SERCA directs cell migration and branching across species and germ layers

Branching morphogenesis underlies organogenesis in vertebrates and invertebrates, yet is incompletely understood. Here, we show that the sarco-endoplasmic reticulum Ca2+ reuptake pump (SERCA) directs budding across germ layers and species. Clonal knockdown demonstrated a cell-autonomous role for SERCA in Drosophila air sac budding. Live imaging of Drosophila tracheogenesis revealed elevated Ca2+ levels in migratory tip cells as they form branches. SERCA blockade abolished this Ca2+ differential, aborting both cell migration and new branching. Activating protein kinase C (PKC) rescued Ca2+ in tip cells and restored cell migration and branching. Likewise, inhibiting SERCA abolished mammalian epithelial budding, PKC activation rescued budding, while morphogens did not. Mesoderm (zebrafish angiogenesis) and ectoderm (Drosophila nervous system) behaved similarly, suggesting a conserved requirement for cell-autonomous Ca2+ signaling, established by SERCA, in iterative budding

Developing a core outcome set for the health outcomes for children and adults with congenital oesophageal atresia and/or tracheo-oesophageal fistula: OCELOT task group study protocol

Introduction: Heterogeneity in reported outcomes of infants with oesophageal atresia (OA) with or without tracheo-oesophageal fistula (TOF) prevents effective data pooling. Core outcome sets (COS) have been developed for many conditions to standardise outcome reporting, facilitate meta-analysis and improve the relevance of research for patients and families. Our aim is to develop an internationally-agreed, comprehensive COS for OA-TOF, relevant from birth through to transition and adulthood. Methods and analysis: A long list of outcomes will be generated using (1) a systematic review of existing studies on OA-TOF and (2) qualitative research with children (patients), adults (patients) and families involving focus groups, semistructured interviews and self-reported outcome activity packs. A two-phase Delphi survey will then be completed by four key stakeholder groups: (1) patients (paediatric and adult); (2) families; (3) healthcare professionals; and (4) researchers. Phase I will include stakeholders individually rating the importance and relevance of each long-listed outcome using a 9-point Likert scale, with the option to suggest additional outcomes not already included. During phase II, stakeholders will review summarised results from phase I relative to their own initial score and then will be asked to rescore the outcome based on this information. Responses from phase II will be summarised using descriptive statistics and a predefined definition of consensus for inclusion or exclusion of outcomes. Following the Delphi process, stakeholder experts will be invited to review data at a consensus meeting and agree on a COS for OA-TOF. Ethics and dissemination: Ethical approval was sought through the Health Research Authority via the Integrated Research Application System, registration no. 297026. However, approval was deemed not to be required, so study sponsorship and oversight were provided by Alder Hey Children’s NHS Foundation Trust. The study has been prospectively registered with the COMET Initiative. The study will be published in an open access forum

Developing a core outcome set for the health outcomes for children and adults with congenital oesophageal atresia and/or tracheo-oesophageal fistula:OCELOT task group study protocol

Introduction Heterogeneity in reported outcomes of infants with oesophageal atresia (OA) with or without tracheo-oesophageal fistula (TOF) prevents effective data pooling. Core outcome sets (COS) have been developed for many conditions to standardise outcome reporting, facilitate meta-analysis and improve the relevance of research for patients and families. Our aim is to develop an internationally-agreed, comprehensive COS for OA-TOF, relevant from birth through to transition and adulthood. Methods and analysis A long list of outcomes will be generated using (1) a systematic review of existing studies on OA-TOF and (2) qualitative research with children (patients), adults (patients) and families involving focus groups, semistructured interviews and self-reported outcome activity packs. A two-phase Delphi survey will then be completed by four key stakeholder groups: (1) patients (paediatric and adult); (2) families; (3) healthcare professionals; and (4) researchers. Phase I will include stakeholders individually rating the importance and relevance of each long-listed outcome using a 9-point Likert scale, with the option to suggest additional outcomes not already included. During phase II, stakeholders will review summarised results from phase I relative to their own initial score and then will be asked to rescore the outcome based on this information. Responses from phase II will be summarised using descriptive statistics and a predefined definition of consensus for inclusion or exclusion of outcomes. Following the Delphi process, stakeholder experts will be invited to review data at a consensus meeting and agree on a COS for OA-TOF. Ethics and dissemination Ethical approval was sought through the Health Research Authority via the Integrated Research Application System, registration no. 297026. However, approval was deemed not to be required, so study sponsorship and oversight were provided by Alder Hey Children’s NHS Foundation Trust. The study has been prospectively registered with the COMET Initiative. The study will be published in an open access forum.</p

UK neonatal stoma practice: a population study

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Timing of neonatal stoma closure: a survey of health professional perspectives and current practice

Optimal timing for neonatal stoma closure remains unclear. In this study, we aimed to establish current practice and illustrate multidisciplinary perspectives on timing of stoma closure using an online survey sent to all 27 UK neonatal surgical units, as part of a research programme to determine the feasibility of a clinical trial comparing ‘early’ and ‘late’ stoma closure. 166 responses from all 27 units demonstrated concordance of opinion in target time for closure (6 weeks most commonly stated across scenarios), although there was a high variability in practice. A sizeable proportion (41%) of respondents use weight, rather than time, to determine when to close a neonatal stoma. Thematic analysis of free text responses identified nine key themes influencing decision-making; most related to nutrition, growth and stoma complications. These data provide an overview of current practice that is critical to informing an acceptable trial design.<br/

dPNUTS is a nuclear protein that colocalises with transcriptionally active RNAPII on salivary gland polytene chromosomes.

<p>A) Distribution of <i>dPNUTS</i> transcripts detected by RNA <i>in situ</i> hybridization; <i>dPNUTS</i> transcripts are maternally provided (top left) and are ubiquitously distributed in embryos at cellularisation (top right). At gastrulation, d<i>PNUTS</i> mRNA levels are enriched in the germband and in the fore- and hind-gut (fg and hg, respectively). Later, d<i>PNUTS</i> is highly expressed in the brain (br) and ventral nerve cord (vnc). Embryonic stage and approximate age, hours post fertilization (hpf), are indicated. B) 3^rd instar wing discs stained to reveal the distribution of ectopically expressed Myc-tagged dPNUTS (green in merge), Histone H3S10ph (red in merge, marking mitotic nuclei) and DNA. C) Images of whole mount salivary gland and magnified images of an individual nucleus (below), stained to show the localization of Myc-tagged dPNUTS (green in merge) and DNA (magenta in merge). D) Line scans of images in C) reveal that Myc-tagged dPNUTS is localised to interbands that stain weakly for DNA. Fluorescence intensity of anti-Myc antibody and TOPRO-3 staining was measured along a line through the indicated chromosomal region in the images shown. The profile plot below shows that the peaks of Myc-PNUTS and DNA of staining do not overlap. E) Polytene chromosomes from salivary gland squashes showing that dPNUTS localises to a number of discrete bands that are broadly distributed. F) Merging of the green signal representing dPNUTS with the red signal representing RNAPII Ser2-P (H5) identifies sites where these two proteins co-localize (example indicated with arrow). The relative signals of dPNUTS and RNAPII Ser2-P vary between sites, but the majority dPNUTS loci colocalize with RNAPII Ser2-P staining (star indicates example where only dPNUTS staining is visible).</p

dPNUTS binds to and co-localises with PP1 on chromosomes.

<p>A) Predicted domain structure of dPNUTS and dPNUTS-S proteins, indicating the position of the putative PP1-binding motif (residues 722–726), which is located in the shortest yeast two-hybrid interacting clone (SIC) of dPNUTS. B) Beta-galactosidase assays showing binding of dPNUTS but not dPNUTS-S to all four <i>D. melanogaster</i> PP1 isoforms in the yeast two-hybrid system. C) dPNUTS^WT, but not dPNUTS^W726A, co-precipitates PP1 from nuclear extracts from adult flies. <i>da-GAL4 UAS-HM-dPNUTS^WT</i> and <i>da-GAL4 UAS-HM-dPNUTS^W726A</i> fly extracts were subjected to immunoprecipitation (IP) with Myc antibodies followed by immunoblotting with PP1 antibodies. Blots of total lysates confirmed levels of HM-tagged dPNUTS and PP1. D) dPNUTS and PP1 colocalise at many sites on polytene chromosomes. Inset is an enlarged view of the end of the X chromosome where this is clearly visible. Plot of fluorescence intensity of anti-PP1 and dPNUTS antibody staining, measured along a line through the indicated chromosomal region, reveal the degree of colocalisation between PP1 and dPNUTS.</p