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.

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

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Fluorescence spectroscopy, one of the most informative and sensitive analytical techniques, has played and continues to play key roles in modern research. Indeed, unraveling the inner workings of biomolecules, cells and organisms relied on the development of fluorescence-based tools. As many of the players in these sophisticated interactions and exceedingly complex systems are not inherently emissive, researchers have relied on synthesizing fluorescent analogs of the building blocks found in biological macromolecules. These are the constituents of the cell surface and cell membrane, as well as proteins and nucleic acids. This review article is dedicated to emissive analogs of these relatively small molecules.

For organizational purposes, we have arbitrarily selected to approach these diverse families of biomolecules by imagining “a journey into the center of the cell”. Approaching the exterior of a cell, one first encounters oligosaccharides that decorate the cell surface and are involved in cell recognition and signaling. Next, we arrive at the cell membrane itself. This semi-permeable envelope sets the cell boundaries and regulates its traffic. Several types of building blocks assemble this membrane, most notably among them are the phospholipids. Upon entering the cell, the cytosol reveals a plethora of small and large molecules, including proteins, as well as soluble RNA molecules and RNA-rich ribosomes. Within the cytosol of eukaryotes and prokaryotes lies the nucleus or nucleoid, respectively. This membrane-enclosed control center contains most of the cells’ genetic material. DNA, the cellular blueprint, is permanently found in the nucleus, which also hosts diverse RNA molecules. Accordingly, we first discuss emissive carbohydrate derivatives. We then present fluorescent membrane constituents, followed by emissive amino acids. Our journey ends by focusing on emissive analogs of nucleosides and nucleotides, the building blocks of nucleic acids.

The common biomolecular building blocks, excluding a few amino acids, lack appreciably useful fluorescence properties. This implies that structural modifications are required to impart such photophysical features. Ideally, a designer probe should closely resemble its natural counterpart in size and shape without the loss of the original function (a feature we refer to as “isomorphicity”). This presents a fundamental predicament, as any modification attempting to alter the electronic nature of a molecule, typically by including aromatic residues or extending conjugation, will also alter its steric bulk and therefore the interactions with its surroundings.

Clearly not all biomolecular building blocks can or need to accommodate strict isomorphic design criteria. The heterocycles found in nucleosides already provide a platform that facilitates the extension of π-conjugation, which is also true for some aromatic amino acids. In contrast, employing fluorescence spectroscopy to membrane research requires very creative probe designs. Saccharides can be viewed as the most restrictive in this context, as no chemical modification is conceivable without a major structural disruption and likely loss of function. Such aliphatic biomolecules accommodate labeling only, where an established fluorophore is covalently conjugated to provide an emissive derivative. We therefore reserve the term probe to molecular designs that are expected to furnish useful modified biomolecules capable of reliable reporting. Understandably, fluorescent probes must meet the most stringent isomorphic design principles to ensure a biologically meaningful read-out. The isomorphic design principle is therefore a central theme of this review.

This article focuses on designing fluorescent probes for the four major families of macromolecular building blocks discussed above. Although not necessarily in chronological order, it spans roughly four decades of probe design with emphasis, when justified, on recent contributions. As the reader may imagine, this topic encapsulates a vast research field and cannot be comprehensively reviewed within the space limitation of Chemical Reviews. Nevertheless, we have attempted to summarize the most important and general contributions discussing fluorescent probes that were designed to shed light on biological processes and refer the reader to other resources.1 Although a few examples have found their way into the text, we do not generally address here the development of small molecule fluorophores and sensors that are not part of biomolecular assemblies. We open this article with a brief overview of the key features of fluorescence spectroscopy, where essential theoretical, experimental, and practical elements are discussed.

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Fluorescent Analogs of Biomolecular Building Blocks: Design, Properties, and Applications

Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Insights into the Mode of Action of Chitosan as an Antibacterial Compound

Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.

/pdf/enzyme-immobilization-by-adsorption-a-review-24ulf3yy9j.pdf

Enzyme immobilization by adsorption: a review

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions.

Probing the Modes of Antibacterial Activity of Chitosan. Effects of pH and Molecular Weight on Chitosan Interactions with Membrane Lipids in Langmuir Films

The influence of fatty acids on model cholesterol/phospholipid membranes

The study on the interaction between phytosterols and phospholipids in model membranes.

In this work, we have investigated the condensing and ordering effect induced by cholesterol on phosphatidylcholines (PCs). To perform the studies systematically, for the experiments we have selected phospholipids differing only in the number of cis monounsaturated chains (1,2-distearoyl-sn-glycero-3-phosphocholine − DSPC, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine − SOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine − DOPC) or in the length (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine − POPC vs SOPC) of sn-1 acyl chain. Because the cholesterol concentration in mammalian membranes can be as high as 70 mol % of total lipids, the investigations were performed in a wide range of the sterol content. The results of the Langmuir monolayer experiments evidence that the relation between the structure of hydrophobic part of PC and the magnitude of the effects induced by cholesterol found at lower sterol content is different from that observed at higher sterol concentration. At a lower concentration of sterol...

Paweł Wydro

Papers

Chitosan as a lipid binder: a langmuir monolayer study of chitosan-lipid interactions.

Probing the Modes of Antibacterial Activity of Chitosan. Effects of pH and Molecular Weight on Chitosan Interactions with Membrane Lipids in Langmuir Films

The influence of fatty acids on model cholesterol/phospholipid membranes

The study on the interaction between phytosterols and phospholipids in model membranes.

Variations in the condensing effect of cholesterol on saturated versus unsaturated phosphatidylcholines at low and high sterol concentration.