Adult zebrafish show a remarkable capacity to regenerate their spinal column after injury, an ability that stands in stark contrast to the limited repair that occurs within the mammalian CNS ...post-injury. The reasons for this interspecies difference in regenerative capacity remain unclear. Here we demonstrate a novel role for Fgf signaling during glial cell morphogenesis in promoting axonal regeneration after spinal cord injury. Zebrafish glia are induced by Fgf signaling, to form an elongated bipolar morphology that forms a bridge between the two sides of the resected spinal cord, over which regenerating axons actively migrate. Loss of Fgf function inhibits formation of this "glial bridge" and prevents axon regeneration. Despite the poor potential for mammalian axonal regeneration, primate astrocytes activated by Fgf signaling adopt a similar morphology to that induced in zebrafish glia. This suggests that differential Fgf regulation, rather than intrinsic cell differences, underlie the distinct responses of mammalian and zebrafish glia to injury.
Summary Background Perioperative respiratory adverse events (PRAE) are the most common critical incidents in paediatric anaesthesia and occur more often in infants. Use of laryngeal mask airways ...(LMAs) is associated with reduced PRAE compared with endotracheal tubes in older children (>1 year). We aimed to evaluate the effect of these devices on the incidence of PRAE in infants. Methods We did a randomised controlled trial at the Princess Margaret Hospital for Children in Perth (WA, Australia) by recruiting infants (aged 0–12 months) undergoing general (with or without regional or local) anaesthesia with anticipated fentanyl dose 1 μg/kg or lower for minor elective surgery. We excluded patients contraindicated for LMA or endotracheal tube; who had known cardiac disease or airway or thoracic malformations; who were receiving midazolam premedication; who were undergoing airway, thoracic, or abdomen surgery at the time of participation; and if the parents did not speak English. Written parental or guardian consent was obtained before enrolment. Participants were randomly assigned (1:1), by computer-generated variable block randomisation, to receive an LMA (PRO-Breathe, Well Lead Medical Co Ltd, Panyu, China) or an endotracheal tube (Microcuff, Halyard Health Inc, Atlanta, GA, USA). Sealed randomisation envelopes were used to conceal device assignment. An interim analysis was planned once half the number of infants needed (145) had been recruited. The primary outcome was incidence of PRAE, assessed in the intention-to-treat population. The institutional ethics committee at the Princess Margaret Hospital for Children granted ethical approval (1786/EP). The trial is registered with the Australian New Zealand Clinical Trials Registry (ACTRN12610000250033). Findings The trial began on July 8, 2010, and was ended early on May 7, 2015, after the interim analysis results met the study stopping rules. During this time, 239 infants were assessed and 181 eligible infants were randomly assigned to receive an LMA (n=85) or an endotracheal tube (n=95). Four infants were not included in the analysis (two due to cancelled procedures, one did not meet inclusion criteria, and one with missing dataset). In the intention-to-treat analysis, PRAE occurred in 50 (53%) infants in the endotracheal tube group and in 15 (18%) infants in the LMA group (risk ratio RR 2·94, 95% CI 1·79–4·83, p<0·0001). Laryngospasm and bronchospasm (major PRAE) were recorded in 18 (19%) infants in the endotracheal tube group and in three (4%) infants in the LMA group (RR 5·30, 95% CI 1·62–17·35, p=0·002). No deaths were reported. Interpretation In infants undergoing minor elective procedures, LMAs were associated with clinically significantly fewer PRAE and lower occurrence of major PRAE (laryngospasm and bronchospasm) than endotracheal tubes. This difference should be a consideration in airway device selection. Funding Princess Margaret Hospital Foundation, National Health and Australian Medical Research Council, Stan Perron Charitable Trust, and Callahan Estate.
Herein, we leveraged lithium salt doping of linear and cyclic block polymers (BPs) in thin film geometries to demonstrate how BP architecture influences self-assembled nanofeature sizes and ...interfacial widths in nanostructured thin films as a function of segregation strength. To mitigate potential issues with sample-to-sample variability and time-consuming synthesis, we used a single linear BP specimen that was ring-closed to generate an analogous cyclic BP, and we mixed the cyclic and/or parent linear BP with a lithium salt to modulate effective segregation strength (χeff N). Based on X-ray reflectivity analyses, cyclic polystyrene-block-polyoligo(ethylene glycol) methacrylate BP thin film specimens had ∼20% smaller domain spacings than their linear counterparts at all χeff Ns and lower absolute sensitivities to changes in χeff at identical molecular weights. We also report the first direct measurements of interfacial widths in cyclic BP assemblies, which quantitatively demonstrated that interfacial mixing in cyclic BPs was greater relative to linear BPs. Furthermore, the trends in domain characteristics with increasing salt concentration qualitatively agreed with results from molecular dynamics (MD) simulations with increasing χ, despite the fact that salt species were not explicitly included in the MD simulations. Our results underscore the utility of lithium salt doping to explore the BP phase behavior of synthetically challenging macromolecules and demonstrate key architecture/segregation strength relationships in cyclic BP thin films, which provides useful information to further evaluate cyclic BP suitability for nanoscale patterning and templating applications.
The embryonic notochord is a flexible structure present during development that serves as scaffold for formation of the vertebrate spine. This rod-like organ is thought to have evolved in ...non-vertebrate chordates to facilitate locomotion by providing a rigid but flexible midline structure against which the axial muscles can contract. This hydrostatic “skeleton” is exposed to a variety of mechanical forces during oscillation of the body. There is evidence that caveolae, submicroscopic cup-shaped plasma membrane pits, can buffer tension in cells that undergo high levels of mechanical stress. Indeed, caveolae are particularly abundant in the embryonic notochord. In this study, we used the CRISPR/Cas9 system to generate a mutant zebrafish line lacking Cavin1b, a coat protein required for caveola formation. Our cavin1b−/− zebrafish line exhibits reduced locomotor capacity and prominent notochord lesions characterized by necrotic, damaged, and membrane-permeable cells. Notochord diameter and body length are reduced, but remarkably, the mutants recover and are homozygous viable. By manipulating mechanical stress using a number of different assays, we show that progression of lesion severity in the mutant notochord is directly dependent on locomotion. We also demonstrate changes in caveola morphology in vivo in response to mechanical stress. Finally, induction of a catastrophic collapse of live cavin1b−/− mutant notochord cells provides the first real-time observation of caveolae mediating cellular mechanoprotection.
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•Zebrafish Cavin1b deletion induces notochord lesions and locomotor defects•Notochord lesions are damaged and membrane-permeable cells•The remaining cavin1b mutant caveolae are morphologically dysmorphic•Mechanical stress leads to cavin1b mutant notochord cell collapse
The notochord cell membrane is characterized by an abundance of caveolae. Lim et al. show that caveolae and Cavin1b, a coat protein required for notochord caveola formation, mediate embryonic notochord cell fragility during zebrafish early locomotion. These findings suggest a role for caveolae in conveying notochord cell mechanoprotection.
We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable ...GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
One of the central questions of developmental biology is how cells of equivalent potential-an equivalence group-come to adopt specific cellular fates. In this study we have used a combination of live ...imaging, single cell lineage analyses, and perturbation of specific signaling pathways to dissect the specification of the adaxial cells of the zebrafish embryo. We show that the adaxial cells are myogenic precursors that form a cell fate equivalence group of approximately 20 cells that consequently give rise to two distinct sub-types of muscle fibers: the superficial slow muscle fibers (SSFs) and muscle pioneer cells (MPs), distinguished by specific gene expression and cell behaviors. Using a combination of live imaging, retrospective and indicative fate mapping, and genetic studies, we show that MP and SSF precursors segregate at the beginning of segmentation and that they arise from distinct regions along the anterior-posterior (AP) and dorsal-ventral (DV) axes of the adaxial cell compartment. FGF signaling restricts MP cell fate in the anterior-most adaxial cells in each somite, while BMP signaling restricts this fate to the middle of the DV axis. Thus our results reveal that the synergistic actions of HH, FGF, and BMP signaling independently create a three-dimensional (3D) signaling milieu that coordinates cell fate within the adaxial cell equivalence group.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Skeletal muscle is an example of a tissue that deploys a self-renewing stem cell, the satellite cell, to effect regeneration. Recent in vitro studies have highlighted a role for asymmetric divisions ...in renewing rare "immortal" stem cells and generating a clonal population of differentiation-competent myoblasts. However, this model currently lacks in vivo validation. We define a zebrafish muscle stem cell population analogous to the mammalian satellite cell and image the entire process of muscle regeneration from injury to fiber replacement in vivo. This analysis reveals complex interactions between satellite cells and both injured and uninjured fibers and provides in vivo evidence for the asymmetric division of satellite cells driving both self-renewal and regeneration via a clonally restricted progenitor pool.
IMPORTANCE: Tonsillectomy is a common pediatric procedure for the treatment of sleep-disordered breathing and chronic tonsillitis. Up to half of children having this procedure experience a ...perioperative respiratory adverse event. OBJECTIVE: To determine whether inhaled albuterol sulfate (salbutamol sulfate) premedication decreases the risk of perioperative respiratory adverse events in children undergoing anesthesia for tonsillectomy. DESIGN, SETTING, AND PARTICIPANTS: A randomized, triple-blind, placebo-controlled trial (the Reducing Anesthetic Complications in Children Undergoing Tonsillectomies REACT trial) was conducted at Perth Children’s Hospital (formerly Princess Margaret Hospital for Children), the only tertiary pediatric hospital in Western Australia. Participants included 484 children aged 0 to 8 years who were undergoing anesthesia for tonsillectomy. The study was conducted between July 15, 2014, and May 18, 2017. INTERVENTIONS: Participants were randomized to receive either albuterol (2 actuations, 200 μg) or placebo before their surgery. MAIN OUTCOMES AND MEASURES: Occurrence of perioperative respiratory adverse events (bronchospasm, laryngospasm, airway obstruction, desaturation, coughing, and stridor) until discharge from the postanesthesia care unit. RESULTS: Of 484 randomized children (median range age, 5.6 1.6-8.9 years; 285 58.9% boys), 479 data sets were available for intention-to-treat analysis. Perioperative respiratory adverse events occurred in 67 of 241 children (27.8%) receiving albuterol and 114 of 238 children (47.9%) receiving placebo. After adjusting for age, type of airway device, and severity of obstructive sleep apnea in a binary logistic regression model, the likelihood of perioperative respiratory adverse events remained significantly higher in the placebo group compared with the albuterol group (odds ratio, 2.8; 95% CI, 1.9-4.2; P < .001). Significant differences were seen in children receiving placebo vs albuterol in laryngospasm (28 11.8% vs 12 5.0%; P = .009), coughing (79 33.2% vs 27 11.2%; P < .001), and oxygen desaturation (54 22.7% vs 36 14.9%; P = .03). CONCLUSIONS AND RELEVANCE: Albuterol premedication administered before tonsillectomy under general anesthesia in young children resulted in a clinically significant reduction in rates of perioperative respiratory adverse events compared with the rates in children who received placebo. Premedication with albuterol should be considered for children undergoing tonsillectomy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry identifier: ACTRN12614000739617
Self-assembly is one of the most promising strategies for making functional materials at the nanoscale, yet new design principles for making self-limiting architectures, rather than spatially ...unlimited periodic lattice structures, are needed. To address this challenge, we explore the tradeoffs between addressable assembly and self-closing assembly of a specific class of self-limiting structures: cylindrical tubules. We make triangular subunits using DNA origami that have specific, valence-limited interactions and designed binding angles, and we study their assembly into tubules that have a self-limited width that is much larger than the size of an individual subunit. In the simplest case, the tubules are assembled from a single component by geometrically programming the dihedral angles between neighboring subunits. We show that the tubules can reach many micrometers in length and that their average width can be prescribed through the dihedral angles. We find that there is a distribution in the width and the chirality of the tubules, which we rationalize by developing a model that considers the finite bending rigidity of the assembled structure as well as the mechanism of self-closure. Finally, we demonstrate that the distributions of tubules can be further sculpted by increasing the number of subunit species, thereby increasing the assembly complexity, and demonstrate that using two subunit species successfully reduces the number of available end states by half. These results help to shed light on the roles of assembly complexity and geometry in self-limited assembly and could be extended to other self-limiting architectures, such as shells, toroids, or triply periodic frameworks.
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