| 1. |
- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)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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| 2. |
- Hao, Dong-Xia, et al.
(författare)
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Residue-level elucidation of the ligand-induced protein binding on phenyl-argarose microspheres by NMR hydrogen/deuterium exchange technique
- 2012
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-683X .- 1744-6848. ; 8:23, s. 6248-6255
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Tidskriftsartikel (refereegranskat)abstract
- Protein-ligand interactions on liquid-solid interfaces governed the design of functional biomaterials. However, accurate residue details of ligand induced protein binding and unfolding on an interface were still unknown by the current ensemble of protein structure characterizations. Here, a hydrogen/deuterium (H/D) approach coupled with analysis of NMR TOCSY spectra and the solvent accessible surface area (SASA) was designed to enable residue level understanding of lysozyme adsorbed at a phenyl-ligand modified surface. Results showed that the binding sites and unfolding of lysozyme molecules on phenyl-agarose microspheres demonstrated significant ligand-density dependence and protein-coverage dependence. Either increasing ligand density or decreasing adsorption coverage would lead to more binding sites and unfolding of the protein molecules. With the multipoint adsorption strengthening, the protein molecule changed from lying end-on to side-on. Finally, Molecular Dock simulation was utilized to evaluate the NMR determined binding sites based on energy ranking of the binding. It confirmed that this NMR approach represents a reliable route to in silico abundant residue-level structural information during protein interaction with biomaterials.
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| 3. |
- Zhao, Xi, et al.
(författare)
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Preparation of uniform and large sized agarose microspheres by an improved membrane emulsification technique
- 2014
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Ingår i: Powder Technology. - : Elsevier BV. - 0032-5910 .- 1873-328X. ; 253, s. 444-452
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Tidskriftsartikel (refereegranskat)abstract
- The SPG (Shirasu porous-glass) membrane emulsification technique has been subject to much attention for the preparation of uniform emulsions. However, so far primarily used for the production of droplets with sizes below approximately 60 mu m. A production bottleneck occurred if the desired size was further increased, especially when highly viscous dispersed phases were involved. To this end, an improved membrane emulsification technique was proposed and has been applied to the preparation of large agarose microspheres, with a size of around 90 mu m and with a narrow size distribution. The effects of important emulsification parameters, including the pore size of the SPG membrane, the operating pressure, the stirring rate of the continuous phase, the composition of the continuous oil phase, and the concentration of agarose in the dispersed water phase, have been extensively studied. Under optimum conditions, uniform-size agarose microspheres with an average diameter of 93 pm and a size distribution index of 0.65 were successfully prepared. The average particle size of the home-made agarose microspheres was almost identical to that of the commercial product Sepharose 4 Fast Flow (4FF), which is produced by mechanical stirring and an additional sieving process. However, the size distribution of the former was much narrower than that of the latter. Therefore, the improved membrane emulsification technique presented here is promising for the application of high viscosity systems such as agarose solutions, and the production scale can be further enhanced by increasing the number of membrane units attached to the experimental apparatus. (C) 2013 Elsevier B.V. All rights reserved.
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