VEGF-A and neuropilin 1 (NRP1) shape axon projections in the developing CNS via dual roles in neurons and blood vessels

We thank Vann Bennett and Daizing Zhou (Department of Biochemistry, Duke University Medical Center) for the design and generation of the Brn3bCre knock-in mice. We are grateful to Bennett Alakakone and Susan Reijntjes for help with preliminary experiments and to Anastasia Lampropoulou for preparing tissue culture media. We thank the staff of the Biological Resource Unit at the UCL Institute of Ophthalmology and the University of Aberdeen Institute of Medical Sciences Microscopy and Histology Facility and Medical Research Facility for technical assistance. Funding This research was funded by project grants from the Wellcome Trust [085476/A/08/Z to L.E., C.R.] and Biotechnology and Biological Sciences Research Council (BBSRC) [BB/J00815X/1 to L.E.; BB/J00930X/1 to C.R.] and a Wellcome Trust PhD Fellowship [092839/Z/10/Z to M.T.]. Deposited in PMC for immediate release.Peer reviewedPublisher PD

DSCAM promotes axon fasciculation and growth in the developing optic pathway

Acknowledgments We thank Drs. Robert Burgess, Carol Mason, and Eloisa Herrera for helpful discussions; Dr. Thomas Theil for his invaluable advice on the slice culture methods; Francesca Lamb and Emma Smith for technical assistance; and the Institute of Medical Sciences Microscopy and Imaging Facility for assistance with confocal microscopy. This work was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) doctoral training award studentship and a BBSRC project grant (BB/J00815X/1). Freely available online through the PNAS open access option.Peer reviewedPublisher PD

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium

PURPOSE: To investigate the roles of intracellular signaling elicited by Hedgehog (Hh) ligands in corneal maintenance and wound healing. METHODS: The expression of Hedgehog pathway components in the cornea was assayed by immunohistochemistry, western blot and reverse-transcription polymerase chain reaction (RT-PCR), in wild-type mice and mice that were heterozygous null for the gene encoding the transcription factor, paired box gene 6 (Pax6). Corneal epithelial wound healing and cell migration assays were performed after pharmacological upregulation and downregulation of the hedgehog pathway. Reporter mice, mosaic for expression of the gene encoding β-galactosidase (LacZ), were crossed to Pax6(+/-) mice, mice heterozygous for the gene encoding GLI-Kruppel family member GLI3, and Pax6(+/-)Gli3(+/-) double heterozygotes, to assay patterns of cell migration and corneal epithelial organization in vivo. RESULTS: Corneal epithelial wound healing rates increased in response to application of Sonic hedgehog (Shh), but only in mice with wild-type Pax6 dosage. Downregulation of Hedgehog signalling inhibited corneal epithelial cell proliferation. Pax6(+/-) corneal epithelia showed increased proliferation in response to exogenous Shh, but not increased migration. Desert hedgehog (Dhh) was shown to be the major endogenous ligand, with Shh detectable only by RT-PCR and only after epithelial wounding. The activity of phosphatidylinositol-3-OH kinase-γ (PI3Kγ) was not required for the increased migration response in response to Shh. Nuclear expression of the activator form of the transcription factor Gli3 (which mediates Hh signalling) was reduced in Pax6(+/-) corneal epithelia. Pax6(+/-)Gli3(+/-) double heterozygotes showed highly disrupted patterns of clonal arrangement of cells in the corneal epithelium. CONCLUSIONS: The data show key roles for endogenous Dhh signalling in maintenance and regeneration of the corneal epithelium, demonstrate an interaction between Pax6 and Hh signalling in the corneal epithelium, and show that failure of Hh signalling pathways is a feature of Pax6(+/-) corneal disease that cannot be remedied pharmacologically by addition of the ligands

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Determining the roles of DSCAM and SDK proteins in vertebrate visual system development

Axons are directed along stereotypic pathways to their targets by cues arrayed in the extracellular environment. Identifying the cellular and molecular nature of these signals is of high interest and the developing optic pathway is a useful model system for achieving this. Although previous studies have identified several molecules essential for optic pathway formation, in vivo only subsets of retinal axons rely on them. I focused on the Dscam (Down’s syndrome cell adhesion molecule) and Sidekick (Sdk) cell adhesion molecules for potentially playing crucial roles in this system. In situ hybridisation in the embryonic mouse visual system showed Dscam and Sdk-1 expression in the RGC layer of the retina, along the optic pathway and in the visual targets. Sdk-2 was detected in the glia of the optic nerve and optic chiasm, marking the pathway that RGC axons follow, but not in RGCs. No DscamL1 was detected in RGCs or the optic pathway at the stages investigated and it was discounted from future analysis. In vitro, DSCAM promoted RGC axon outgrowth, whereas SDK 1 was inhibitory. SDK 2 had no effect on RGC axon outgrowth, suggesting it does not play a direct role in their pathfinding. Repeating this assay using retinal explants from the Dscamdel17 mouse mutant, showed that DSCAM enhanced retinal axon outgrowth, at least in part, through homophilic interactions. Analysis of visual system development in Dscam mutants showed DSCAM involvement in RGC axon fasciculation and in enhancing their growth, particularly within the ipsilateral optic tract. Retinal cell counts revealed that DSCAM played diverse roles in controlling cell number. Pre- and postnatal retinas lacking DSCAM contained more RGCs and mitotic cells. Postnatally, Dscam-/- retinas also show decreased cell death. In many cases, defect severity was dose-dependent, with an intermediate phenotype in the heterozygous mice, implicating DSCAM in the neurological defects of Downs’ Syndrome patients.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Use of Coefficient of Variation in Assessing Variability of Quantitative Assays

We have derived the mathematical relationship between the coefficient of variation associated with repeated measurements from quantitative assays and the expected fraction of pairs of those measurements that differ by at least some given factor, i.e., the expected frequency of disparate results that are due to assay variability rather than true differences. Knowledge of this frequency helps determine what magnitudes of differences can be expected by chance alone when the particular coefficient of variation is in effect. This frequency is an operational index of variability in the sense that it indicates the probability of observing a particular disparity between two measurements under the assumption that they measure the same quantity. Thus the frequency or probability becomes the basis for assessing if an assay is sufficiently precise. This assessment also provides a standard for determining if two assay results for the same subject, separated by an intervention such as vaccination or infection, differ by more than expected from the variation of the assay, thus indicating an intervention effect. Data from an international collaborative study are used to illustrate the application of this proposed interpretation of the coefficient of variation, and they also provide support for the assumptions used in the mathematical derivation

Cell autonomy of DSCAM function in retinal development

Cell adhesion molecules (CAMs) provide identifying cues by which neural architecture is sculpted. The Down Syndrome Cell Adhesion Molecule (DSCAM) is required for many neurodevelopmental processes in different species and also has several potential mechanisms of activity, including homophilic adhesion, homophilic repulsion and heterophilic interactions. In the mouse retina, Dscam is expressed in many, but not all neuronal subtypes. Mutations in Dscam cause the fasciculation of dendrites of neighboring homotypic neurons, indicating a role in self-avoidance among cells of a given type, a disruption of the non-random patterning of their cell bodies, and a decrease in developmental cell death in affected cell populations. In order to address how DSCAM facilitates retinal pattering, we developed a conditional allele of Dscam to use alongside existing Dscam mutant mouse strains. Conditional deletion of Dscam reproduces cell spacing, cell number and dendrite arborization defects. Inducible deletion of Dscam and retinal ganglion cell depletion in Brn3b mutant retinas both indicate that these DSCAM-mediated phenotypes can occur independently. In chimeric retinas, in which wild type and Dscam mutant cells are comingled, Dscam mutant cells entangle adjacent wild type cells of the same type, as if both cells were lacking Dscam, consistent with DSCAM-dependent cell spacing and neurite arborization being mediated through homophilic binding cell-to-cell. Deletion of Dscam in specific cell types causes cell-type-autonomous cell body spacing defects, indicating that DSCAM mediates arborization and spacing by acting within given cell types. We also examine the cell autonomy of DSCAM in laminar stratification and find that laminar disorganization can be caused in a non-cell autonomous fashion. Finally, we find Dscam dosage-dependent defects in developmental cell death and amacrine cell spacing, relevant to the increased cell death and other disorders observed in Down syndrome mouse models and human patients, in which Dscam is present in three copies. ► Conditional allele developed to test DSCAM cell autonomy. ► DSCAM‐mediated processes were genetically separated and are distinct. ► Dscam regulates developmental cell death and spacing in a dose dependent manner. ► DSCAM deficient cells induce mutant phenotype on adjacent homotypic wild type cells. ► DSCAM mediates arborization and spacing by acting within cell types

Interaction between hedgehog signalling and PAX6 dosage mediates maintenance and regeneration of the corneal epithelium

Context: Coronal magnetic null points have been implicated as possible locations for localised heating events in 2D models. We investigate this possibility about fully 3D null points. Aims: We investigate the nature of the fast magnetoacoustic wave about a fully 3D magnetic null point, with a specific interest in its propagation, and we look for evidence of MHD mode coupling and/or conversion to the Alfv\'en mode. Methods: A special fieldline and flux-based coordinate system was constructed to permit the introduction of a pure fast magnetoacoustic wave in the vicinity of proper and improper 3D null points. We considered the ideal, {\beta} = 0, MHD equations, which are solved using the LARE3D numerical code. The constituent modes of the resulting wave were isolated and identified using the special coordinate system. Numerical results were supported by analytical work derived from perturbation theory and a linear implementation of the WKB method. Results: An initially pure fast wave is found to be permanently decoupled from the Alfv\'en mode both linearly and nonlinearly for both proper and improper 3D null points. The pure fast mode also generates and sustains a nonlinear disturbance aligned along the equilibrium magnetic field. The resulting pure fast magnetoacoustic pulse has transient behaviour, which is found to be governed by the (equilibrium) Alfv\'en-speed profile, and a refraction effect focuses all the wave energy towards the null point. Conclusions: Thus, the main results from previous 2D work do indeed carry over to the fully 3D magnetic null points andComment: 11 pages, 6 figure

Establishment of Diagnostic Cutoff Points for Levels of Serum Antibodies to Pertussis Toxin, Filamentous Hemagglutinin, and Fimbriae in Adolescents and Adults in the United States

Numerous reports have documented that serologic methods are much more sensitive than culture for the diagnosis of pertussis in adolescents and adults. However, a standardized serologic test for pertussis is not routinely available to most clinicians, and the serologic test levels or cutoff points correlated with diseases have not been determined. The goal of the present study was to examine the distribution of immunoglobulin G (IgG) levels against three Bordetella pertussis antigens (pertussis toxin [PT], filamentous hemagglutinin [FHA], and fimbria types 2 and 3 [FIM]) and to determine population-based antibody levels for the purpose of establishing such diagnostic cutoff points. Enzyme-linked immunosorbent assays (ELISAs) were performed with sera from >6,000 U.S. residents aged 6 to 49 years who participated in the Third National Health and Nutrition Examination Survey. Mixture models were developed to identify hypothesized exposure groups and establish diagnostic cutoffs. Quantifiable (>20 ELISA units/ml [EU]) anti-FHA and anti-FIM IgG antibodies were common (65 and 62% of individuals, respectively), but quantifiable anti-PT IgG antibodies were less frequent (16%). Given the distributions of antibody levels, an anti-PT IgG level of ≥94 EU was proposed as the diagnostic cutoff point. Application of this cutoff point to culture-confirmed illness in a prior study investigating cough illness yielded a high diagnostic sensitivity (80%) and specificity (93%). A standardized ELISA for anti-PT IgG with a single serum sample appears to be useful for the identification of recent B. pertussis infection in adolescents and adults with cough illness. The PT cutoff point will be further evaluated in prospective studies of confirmed B. pertussis infection