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

Standardized chemical synthesis of Pseudomonas aeruginosa pyocyanin

Preparation of the toxin pyocyanin from the bacterium Pseudomonas aeruginosa is an exacting procedure. Pyocyanin is expensive to commercially purchase. The sellers do not give out the extraction procedure. Classically, pyocyanin preparation involves complicated multi-step P. aeruginosa culturing and solvent transfer extractions. The chemical synthesis first used (1979) has not been adequately described. We devised an easily reproducible protocol which consistently decreases the time taken for synthesis, extraction and purification of pyocyanin, and increases the pure pyocyanin proportion produced. Our procedure:Involves more purification steps (chloroform/methanol/acidification/ alkalinization).Starts with a different pH (7.4 instead of 7), and lesser concentration of phenazine methosulfate; and retrenches a rotary evaporation step.Removes 2 lyophilization steps, and entails different solvent proportions for thin layer chromatography. As we have extracted pyocyanin both from P. aeruginosa cultures, and via chemical synthesis; we know the procedural and product-quality differences. We endorse the relative ease, safety, and convenience of using the chemical synthesis described here. Crucially, our "naturally endotoxin-free" pyocyanin can be extracted easily without using infectious bacteria. © 2014 Elsevier Ltd. All rights reserved

Th17 pathway suppression by appendicitis and appendectomy protects against colitis

Volume: 26 Special Issue: SI Supplement:

A comprehensive evaluation of bladder cancer epidemiology and outcomes in Australia

OBJECTIVE: To review bladder cancer statistics and management in Australia and identify gaps for future work here. METHODS: Evidence was reviewed from GLOBOCAN 2008v2.0, Pubmed, and conference presentations. We also use data from reports from Cancer Council Australia, State Cancer Councils, and Australian Institute of Health and Welfare. RESULTS: The incidence and mortality rates of bladder cancer in Australia closely parallel those of other developed countries. Bladder cancer was the 8th most common cause of cancer in men, and the 17th most common cause of cancer in women. Bladder cancer was the 13th most common cause of cancer death in men, and the 17th most common cause of cancer death in women. We briefly review the evidence regarding causality, including nutritional, occupational, and environmental factors. We compare Australian incidence and mortality rates internationally, by state/territory, by socioeconomic strata, and by geographical regions. Importantly, we review evidence on the quality of bladder cancer management in Australia. CONCLUSIONS: The geographical, regional, and socioeconomic differences in Australian bladder cancer statistics may be associated with different patterns of diagnosis and treatment. IMPLICATIONS: The quality of bladder cancer surveillance and cystectomies in Australia requires improvement to conform to global standards and to improve decreasing survival rate

Poor survival of females with bladder cancer is limited to those aged 70 years or over: a population-wide linkage study, New South Wales, Australia

Abstract Although men are diagnosed with bladder cancer (BC) with a rate three times higher than women, women experience poorer survival. The cause of this gender difference is not clear. The aim of this study was to investigate the discrepancy in survival from BC by gender and explore potential explanations for the difference using a population-wide linkage study. Using the New South Wales (NSW) Central Cancer Registry, all invasive BC cases diagnosed between 2001 and 2009 were identified. Records were linked to the NSW Admitted Patient Data Collection (APDC), to retrieve treatment details, and to the Registry of Births Deaths and Marriages and Australian Bureau of Statistics to obtain death details. A total of 5377 new cases of BC were identified. No differences were identified in the proportions of patients presenting at different stages between genders. However, disease-specific survival (DSS) was worse for females compared to males with localized and regional disease (P < 0.05). This difference was only apparent in individuals aged ≥70 years and no difference was identified in those younger. Multivariable Cox-regression analysis of the cohort of individuals aged ≥70 years revealed that stage, age, comorbidity, and sex remained independent variables (P < 0.05) predicting DSS. In a population wide analysis, females aged 70 years or more suffer worse DSS compared to males. The differences are not accounted for by stage at presentation or comorbidity and are independent of age. BC in postmenopausal females may be biologically more aggressive

Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione

Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37°C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O 2-dependent. The formation of H2O2 as an intermediate and the thiol group in GSH seem to be important in this reaction

The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells

10.1111/liv.12231Liver International3391386-1397LIIN