PKR deficiency alters E. coli-induced sickness behaviors but does not exacerbate neuroimmune responses or bacterial load

published_or_final_versio

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Retinal Microvascular Abnormalities Predict Clinical Outcomes in Patients with Heart Failure

Background: Narrower retinal arterioles and wider retinal venules have been associated with the incidence of heart failure (HF). However, whether they are predictive of the prognosis of heart failure (HF) is unclear. We aimed to explore the role of retinal vessel calibers in predicting long-term clinical outcomes of HF. Methods: This is a prospective, single-center, observational study that surveyed patients in a tertiary referral hospital for the treatment of HF. Retinal vessel caliber was graded using retinal photography. The primary endpoint was the composite endpoint of HF rehospitalization and mortality at 12 months. Results: There were 55 patients with chronic HF included in the final analysis. At 12 months, the cumulative incidence of the primary endpoint, HF rehospitalization, and mortality tended to be higher with the widening of the central retinal venular equivalent (CRVE) (p for non-linearity = 0.059) and was significantly increased when CRVE reached a cut-off value (283 μm) (p = 0.011) following adjustment for age, sex, etiology of HF, and diabetes. No association between the central retinal arteriolar equivalent (CRAE) and arteriolar-to-venular caliber ratio (AVR) was found with the clinical outcome in both univariable and multivariable Cox regression. CRAE, CRVE, and AVR had no relationship with the concentration of the N-terminal pro-B-type natriuretic peptide. In addition, CRVE was not associated with cardiac diastolic and systolic function. Conclusions: When the retinal venular caliber widens to a certain point, the composite incidence of HF rehospitalization and mortality significantly increase, suggesting retinal vessel caliber imaging may provide insight into the development of HF

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