Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these ...progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.
Tracking the progeny of single cells is necessary for building lineage trees that recapitulate processes such as embryonic development and stem cell differentiation. In classical lineage tracing ...experiments, cells are fluorescently labelled to allow identification by microscopy of a limited number of cell clones. To track a larger number of clones in complex tissues, fluorescent proteins are now replaced by heritable DNA barcodes that are read using next-generation sequencing. In prospective lineage tracing, unique DNA barcodes are introduced into single cells through genetic manipulation (using, for example, Cre-mediated recombination or CRISPR-Cas9-mediated editing) and tracked over time. Alternatively, in retrospective lineage tracing, naturally occurring somatic mutations can be used as endogenous DNA barcodes. Finally, single-cell mRNA-sequencing datasets that capture different cell states within a developmental or differentiation trajectory can be used to recapitulate lineages. In this Review, we discuss methods for prospective or retrospective lineage tracing and demonstrate how trajectory reconstruction algorithms can be applied to single-cell mRNA-sequencing datasets to infer developmental or differentiation tracks. We discuss how these approaches are used to understand cell fate during embryogenesis, cell differentiation and tissue regeneration.
Within the extensive range of self-propagating pathologic protein aggregates of mammals, prions are the most clearly infectious (e.g., ∼109 lethal doses per milligram). The structures of such lethal ...assemblies of PrP molecules have been poorly understood. Here we report a near-atomic core structure of a brain-derived, fully infectious prion (263K strain). Cryo-electron microscopy showed amyloid fibrils assembled with parallel in-register intermolecular β sheets. Each monomer provides one rung of the ordered fibril core, with N-linked glycans and glycolipid anchors projecting outward. Thus, single monomers form the templating surface for incoming monomers at fibril ends, where prion growth occurs. Comparison to another prion strain (aRML) revealed major differences in fibril morphology but, like 263K, an asymmetric fibril cross-section without paired protofilaments. These findings provide structural insights into prion propagation, strains, species barriers, and membrane pathogenesis. This structure also helps frame considerations of factors influencing the relative transmissibility of other pathologic amyloids.
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•Cryo-EM reveals parallel in-register structure for an infectious brain-derived prion•N-linked glycans and GPI anchor project outward from the fibril core•Comparison to another prion strain reveals distinct conformational templates•In silico modeling suggests a structural basis for a prion transmission barrier
Kraus et al. provide a near-atomic-resolution structure of a brain-derived prion solved by cryo-electron microscopy. The parallel in-register assembly of prion protein monomers within the prion fibril, together with observed strain-dependent differences in fibril morphology, provide a structural foundation for understanding prion replication, strains, species barriers, and membrane pathogenesis.
The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates ...using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.
Haematopoietic stem cells (HSCs) are generated from haemogenic endothelial (HE) cells via the formation of intra-aortic haematopoietic clusters (IAHCs) in vertebrate embryos. The molecular events ...controlling endothelial specification, endothelial-to-haematopoietic transition (EHT) and IAHC formation, as it occurs in vivo inside the aorta, are still poorly understood. To gain insight in these processes, we performed single-cell RNA-sequencing of non-HE cells, HE cells, cells undergoing EHT, IAHC cells, and whole IAHCs isolated from mouse embryo aortas. Our analysis identified the genes and transcription factor networks activated during the endothelial-to-haematopoietic switch and IAHC cell maturation toward an HSC fate. Our study provides an unprecedented complete resource to study in depth HSC generation in vivo. It will pave the way for improving HSC production in vitro to address the growing need for tailor-made HSCs to treat patients with blood-related disorders.
Abstract Background context Spine surgery is usually associated with large amount of blood loss, necessitating blood transfusions. Blood loss-associated morbidity can be because of direct risks, such ...as hypotension and organ damage, or as a result of blood transfusions. The antifibrinolytic, tranexamic acid (TXA), is a lysine analog that inhibits activation of plasminogen and has shown to be beneficial in reducing surgical blood loss. Purpose To consolidate the findings of randomized controlled trials (RCTs) investigating the use of TXA on surgical bleeding in spine surgery. Study design A metaanalysis. Study sample Randomized controlled trials investigating the effectiveness of intravenous TXA in reducing blood loss in spine surgery, compared with a placebo/no treatment group. Methods MEDLINE, Embase, Cochrane controlled trials register, and Google Scholar were used to identify RCTs published before January 2014 that examined the effectiveness of intravenous TXA on reduction of blood loss and blood transfusions, compared with a placebo/no treatment group in spine surgery. Metaanalysis was performed using RevMan 5. Weighted mean difference with 95% confidence intervals was used to summarize the findings across the trials for continuous outcomes. Dichotomous data were expressed as risk ratios with 95% confidence intervals. A p<.05 was considered statistically significant. Results Eleven RCTs were included for TXA (644 total patients). Tranexamic acid reduced intraoperative, postoperative, and total blood loss by an average of 219 mL (−322, −116, p<.05), 119 mL (−141, −98, p<.05), and 202 mL (−299, −105, p<.05), respectively. Tranexamic acid led to a reduction in proportion of patients who received a blood transfusion (risk ratio 0.67 0.54, 0.83, p<.05) relative to placebo. There was one myocardial infarction (MI) in the TXA group and one deep vein thrombosis (DVT) in placebo. Conclusions Tranexamic acid reduces surgical bleeding and transfusion requirements in patients undergoing spine surgery. Tranexamic acid does not appear to be associated with an increased incidence of pulmonary embolism, DVT, or MI.
This volume introduces two of the earliest writings about Vietnam to appear in the English language. The reports come from narrators with different interests who are viewing different parts of ...Vietnam at an early stage of European involvement in the region. This volume introduces two of the earliest writings about Vietnam to appear in the English language. The reports come from narrators with different interests who are viewing different parts of Vietnam at an early stage of European involvement in the region.
The prion (infectious protein) concept has evolved with the discovery of new self-propagating protein states in organisms as diverse as mammals and fungi. The infectious agent of the mammalian ...transmissible spongiform encephalopathies (TSE) has long been considered the prototypical prion, and recent cell-free propagation and biophysical analyses of TSE infectivity have now firmly established its prion credentials. Other disease-associated protein aggregates, such as some amyloids, can also have prion-like characteristics under certain experimental conditions. However, most amyloids appear to lack the natural transmissibility of TSE prions. One feature that distinguishes the latter from the former is the glycophosphatidylinositol membrane anchor on prion protein, the molecule that is corrupted in TSE diseases. The presence of this anchor profoundly affects TSE pathogenesis, which involves major membrane distortions in the brain, and may be a key reason for the greater neurovirulence of TSE prions relative to many other autocatalytic protein aggregates.
Brace treatment is the most common nonoperative treatment for the prevention of curve progression in adolescent idiopathic scoliosis. The success reported in level 1 and 2 clinical trials is ...approximately 75%. The aim of this review was to identify the main risk factors that significantly reduce success rate of brace treatment.
A literature search using the MEDLINE and Embase databases was conducted. Studies were included if they identified specific risk factor(s) for curve progression. Studies that looked at nighttime braces, superiority of one type of brace over another, the effect of physical therapy on brace performance, cadaver or nonhuman studies were excluded. A total of 1,022 articles were identified of which 25 met all of the inclusion criteria. Seven risk factors were identified: Poor brace compliance (eight studies), lack of skeletal maturity (six studies), Cobb angle over a certain threshold (six studies), poor in-brace correction (three studies), vertebral rotation (four studies), osteopenia (two studies), and thoracic curve type (two studies). Three risk factors were highly repeated in the literature which identified specific subgroups of patients who have a much higher risk to fail brace treatment and to progress to fusion. This data demonstrates that 60% to 70% of the patients referred to bracing are Risser 0 and 30% to 70% of this group will not wear the brace enough to ensure treatment efficacy. Furthermore, Risser 0 patients who reach the accelerated growth phase with a curve ≥40° are at 70% to 100% risk of curve progression to the fusion surgical threshold despite proper brace wear. Skeletally immature patients with relatively large magnitude scoliosis who are noncompliant are at a higher risk of failing brace treatment.
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