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

Mono- or polynuclear surface species: Models and tendencies with cobalt and rhodium

Models of surface reactions of cobalt and rhodium carbonyls with oxide or thiolate type supports were prepared and characterized. For Co carbonyls it was found that simple noncarbonyl ligands that can be formed in reductive CO reactions may promote clusterification under conditions where the support alone causes declusterification. Model complexes of the interaction of rhodium with silica and aromatic thiolate supports were structurally characterized by X-ray diffraction. Model compounds indicate that organic thiolate supports promote clusterification with cobalt and declusterification with rhodium. \ua9 1992

Synthesis and X-ray powder diffraction characterization of (OC)(2)RhCl2Rh(cod) (cod = cycloocta-1,4-diene)

In order to elucidate the nature and the structure of the elusive (OC)(2)Rh(Ph3SiO)(2)Rh(cod) (cod)=cycloocta-1,5-diene) complex, an important model compound for surface catalysis, (OC)(2)RhCl2Rh(cod) has been synthesized, and structurally characterized by ab initio X-ray powder diffraction. Crystals of (OC)(2)RhCl2Rh(cod) are monoclinic, space group P2(1)/c, a = 6.659(1), b = 12.274(1) and c = 16.096(1) Angstrom, beta = 92.176(5)degrees, Z = 4, rho(calc),,,, = 2.209 g cm(-3). The structure has been solved, from powder diffraction data only, by Patterson and Fourier-difference methods and has been ultimately refined, by the Rietveld method, down to R-p, = 0.116 and R-wp, = 0.154 for 4050 data points collected in the 12-93 degrees (2 theta) range. The molecule contains two square-planar rhodium atoms, one bearing two terminal carbonyls and the other bound to the chelating cod fragment, and two chlorine atoms bridging the Rh ... Rh vector. The Rh2Cl2, core is markedly non-planar, the dihedral angle about the Cl ... Cl hinge being 135.4(6)degrees

Heterobimetallic cyclosiloxanolate sandwich clusters: Na [\uf0686-cyclo(PhSiO2)6]2[Fe(OR)]2Ni4(\uf06d6-Cl) (R=H. Me)

The heterobimetallic cyclosiloxanolate sandwich clusters Na{[\u3b76-cyclo (PhSiO2)6]2[Fe(OR)]2Ni 4(\u3bc6-Cl)} (R = H, Me) (1) were prepared from Na2{[(PhSiO2)6]2Na 4Ni4(OH)2} in the form of solvates. The new clusters 1 were characterized by spectra (UV-VIS, IR, 1H-NMR), cyclic voltammetry. conductivity, magnetic susceptibility, and single-crystal X-ray diffraction

Heterobimetallic cyclosiloxanolate sandwich clusters: Na{[η6-cyclo(PhSiO2)6]2[Fe(OR)]2Ni4(μ6-Cl)} (R = H, Me)

The heterobimetallic cyclosiloxanolate sandwich clusters Na{[η6-cyclo(PhSiO2)6]2[Fe(OR)]2Ni4(μ6-Cl)} (R = H, Me) (1) were prepd. from Na2{[(PhSiO2)6]2Na4Ni4(OH)2} as solvates. The new clusters 1 were characterized by spectra (UV-visible, IR, 1H-NMR), cyclic voltammetry, cond., magnetic susceptibility, and single-crystal x-ray diffraction for 1·10MeOH (R = Me)

Bimetallic siloxane cluster of higher valent transition metals: Na{[η\u3csup\u3e6\u3c/sup\u3e-cyclo-(PhSiO\u3csub\u3e2\u3c/sub\u3e)\u3csub\u3e6\u3c/sub\u3e]\u3csub\u3e2\u3c/sub\u3eCo\u3csub\u3e2\u3c/sub\u3eNi\u3csub\u3e4\u3c/sub\u3e(μ\u3csub\u3e6\u3c/sub\u3e-Cl)}

The bimetallic siloxane cluster / cage compound Na{[η6-cyclo-(PhSiO2)6]2Co2Ni4(μ6-Cl)} was preparred and characterized by single crystal X-ray diffraction, spectroscopic, magnetic and electrochemical methods