Massive rearrangements of cellular MicroRNA signatures are key drivers of hepatocyte dedifferentiation

Hepatocytes are dynamic cells that, upon injury, can alternate between nondividing differentiated and dedifferentiated proliferating states in vivo . However, in two‐dimensional cultures, primary human hepatocytes (PHHs) rapidly dedifferentiate, resulting in loss of hepatic functions that significantly limits their usefulness as an in vitro model of liver biology, liver diseases, as well as drug metabolism and toxicity. Thus, understanding the underlying mechanisms and stalling of the dedifferentiation process would be highly beneficial to establish more‐accurate and relevant long‐term in vitro hepatocyte models. Here, we present comprehensive analyses of whole proteome and transcriptome dynamics during the initiation of dedifferentiation during the first 24 hours of culture. We report that early major rearrangements of the noncoding transcriptome, hallmarked by increased expression of small nucleolar RNAs, long noncoding RNAs, microRNAs (miRNAs), and ribosomal genes, precede most changes in coding genes during dedifferentiation of PHHs, and we speculated that these modulations could drive the hepatic dedifferentiation process. To functionally test this hypothesis, we globally inhibited the miRNA machinery using two established chemically distinct compounds, acriflavine and poly‐l ‐lysine. These inhibition experiments resulted in a significantly impaired miRNA response and, most important, in a pronounced reduction in the down‐regulation of hepatic genes with importance for liver function. Thus, we provide strong evidence for the importance of noncoding RNAs, in particular, miRNAs, in hepatic dedifferentiation, which can aid the development of more‐efficient differentiation protocols for stem‐cell‐derived hepatocytes and broaden our understanding of the dynamic properties of hepatocytes with respect to liver regeneration. Conclusion: miRNAs are important drivers of hepatic dedifferentiation, and our results provide valuable information regarding the mechanisms behind liver regeneration and possibilities to inhibit dedifferentiation in vitro

Prophage exotoxins enhance colonization fitness in epidemic scarlet fever-causing Streptococcus pyogenes

Abstract: The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins

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

Comparison of Human Primary with Human iPS Cell-Derived Dopaminergic Neuron Grafts in the Rat Model for Parkinson's Disease

Neuronal degeneration within the substantia nigra and the loss of the dopaminergic nigro-striatal pathway are the major hallmarks of Parkinson’s disease (PD). Grafts of foetal ventral mesencephalic (VM) dopaminergic (DA) neurons into the striatum have been shown to be able to restore striatal dopamine levels and to improve overall PD symptoms. However, human foetus-derived cell grafts are not feasible for clinical application. Autologous induced pluripotent stem cell (iPS cell)-derived DA neurons are emerging as an unprecedented alternative. In this review, we summarize and compare the efficacy of human iPS cell-derived DA neuron grafts to restore normal behaviour in a rat model for PD with that of human foetal primary DA neurons. The differences we observed in the efficacy to restore normal function between the 2 types of DA neuron grafts could be ascribed to intrinsic properties of the iPS cell-derived DA neurons that critically affected survival and proper neurite extension in the striatum after implantation