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- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- 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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased 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. 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. 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.
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- Russo, Giovanni, et al.
(author)
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Tuning Pluronic F127 phase transitions by adding physiological amounts of salts : A rheology, SAXS, and NMR investigation
- 2024
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In: European Polymer Journal. - 0014-3057. ; 204
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Journal article (peer-reviewed)abstract
- Specific ion (Hofmeister) effects in colloid and biological systems represented a scientific challenge for more than 100 years. Recently, possible applications, based on their rationalization, are emerging. Here, Cl-, SO42-, SCN- anions and Na+, K+, Mg2+ cations are added at physiological concentration (∼0.15 mol/kg) to Pluronic F127 20 wt% aqueous solutions to suitably tune phase transitions for a smart drug delivery platform. Rheological measurements, along with SAXS and NMR self-diffusion experiments, are used to carefully characterize the prepared F127/salt-based formulations. The critical micellar temperature (cmt), the hard-gel formation temperature (Thg), liquid crystal structures, and self-diffusion coefficients are determined. The cmt and Thg values of F127/salt formulations are lower than that of F127 20 wt% sample, following an anionic Hofmeister series: SO42- < Cl- < SCN-. All added salts significantly increase storage modulus and complex viscosity with maximum values occurring at T around 40 °C. SAXS data confirm that added salts preserve cubic liquid crystal phases. NMR self-diffusion analysis highlights that the intermolecular interactions and mobility of F127 unimers/aggregates are ion specific at 16 °C but not at 40 °C. These findings suggest that F127/salt-based formulations may constitute a versatile thermosensitive platform for drug delivery able to assure sustained release in topical or surgery administrations, in the range of temperatures 30–45 °C.
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