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

Soluble and Bound Hydroxycinnamates in Coffee Pulp (Coffea arabica) from Seven Cultivars at Three Ripening Stages

The contents of soluble and bound hydroxycinnamates (HCAs) were analyzed in coffee pulp (CP) of seven cultivars of Coffea arabica at three different ripening stages. Methodologies for the extraction and analysis of HCAs were evaluated and improved. HCAs were present mainly in the soluble fraction (68–97%). Chlorogenic acid was the main phenolic acid (94–98%) in the soluble fraction, whereas caffeic acid was the most abundant HCA found in the bound fraction (72–88%). Small amounts of free and bound ferulic and <i>p</i>-coumaric acids were also detected. The content of total HCAs in CP reached the maximum concentration at the semiripe stage (7.4–25.5 mg/g CP, dw) but decreased at the ripe stage for six of the seven cultivars. These findings suggest that unripe or semiripe coffee cherries, considered as defective cherries, are a potential inexpensive source of phenolic compounds, such as chlorogenic and caffeic acids

Avelumab First-line Maintenance for Advanced Urothelial Carcinoma: Analysis from JAVELIN Bladder 100 by Duration of First-line Chemotherapy and Interval Before Maintenance

Background: In the JAVELIN Bladder 100 phase 3 trial, avelumab first-line maintenance&nbsp;+&nbsp;best supportive care (BSC) prolonged overall survival (OS) and progression-free survival (PFS) versus BSC alone in patients with advanced urothelial carcinoma (advanced UC) without progression after first-line platinum-based chemotherapy. Objective: To report post hoc analyses of subgroups defined by the duration of first-line chemotherapy and interval before maintenance. Design, setting, and participants: Patients with advanced UC without progression after four to six cycles of platinum-based chemotherapy and a 4-10-wk interval after chemotherapy (n&nbsp;=&nbsp;700) were randomized to receive avelumab&nbsp;+&nbsp;BSC or BSC alone. Subgroups were defined by duration (quartile [Q]) and estimated number of cycles of chemotherapy, and interval between chemotherapy and maintenance. The median follow-up was &gt;19 mo in both arms. Outcome measurements and statistical analysis: OS (primary endpoint), PFS, and safety were assessed. Results and limitations: Hazard ratios (95% confidence interval) for OS with avelumab&nbsp;+&nbsp;BSC versus BSC alone were as follows: by chemotherapy duration-Q3: 0.63 (0.39-1.00); by number of cycles-four cycles: 0.69 (0.48-1.00), five cycles: 0.98 (0.57-1.71), and six cycles: 0.66 (0.47-0.92); and by interval-4-&lt;6 wk: 0.75 (0.54-1.04), 6-&lt;8 wk: 0.67 (0.43-1.06), and 8-10 wk: 0.69 (0.47-1.02). Results were similar for PFS. Safety was similar across subgroups. All analyses were exploratory. Conclusions: Post hoc analyses of OS and PFS in subgroups defined by first-line chemotherapy duration and interval before maintenance were generally consistent with the results in the overall population, with similar safety findings. Prospective trials are warranted to confirm these findings. Patient summary: Avelumab maintenance treatment helped patients with advanced urothelial cancer without disease progression after at least four cycles of prior chemotherapy, and who started maintenance treatment at least 4 wk after chemotherapy, to live longer

Quantum features of a barely bound molecular dopant: Cs2( 3Σu) in bosonic helium droplets of variable size

We present in this work the study of small 4He N-Cs2(3Σu) aggregates (2 ≥ N ≥ 30) through combined variational, diffusion Monte Carlo (DMC), and path integral Monte Carlo (PIMC) calculations. The full surface is modeled as an addition of He-Cs2 interactions and He-He potentials. Given the negligible strength and large range of the He-Cs2 interaction as compared with the one for He-He, a propensity of the helium atoms to pack themselves together, leaving outside the molecular dopant is to be expected. DMC calculations determine the onset of helium gathering at N = 3. To analyze energetic and structural properties as a function of N, PIMC calculations with no bosonic exchange, i.e., Boltzmann statistics, at low temperatures are carried out. At T = 0.1 K, although acceptable one-particle He-Cs2 distributions are obtained, two-particle He-He distributions are not well described, indicating that the proper symmetry should be taken into account. PIMC distributions at T = 1 K already compare well with DMC ones and show minor exchange effects, although binding energies are still far from having converged in terms of the number of quantum beads. As N increases, the He-He PIMC pair correlation function shows a clear tendency to coincide with the experimental boson-liquid helium one at that temperature. It supports the picture of a helium droplet which carries the molecular impurity on its surface, as found earlier for other triplet dimers. © 2011 American Chemical Society.This work has been supported by by DGICYT, Spain, Grant Nos. FIS2007-62006 and FIS2010-18132.Peer Reviewe