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 community approach to mortality prediction in sepsis via gene expression analysis.

Improved risk stratification and prognosis prediction in sepsis is a critical unmet need. Clinical severity scores and available assays such as blood lactate reflect global illness severity with suboptimal performance, and do not specifically reveal the underlying dysregulation of sepsis. Here, we present prognostic models for 30-day mortality generated independently by three scientific groups by using 12 discovery cohorts containing transcriptomic data collected from primarily community-onset sepsis patients. Predictive performance is validated in five cohorts of community-onset sepsis patients in which the models show summary AUROCs ranging from 0.765-0.89. Similar performance is observed in four cohorts of hospital-acquired sepsis. Combining the new gene-expression-based prognostic models with prior clinical severity scores leads to significant improvement in prediction of 30-day mortality as measured via AUROC and net reclassification improvement index These models provide an opportunity to develop molecular bedside tests that may improve risk stratification and mortality prediction in patients with sepsis.y NIGMS Glue Grant Legacy Award R24GM102656. J.F.B.-M., R.A., and E.T. were supported by Instituto de Salud Carlos III (grants EMER07/050, PI13/02110, PI16/01156). R.J.L. was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR001417. The CAPSOD study was supported by NIH (U01AI066569, P20RR016480, HHSN266200400064C). P.K. is supported by grants from Bill Melinda Gates Foundation, R01 AI125197-01, 1U19AI109662, and U19AI057229, outside the submitted work. The GAinS study was supported by the National Institute for Health Research through the Comprehensive Clinical Research Network for patient recruitment; Wellcome Trust (Grants 074318 [to J.C.K.], and 090532/Z/09/Z [core facilities Wellcome Trust Centre for Human Genetics including High-Throughput Genomics Group]); European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013)/ERC Grant agreement no. 281824 (to J.C.K.), the Medical Research Council (98082 [to J.C.K.]); UK Intensive Care Society; and NIHR Oxford Biomedical Research Centre. The Duke HAI study was supported by a research agreement between Duke University and Novartis Vaccines and Diagnostics, Inc. According to the terms of the agreement, representatives of the sponsor had an opportunity to review and comment on a draft of the manuscript. The authors had full control of the analyses, the preparation of the manuscript, and the decision to submit the manuscript for publication. For the University of Florida ‘P50’ Study, data were obtained from the Sepsis and Critically Illness Research Center (SCIRC) at the University of Florida College of Medicine, which is supported in part by NIGMS P50 GM111152. This work was supported by Defense Advanced Research Projects Agency and the Army Research Office through Grant W911NF-15-1-0107.