Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

A Modified Calculation Improves the Accuracy of Predicted Postoperative Lung Function Values in Lung Cancer Patients

Purpose Preoperative pulmonary function testing is mandatory for non-small cell lung cancer (NSCLC) surgery. The predicted postoperative FEV1 (ppoFEV1) is used for further risk stratification. We compared the ppoFEV1 with the postoperative FEV1 (postFEV1) in order to improve the calculation of the ppoFEV1. Methods 87 patients voluntarily received an FEV1 assessment 1 year after surgery. ppoFEV1 was calculated according to the Brunelli calculation. Baseline characteristics and surgical procedure were compared in a uni- and multivariate analysis between different accuracy levels of the ppoFEV1. Parameters which remained significant in the multinominal regression analysis were evaluated for a modification of the ppoFEV1 calculation. Results Independent factors for a more inaccurate ppoFEV1 were preoperative active smoking (odds ratio (OR) 4.1, confidence interval (CI) 3.6-6.41; p = 0.01), packyears (OR 4.1, CI 3.6-6.41; p = 0.008), younger age (OR 1.1, CI 1.01-1.12; p = 0.03), and patients undergoing pneumectomy (OR 5.55, CI 1.35-23.6; p = 0.01). For the customized ppoFEV1 we excluded pneumonectomies. For patients < 60 years, an additional lung segment was added to the calculation. ppoFEV1 = preFEV1 x 1 - (Lung segments resected+1/Total number of segments). For actively smoking patients with more than 30 packyears we subtracted one lung segment from the calculation ppoFEV1 = PreFEV1 x 1 - (Lung segments resected-1/Total number of segments). Conclusion We were able to enhance the predictability of the ppoFEV1 with modifications. The modified ppoFEV1 (1.828 1 +/- 0.479 1) closely approximates the postFEV1 of 1.823 1 +/- 0.476 1, (0.27%) while the original ppoFEV1 calculation is at 1.78 1 +/- 0.53 (2.19%). However, if patients require pneumectomy, more complex techniques to determine the ppoFEV1 should be included to stratify risk

SHP-1 and FcγRIIB mediate impairment of IgG bead-phagosomes maturation by TDM.

<p>DexTR-labelled WT untreated (A, B and C), SHP-1 siRNA, scramble siRNA-transfected (B) or FcγRIIB KO (C) BMDMs were either treated with 11 μM SHP-1 inhibitor (A) prior incubation with IgG or IgG TDM beads (ratio 1:5) or were directly incubated with beads (B and C) for 30 min. (A, B and C) The graphs indicate quantification by CLSM of the percentage of bead-phagosomes colocalising with dexTR. (B) 2 μg of protein extracts from SHP-1 siRNA or scramble siRNA-transfected BMDMs were assessed for SHP-1 expression by Western blot (loading control: β-actin). Data are expressed as means % of dexTR-positive bead-phagosomes and combined from two (A and B) and three (C) independent experiments done in triplicate ± interquartile ranges (Kruskal-Wallis test followed by Dunns post-test: *<i>p</i><0.05; *** <i>p</i><0.001). Western blot (C) is representative for two independent experiments.</p

Mincle signalling inhibits IgG bead-phagosome maturation.

<p>(A) DexTR-labelled WT and Mincle KO BMDMs were incubated with IgG or IgG TDM (ratio 1:5) for 30 min. In (B), the same procedure as (A) was applied but WT BMDMs were treated with 200nM Syk inhibitor or medium for 30 min, prior to bead addition. The graphs show the quantification by CLSM of the percentage of bead-phagosomes colocalising with dexTR. Data are expressed as medians % of dexTR-positive bead-phagosomes and combined from three independent experiments done in triplicate ± interquartile ranges (Kruskal-Wallis test followed by Dunns post-test: **<i>p</i><0.01).</p

Trehalose dimycolate interferes with FcγR-mediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6’-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5’ phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

International audienceIn 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field