NS5A inhibitors unmask differences in functional replicase complex half-life between different hepatitis C virus strains

Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOSHCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPORHCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOSH77S.3 and the LPORH77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOSand LPORviruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs

Automated reconstruction of whole-embryo cell lineages by learning from sparse annotations

We present a method to automatically identify and track nuclei in time-lapse microscopy recordings of entire developing embryos. The method combines deep learning and global optimization. On a mouse dataset, it reconstructs 75.8% of cell lineages spanning 1 h, as compared to 31.8% for the competing method. Our approach improves understanding of where and when cell fate decisions are made in developing embryos, tissues, and organs

Automated Reconstruction of Whole-Embryo Cell Lineages by Learning from Sparse Annotations

Abstract We present a method for automated nucleus identification and tracking in time-lapse microscopy recordings of entire developing embryos. Our method combines deep learning and global optimization to enable complete lineage reconstruction from sparse point annotations, and uses parallelization to process multi-terabyte light-sheet recordings, which we demonstrate on three common model organisms: mouse, zebrafish, Drosophila . On the most difficult dataset (mouse), our method correctly reconstructs 75.8% of cell lineages spanning 1 hour, compared to 31.8% for the previous state of the art, thus enabling biologists to determine where and when cell fate decisions are made in developing embryos, tissues, and organs

Narrative vigilance: the analysis of stories in health care

The idea of narrative has been widely discussed in the recent health care literature, including nursing, and has been portrayed as a resource for both clinical work and research studies. However, the use of the term 'narrative' is inconsistent, and various assumptions are made about the nature (and functions) of narrative: narrative as a naive account of events; narrative as the source of 'subjective truth'; narrative as intrinsically fictional; and narrative as a mode of explanation. All these assumptions have left their mark on the nursing literature, and all of them (in our view) are misconceived. Here, we argue that a failure to distinguish between 'narrative' and 'story' is partly responsible for these misconceptions, and we offer an analysis that shows why the distinction between them is essential. In doing so, we borrow the concept of 'narrativity' from literary criticism. Narrativity is something that a text has degrees of, and our proposal is that the elements of narrativity can be 'sorted' roughly into a continuum, at the 'high narrativity' end of which we find 'story'. On our account, 'story' is an interweaving of plot and character, whose organization is designed to elicit a certain emotional response from the reader, while 'narrative' refers to the sequence of events and the (claimed) causal connections between them. We suggest that it is important not to confuse the emotional persuasiveness of the 'story' with the objective accuracy of the 'narrative', and to this end we recommend what might be called 'narrative vigilance'. There is nothing intrinsically authentic, or sacrosanct, or emancipatory, or paradigmatic about narrative itself, even though the recent health care literature has had a marked tendency to romanticize it