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

Chronic impact of tetracycline on the biodegradation of an organic substrate mixture under anaerobic conditions

The study evaluates the chronic impact of the antibiotic tetracycline on the biodegradation of organic substrate under anaerobic conditions. The experiments involved an anaerobic sequencing batch reactor fed with a synthetic substrate mixture including glucose, starch and volatile fatty acids, and operated in a sequence of different phases with gradually increasing tetracycline doses of 1.65–8.5 mg/L, for more than five months. Tetracycline exerted a terminal/lethal effect at 8.5 mg/L on the microbial community under anaerobic conditions, which caused the inhibition of substrate/COD utilization and biogas generation and leading to a total collapse of the reactor. The microbial activity could not be recovered and re-started within a period of more than 10 days, even after stopping tetracycline dosing. At lower doses, substrate utilization was not affected but a reduction of 10–20% was observed in the biogas/methane generation, suggesting that substrate utilization of tetracycline to the biomass was limiting their bioavailability. During the experiments, tetracycline was partially removed either through biodegradation or conversion into its by-products. The adverse long-term impact was quite variable for fermenting heterotrophic and methanogenic fractions of the microbial community based on changes inflicted on the composition of remaining/residual organic substrate.This study was funded by The Turkish Academy of Sciences (TUBA). It was also this work was partly supported by the Generalitat de Catalunya (Consolidated Research Group: Water and Soil Quality Unit 2009-SGR-965).Peer reviewe

WASTE-WATER MANAGEMENT FOR ISTANBUL - BASIS FOR TREATMENT AND DISPOSAL

A number of studies have so far been conducted to assess the magnitude of pollution generated by domestic and industrial sources in the Istanbul Metropolitan Area. They indicate that a management scheme for wastewater treatment and disposal should involve a total discharge of 15.4 m3 s-1 with a potential pollution load of 330 tons BOD5 day-1 for 1990. A scheme of this magnitude inevitably requires careful evaluation of receiving water characteristics, both from an oceanographic and a quality standpoint

Pollution profile and biodegradation characteristics of fur‐suede processing effluents

This study investigated the effect of stream segregation on the biodegradation characteristics of wastewaters generated by fur-suede processing. It was conducted on a plant located in an organized industrial district in Turkey. A detailed in-plant analysis of the process profile and the resulting pollution profile in terms of significant parameters indicated the characteristics of a strong wastewater with a maximum total COD of 4285 mg L-1, despite the excessive wastewater generation of 205 m(3) (ton skin)(-1). Respirometric analysis by model calibration yielded slow biodegradation kinetics and showed that around 50% of the particulate organics were utilized at a rate similar to that of endogenous respiration. A similar analysis on the segregated wastewater streams suggested that biodegradation of the plant effluent is controlled largely by the initial washing/pickling operations. The effect of other effluent streams was not significant due to their relatively low contribution to the overall organic load. The respirometric tests showed that the biodegradation kinetics of the joint treatment plant influent of the district were substantially improved and exhibited typical levels reported for tannery wastewater, so that the inhibitory impact was suppressed to a great extent by dilution and mixing with effluents of the other plants. The chemical treatment step in the joint treatment plant removed the majority of the particulate organics so that 80% of the available COD was utilized in the oxygen uptake rate (OUR) test, a ratio quite compatible with the biodegradable COD fractions of tannery wastewater. Consequently, process kinetics and especially the hydrolysis rate appeared to be significantly improved.Corlu Leather Industrial Organized DistrictThe support provided the management of Corlu Leather Industrial Organized District for the study and the technical assistance of Professor Ozlem Karahan and Asli Ciggin in the experimental work are gratefully acknowledged

p53 represses autophagy in a cell cycle-dependent fashion

: Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-alpha), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-X(L) with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed