungal Glucosylceramides

Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants

The evolution of desaturases.

Similarities and differences in lipid metabolism of chloroplasts isolated from 18:3 and 16:3 plants.