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

Information Disclosure Ranking, Industry Production Market Competition, and Mispricing: An Empirical Analysis

Improving the transparency of corporate information disclosure is a key principle of corporate governance in Taiwan. This study uses the information disclosure assessment system established by the information disclosure and transparency ranking system to explore whether information transparency can reduce the degree of mispricing. The study uses the data of 10,686 listed companies in Taiwan for the period from 2005 to 2014. We find that a higher information disclosure ranking (IDR) of rated companies corresponds to a more substantial reduction in the degree of mispricing. Moreover, we discover that product market competition affects mispricing in that smaller degrees of mispricing reflect greater exclusivity; this suggests that lower industry transaction and competition costs lead to less substantial mispricing. Finally, we observe that the effect of information disclosure score on the degree of mispricing is lower in more exclusive industries. Furthermore, a regression process using instrumental variables reveals that IDRs have the significant effect of reducing the degree of mispricing

Distribution of Crystalline Polymer and Fullerene Clusters in Both Horizontal and Vertical Directions of High-Efficiency Bulk Heterojunction Solar Cells

In this study, we used (i) synchrotron grazing-incidence small-/wide-angle X-ray scattering to elucidate the crystallinity of the polymer PBTC₁₂TPD and the sizes of the clusters of the fullerenes PC₆₁BM and ThC₆₁BM and (ii) transmission electron microscopy/electron energy loss spectroscopy to decipher both horizontal and vertical distributions of fullerenes in PBTC₁₂TPD/fullerene films processed with chloroform, chlorobenzene and dichlorobezene. We found that the crystallinity of the polymer and the sizes along with the distributions of the fullerene clusters were critically dependent on the solubility of the polymer in the processing solvent when the solubility of fullerenes is much higher than that of the polymer in the solvent. In particular, with chloroform (CF) as the processing solvent, the polymer and fullerene units in the PBTC₁₂TPD/ThC₆₁BM layer not only give rise to higher crystallinity and a more uniform and finer fullerene cluster dispersion but also formed nanometer scale interpenetrating network structures and presented a gradient in the distribution of the fullerene clusters and polymer, with a higher polymer density near the anode and a higher fullerene density near the cathode. As a result of combined contributions from the enhanced polymer crystallinity, finer and more uniform fullerene dispersion and gradient distributions, both the short current density and the fill factor for the device incorporating the CF-processed active layer increase substantially over that of the device incorporating a dichlorobenzene-processed active layer; the resulting power conversion efficiency of the device incorporating the CF-processed active layer was enhanced by 46% relative to that of the device incorporating a dichlorobenzene-processed active layer

Structural Evolution of Crystalline Conjugated Polymer/Fullerene Domains from Solution to the Solid State in the Presence and Absence of an Additive

The power conversion efficiencies of polymer/fullerene solar cells are critically dependent on the nanometer-scale morphologies of their active layers, which are typically processed from solution. Using synchrotron wide- and small-angle X-ray scattering, we have elucidated the intricate mechanism of the structural transitions from solutions to solid films of the crystalline polymer poly­[bis­(dodecyl)­thiophene-thieno­[3,4-<i>c</i>]­pyrrole-4,6-dione] (PBTTPD) and [6,6]-phenyl-C₇₁-butyric acid methyl ester (PC₇₁BM), including the effect of the solvent additive 1,6-diiodohexane (DIH). We found that the local assembly of rigid-rod PBTTPD segments that formed in solution instantly and then relaxed within several hundred seconds upon cooling to room temperature from 90 °C could re-emerge and develop into seeds for subsequent crystallization of the polymer in the solid films. At room temperature (25 °C), the presence of DIH in chlorobenzene slightly enhanced the formation of local assembly PBTTPD segments in the supersaturated PBTTPD in PBTTPD/PC₇₁BM blend solution. Two cases of films were subsequently developed from these blend solutions with drop-casted and spin-coated methods. For spin-coated thin films (90 nm thick), which evolve quickly, polymer’s crystallinity and the fullerene packing in the solid-state thin films were enhanced in the case of involving DIH. Regarding the effect of DIH for processing the drop-casted thick films (2.5 μm thick), which evolve slowly, DIH has no observable effect on PBTTPD/PC₇₁BM structure. Our results provide some understanding of the mechanism behind the structural development of polymer/fullerene blends upon their transitions from solution to the solid state, as well as the key functions of the additive

Quantitative Characterization and Mechanism of Formation of Multilength-scale Bulk Heterojunction Structures in Highly Efficient Solution-Processed Small-Molecule Organic Solar Cells

In this study we used simultaneous grazing-incidence small- and wide-angle X-ray scattering (GISAXS and GIWAXS, respectively) to probe the multilength-scale structures of thin active layers comprising the linear A–D–A-type π-conjugated donor molecule TBDTCNR and the fullerene acceptor molecule PC₆₁BM for use in solution-processed small-molecule-based organic solar cells (SMOSCs). We found that the pseudo-two-dimensional fractal-like networks in the bulk heterojunction (BHJ) structure were determined by mutual interactions between the small-molecule (SM) crystallites and the nanoscale PC₆₁BM clusters during their formation and phase separation, and deduced quantitatively, at multiple length scales, the BHJ structures comprising these SM crystallites and PC₆₁BM clusters. We also conducted in situ GIWAXS measurements to study the temporal behavior and kinetics of SM crystallization from solution to the solid film state. Our GISAXS/GIWAXS study revealed that the multilength-scale BHJ structures in the thin films could be tuned effectively by varying the amount of incorporated PC₆₁BM and the annealing temperature. This study provides fundamental information relating to the mechanism of formation of hierarchical BHJ structures through relatively rapid crystallization of a highly crystalline SM, as well as the relationships among the hierarchical structure, the photovoltaic performance, and the mechanism of formation, thereby allowing greater control over BHJ structures in SMOSCs with optimized fabrication and performance