Fibrin clot characteristics and anticoagulant response in a SARS-CoV-2-infected endothelial model.

Coronavirus disease 2019 (COVID-19) patients have increased thrombosis risk. With increasing age, there is an increase in COVID-19 severity. Additionally, adults with a history of vasculopathy have the highest thrombotic risk in COVID-19. The mechanisms of these clinical differences in risk remain unclear. Human umbilical vein endothelial cells (HUVECs) were infected with SARS-CoV-2, influenza A/Singapore/6/86 (H1N1) or mock-infected prior to incubation with plasma from healthy children, healthy adults or vasculopathic adults. Fibrin on surface of cells was observed using scanning electron microscopy, and fibrin characteristics were quantified. This experiment was repeated in the presence of bivalirudin, defibrotide, low-molecular-weight-heparin (LMWH) and unfractionated heparin (UFH). Fibrin formed on SARS-CoV-2 infected HUVECs was densely packed and contained more fibrin compared to mock-infected cells. Fibrin generated from child plasma was the thicker than fibrin generated in vasculopathic adult plasma (p = 0.0165). Clot formation was inhibited by LMWH (0.5 U/ml) and UFH (0.1-0.7 U/ml). We show that in the context of the SARS-CoV-2 infection on an endothelial culture, plasma from vasculopathic adults produces fibrin clots with thinner fibrin, indicating that the plasma coagulation system may play a role in determining the thrombotic outcome of SARS-CoV-2 infection. Heparinoid anticoagulants were most effective at preventing clot formation.

Identifying stem cell numbers and functional heterogeneities during postembryonic organ growth.

Uncovering the number of stem cells necessary for organ growth has been challenging in vertebrate systems. Here, we developed a mathematical model characterizing stem cells in the fish gill, an organ displaying non-exhaustive growth. We employ a Markov model, stochastically simulated via an adapted Gillespie algorithm, and further improved through probability theory. The stochastic algorithm produces a simulated dataset for comparison with experimental clonal data by inspecting quantifiable properties. The analytical approach skips the step of artificial data generation and goes directly to the quantification, being more abstract and efficient. We report that a reduced number of stem cells actively contribute to growing and maintaining the gills. The model also highlights a functional heterogeneity among the stem cells involved, where activation and quiescence phases determine their relative growth contribution. Overall, our work presents a method for inferring the number and properties of stem cells required in a lifelong growing system.

Towards spatio-temporally resolved developmental cardiac gene regulatory networks in zebrafish.

Heart formation in the zebrafish involves a rapid, complex series of morphogenetic events in three-dimensional space that spans cardiac lineage specification through to chamber formation and maturation. This process is tightly orchestrated by a cardiac gene regulatory network (GRN), which ensures the precise spatio-temporal deployment of genes critical for heart formation. Alterations of the timing or spatial localisation of gene expression can have a significant impact in cardiac ontogeny and may lead to heart malformations. Hence, a better understanding of the cellular and molecular basis of congenital heart disease relies on understanding the behaviour of cardiac GRNs with precise spatiotemporal resolution. Here, we review the recent technical advances that have expanded our capacity to interrogate the cardiac GRN in zebrafish. In particular, we focus on studies utilising high-throughput technologies to systematically dissect gene expression patterns, both temporally and spatially during heart development.

Stem cell topography splits growth and homeostatic functions in the fish gill.

While lower vertebrates contain adult stem cells (aSCs) that maintain homeostasis and drive un-exhaustive organismal growth, mammalian aSCs display mainly the homeostatic function. Here, we use lineage analysis in the medaka fish gill to address aSCs and report separate stem cell populations for homeostasis and growth. These aSCs are fate-restricted during the entire post-embryonic life and even during re-generation paradigms. We use chimeric animals to demonstrate that p53 mediates growth coordination among fate-restricted aSCs, suggesting a hierarchical organisation among lineages in composite organs like the fish gill. Homeostatic and growth aSCs are clonal but differ in their topology; modifications in tissue architecture can convert the homeostatic zone into a growth zone, indicating a leading role for the physical niche defining stem cell output. We hypothesise that physical niches are main players to restrict aSCs to a homeostatic function in animals with fixed adult size.

TrawlerWeb: an online de novo motif discovery tool for next-generation sequencing datasets.

BACKGROUND: A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57-74, 2012; Nat 507:462-70, 2014; Nat 507:455-61, 2014; Nat 518:317-30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users. RESULTS: We present TrawlerWeb, a web-based version of the Trawler_standalone tool (Nat Methods 4:563-5, 2007; Nat Protoc 5:323-34, 2010), to allow for the identification of enriched motifs in DNA sequences obtained from next-generation sequencing experiments in order to predict their TFBS composition. TrawlerWeb is designed for online queries with standard options common to web-based motif discovery tools. In addition, TrawlerWeb provides three unique new features: 1) TrawlerWeb allows the input of BED files directly generated from NGS experiments, 2) it automatically generates an input-matched biologically relevant background, and 3) it displays resulting conservation scores for each instance of the motif found in the input sequences, which assists the researcher in prioritising the motifs to validate experimentally. Finally, to date, this web-based version of Trawler_standalone remains the fastest online de novo motif discovery tool compared to other popular web-based software, while generating predictions with high accuracy. CONCLUSIONS: TrawlerWeb provides users with a fast, simple and easy-to-use web interface for de novo motif discovery. This will assist in rapidly analysing NGS datasets that are now being routinely generated. TrawlerWeb is freely available and accessible at: http://trawler.erc.monash.edu.au .

Neural stem cells induce the formation of their physical niche during organogenesis.

Most organs rely on stem cells to maintain homeostasis during post-embryonic life. Typically, stem cells of independent lineages work coordinately within mature organs to ensure proper ratios of cell types. Little is known, however, on how these different stem cells locate to forming organs during development. Here we show that neuromasts of the posterior lateral line in medaka are composed of two independent life-long lineages with different embryonic origins. Clonal analysis and 4D imaging revealed a hierarchical organisation with instructing and responding roles: an inner, neural lineage induces the formation of an outer, border cell lineage (nBC) from the skin epithelium. Our results demonstrate that the neural lineage is necessary and sufficient to generate nBCs highlighting self-organisation principles at the level of the entire embryo. We hypothesise that induction of surrounding tissues plays a major role during the establishment of vertebrate stem cell niches.