Behavioral immune landscapes of inflammation.

Transcriptional or proteomic profiling of individual cells have revolutionized interpretation of biological phenomena by providing cellular landscapes of healthy and diseased tissues. These approaches, however, fail to describe dynamic scenarios in which cells can change their biochemical properties and downstream “behavioral” outputs every few seconds or minutes. Here, we used 4D live imaging to record tens to hundreds of morpho-kinetic parameters describing the dynamism of individual leukocytes at sites of active inflammation. By analyzing over 100,000 reconstructions of cell shapes and tracks over time, we obtained behavioral descriptors of individual cells and used these high-dimensional datasets to build behavioral landscapes. These landscapes recognized leukocyte identities in the inflamed skin and trachea, and inside blood vessels uncovered a continuum of neutrophil states, including a large, sessile state that was embraced by the underlying endothelium and associated with pathogenic inflammation. Behavioral in vivo screening of thousands of cells from 24 different mouse mutants identified the kinase Fgr as a driver of this pathogenic state, and genetic or pharmacological interference of Fgr protected from inflammatory injury. Thus, behavioral landscapes report unique biological properties of dynamic environments at high cellular, spatial and temporal resolution.pre-print4302 K

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

VALIDITY OF THE MODIFIED CONCONI TEST FOR DETERMINING VENTILATORY THRESHOLD DURING ON-WATER ROWING

The objectives of this study were to design a field test based on the Conconi protocol to determine the ventilatory threshold of rowers and to test its reliability and validity. A group of sixteen oarsmen completed a modified Conconi test for on-water rowing. The reliability of the detection of the heart rate threshold was evaluated using heart rate breaking point in the Conconi test and retest. Heart rate threshold was detected in 88.8% of cases in the test-retest. The validity of the modified Conconi test was evaluated by comparing the heart rate threshold data acquired with that obtained in a ventilatory threshold test (VT2). No significant differences were found for the values of different intensity parameters i.e. heart rate (HR), oxygen consumption (VO2), stroke rate (SR) and speed (S) between the heart rate threshold and the ventilatory threshold, (170.9 ± 6.8 vs. 169.3 ± 6.4 beats·min-1; 42.0 ± 8.6 vs. 43.5 ± 8.3 ml·kg-1·min-1; 25.8 ± 3.3 vs. 27.0 ± 3.2 strokes·min-1 and 14.4 ± 0.8 vs. 14.6 ± 0.8 km·h-1). The differences in averages obtained in the Conconi test-retest were small with a low standard error of the mean. The reliability data between the Conconi test-retest showed low coefficients of variations (CV) and high intraclass correlation coefficients (ICC). The total errors for the Conconi test-retest are low for the measured variables (1.31 HR, 0.87 VO2, 0.65 SR, and 0.1 S). The Bland- Altman's method for analysis validity showed a strong concordance according to the analyzed variables. We conclude that the modified Conconi test for on-water rowing is a valid and reliable method for the determination of the second ventilatory threshold (VT2)