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

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6−/−) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100. RAS/RAF/MEK/ERK pathway signaling is known to be frequently activated in cancers, in which it regulates cell growth, malignant transformation, drug resistance, and stemness. Using hepatocellular carcinoma as a model system, Chan et al. describe a mechanism by which this oncogenic signaling pathway is regulated by PRMT6 at the post-translational level via arginine methylation

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation

Summary: Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6−/−) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100. : RAS/RAF/MEK/ERK pathway signaling is known to be frequently activated in cancers, in which it regulates cell growth, malignant transformation, drug resistance, and stemness. Using hepatocellular carcinoma as a model system, Chan et al. describe a mechanism by which this oncogenic signaling pathway is regulated by PRMT6 at the post-translational level via arginine methylation. Keywords: arginine methylation, cancer stemness, tumor-initiating cells, HCC, epigenetic

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

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present