Ultrasound Directed Reduction of Colles' type distal radial fractures in ED (UDiReCT): a feasibility randomised controlled trial.

BACKGROUND: There is a high rate of surgical fixation of displaced Colles' type distal radial wrist fractures despite fracture manipulation in the ED. Point-of-care ultrasound has been used to guide ED manipulations but its effect on the quality of fracture reduction or subsequent need for surgical fixation is unknown. This study aims to assess the feasibility of conducting a definitive randomised controlled trial to assess the use of ultrasound to guide these fracture manipulations. METHODS: We conducted a pragmatic randomised controlled feasibility trial in two EDs in England over a 6-month period (7 October 2019 to 6 April 2020). Adult patients with wrist fractures undergoing manipulation in the ED were randomised 1:1 to ultrasound-guided distal radial fracture manipulation or manipulation with sham ultrasound. The primary outcome for this study was trial recruitment rate. Other measures were recorded to assess potential future definitive trial outcomes and feasibility. RESULTS: Of 120 patients meeting inclusion criteria, 48 (40%) were recruited and randomised in the two centres, giving overall recruitment rates of 0.3 and 1.8 participants per week at each site, respectively, and 1 participant per week overall. The most common reason that patients were not included was research staff availability. After 6 weeks, six patients in each group (26% intervention, 24% control) had undergone surgical fixation, with 98% data completeness for this potential definitive trial primary outcome. Randomisation, blinding and data collection processes were effective but there were data limitations in the X-ray assessment of fracture positions. CONCLUSION: A definitive study of a similar design would be feasible within UK ED practice but organisational factors and research staff availability should be considered when estimating the predicted recruitment rate and required sites. 6-week surgical fixation rate was the most reliable outcome measure. TRIAL REGISTRATION: ClinicalTrials.gov (NCT03868696).

The E3 ubiquitin-protein ligase Rbx1 regulates cardiac wall morphogenesis in zebrafish.

Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis.

Endothelial ontogeny and the establishment of vascular heterogeneity.

The establishment of distinct cellular identities was pivotal during the evolution of Metazoa, enabling the emergence of an array of specialized tissues with different functions. In most animals including vertebrates, cell specialization occurs in response to a combination of intrinsic (e.g., cellular ontogeny) and extrinsic (e.g., local environment) factors that drive the acquisition of unique characteristics at the single-cell level. The first functional organ system to form in vertebrates is the cardiovascular system, which is lined by a network of endothelial cells whose organ-specific characteristics have long been recognized. Recent genetic analyses at the single-cell level have revealed that heterogeneity exists not only at the organ level but also between neighboring endothelial cells. Thus, how endothelial heterogeneity is established has become a key question in vascular biology. Drawing upon evidence from multiple organ systems, here we will discuss the role that lineage history may play in establishing endothelial heterogeneity.

An improved zebrafish transcriptome annotation for sensitive and comprehensive detection of cell type-specific genes.

The zebrafish is ideal for studying embryogenesis and is increasingly applied to model human disease. In these contexts, RNA-sequencing (RNA-seq) provides mechanistic insights by identifying transcriptome changes between experimental conditions. Application of RNA-seq relies on accurate transcript annotation for a genome of interest. Here, we find discrepancies in analysis from RNA-seq datasets quantified using Ensembl and RefSeq zebrafish annotations. These issues were due, in part, to variably annotated 3' untranslated regions and thousands of gene models missing from each annotation. Since these discrepancies could compromise downstream analyses and biological reproducibility, we built a more comprehensive zebrafish transcriptome annotation that addresses these deficiencies. Our annotation improves detection of cell type-specific genes in both bulk and single cell RNA-seq datasets, where it also improves resolution of cell clustering. Thus, we demonstrate that our new transcriptome annotation can outperform existing annotations, providing an important resource for zebrafish researchers.

Carpal tunnel decompression in patients with normal nerve conduction studies.

Some patients present with typical clinical features of carpal tunnel syndrome despite normal nerve conduction studies. This study compared the preoperative and 1-year postoperative QuickDASH scores in patients with normal and abnormal nerve conduction studies, who underwent carpal tunnel decompression. Of the 637 patients included in the study, 19 had clinical features of carpal tunnel syndrome but normal nerve conduction studies, and underwent decompression after failure of conservative management. Preoperative QuickDASH scores were comparable in both groups (58 vs 54.8). However, there were significant differences between the normal and abnormal nerve conduction study groups in the QuickDASH at 1 year (34.9 vs 21.5) and change in QuickDASH postoperatively (23.1 vs 33.4). Patients with normal nerve conduction studies had comparable preoperative disability scores compared with those with abnormal studies. Although they had a significant improvement in QuickDASH at 1 year, this was significantly less than those with abnormal nerve conduction studies. Level of evidence: III.

Metabolic modulation regulates cardiac wall morphogenesis in zebrafish.

During cardiac development, cardiomyocytes form complex inner wall structures called trabeculae. Despite significant investigation into this process, the potential role of metabolism has not been addressed. Using single cell resolution imaging in zebrafish, we find that cardiomyocytes seeding the trabecular layer actively change their shape while compact layer cardiomyocytes remain static. We show that Erbb2 signaling, which is required for trabeculation, activates glycolysis to support changes in cardiomyocyte shape and behavior. Pharmacological inhibition of glycolysis impairs cardiac trabeculation, and cardiomyocyte-specific loss- and gain-of-function manipulations of glycolysis decrease and increase trabeculation, respectively. In addition, loss of the glycolytic enzyme pyruvate kinase M2 impairs trabeculation. Experiments with rat neonatal cardiomyocytes in culture further support these observations. Our findings reveal new roles for glycolysis in regulating cardiomyocyte behavior during cardiac wall morphogenesis.

Paraxial Mesoderm Is the Major Source of Lymphatic Endothelium.

Endothelial cells (ECs), which line blood and lymphatic vessels, are generally described to come from the lateral plate mesoderm despite experimental evidence for a broader source of origin, including the paraxial mesoderm (PXM). Current dogma suggests that following specification from mesoderm, local environmental cues establish the distinct molecular and functional characteristics of ECs in different vascular beds. Here we present evidence to challenge this view, showing that lymphatic EC fate is imprinted during transition through the PXM lineage. We show that PXM-derived cells form the lymphatic endothelium of multiple organs and tissues, with a more restricted contribution to blood vessel endothelium. By deleting Prox1 specifically in PXM-derived cells, we show that this lineage is indispensable for lymphatic vessel development. Collectively, our data establish lineage history as a critical determinant of EC specialization, a finding with broad implications for our understanding of vascular development and heterogeneity.

The long-term outcome of simple trapeziectomy.

The aim of this study was to investigate the long-term outcome of simple trapeziectomy by a single surgeon and to compare this with pre-operative function. Two hundred and five patients completed the shortened disabilities of the arm, shoulder and hand questionnaire (QuickDASH) and the EuroQoL five-dimensional questionnaire (EQ-5D) scores at a mean of 8.2 (range 3.5-17) years after simple trapeziectomy. There were no pre-operative scores available, so case controls were selected from our prospective database to compare pre- and post-operative patient-reported outcome measures. The mean QuickDASH score of the post-operative matched group was 37 (SD 17.0) and the mean EQ-5D was 0.56 (SD 0.31). The mean QuickDASH score of the pre-operative group was 54 (SD 17.0). The mean difference in QuickDASH between the pre- and post-operative groups was 17 (95% CI: 8 to 26, p = 0.0003). This study demonstrated a significant and sustained improvement in patient-reported function after simple trapeziectomy. It supports that simple trapeziectomy is a simple, safe and effective treatment for advanced trapeziometacarpal joint arthritis. Level of evidence: IV.

Loss of pyruvate kinase M2 limits growth and triggers innate immune signaling in endothelial cells.

Despite their inherent proximity to circulating oxygen and nutrients, endothelial cells (ECs) oxidize only a minor fraction of glucose in mitochondria, a metabolic specialization that is poorly understood. Here we show that the glycolytic enzyme pyruvate kinase M2 (PKM2) limits glucose oxidation, and maintains the growth and epigenetic state of ECs. We find that loss of PKM2 alters mitochondrial substrate utilization and impairs EC proliferation and migration in vivo. Mechanistically, we show that the NF-κB transcription factor RELB is responsive to PKM2 loss, limiting EC growth through the regulation of P53. Furthermore, S-adenosylmethionine synthesis is impaired in the absence of PKM2, resulting in DNA hypomethylation, de-repression of endogenous retroviral elements (ERVs) and activation of antiviral innate immune signalling. This work reveals the metabolic and functional consequences of glucose oxidation in the endothelium, highlights the importance of PKM2 for endothelial growth and links metabolic dysfunction with autoimmune activation in ECs.

Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish.

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry.

Corrigendum: Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

This corrects the article DOI: 10.1038/nature18614.

CNS-resident progenitors direct the vascularization of neighboring tissues.

Organ growth requires the coordinated invasion and expansion of blood vessel networks directed by tissue-resident cells and morphogenetic cues. A striking example of this intercellular communication is the vascularization of the central nervous system (CNS), which is driven by neuronal progenitors, including neuroepithelial cells and radial glia. Although the importance of neuronal progenitors in vascular development within the CNS is well recognized, how these progenitors regulate the vasculature outside the CNS remains largely unknown. Here we show that CNS-resident radial glia direct the vascularization of neighboring tissues during development. We find that genetic ablation of radial glia in zebrafish larvae leads to a complete loss of the bilateral vertebral arteries (VTAs) that extend along the ventrolateral sides of the spinal cord. Importantly, VTA formation is not affected by ablation of other CNS cell types, and radial glia ablation also compromises the subsequent formation of the peri-neural vascular plexus (PNVP), a vascular network that surrounds the CNS and is critical for CNS angiogenesis. Mechanistically, we find that radial glia control these processes via Vegfab/Vegfr2 signaling: vegfab is expressed by radial glia, and genetic or pharmacological inhibition of Vegfab/Vegfr2 signaling blocks the formation of the VTAs and subsequently of the PNVP. Moreover, mosaic overexpression of Vegfab in radial glia is sufficient to partially rescue the VTA formation defect in vegfab mutants. Thus, our findings identify a critical function for CNS-resident progenitors in the regulation of vascularization outside the CNS, serving as a paradigm for cross-tissue coordination of vascular morphogenesis and growth.

Frameshift indels introduced by genome editing can lead to in-frame exon skipping.

The introduction of frameshift indels by genome editing has emerged as a powerful technique to study the functions of uncharacterized genes in cell lines and model organisms. Such mutations should lead to mRNA degradation owing to nonsense-mediated mRNA decay or the production of severely truncated proteins. Here, we show that frameshift indels engineered by genome editing can also lead to skipping of "multiple of three nucleotides" exons. Such splicing events result in in-frame mRNA that may encode fully or partially functional proteins. We also characterize a segregating nonsense variant (rs2273865) located in a "multiple of three nucleotides" exon of LGALS8 that increases exon skipping in human erythroblast samples. Our results highlight the potentially frequent contribution of exonic splicing regulatory elements and are important for the interpretation of negative results in genome editing experiments. Moreover, they may contribute to a better annotation of loss-of-function mutations in the human genome.

Long-Term Follow-up of Arthrodesis vs Total Joint Arthroplasty for Hallux Rigidus.

BACKGROUND: The optimal operative management of hallux rigidus is still a matter for debate among surgeons. Despite arthrodesis widely considered to be the gold standard treatment, many surgeons advocate arthroplasty as a suitable alternative. There are, however, few long-term or high-quality studies evaluating these modalities. We present the 15-year follow-up of a randomized controlled trial. METHODS: This data is the follow-up to the original study published in 2005. In the original study, 63 patients (77 toes) were recruited to and randomized to have either metatarsophalangeal joint (MTPJ) arthrodesis or arthroplasty. The primary outcome measure was a decrease in pain on a visual analog scale (VAS) at 24 months. In the present study, data were available for all surviving patients (52 patients, 66 toes). Data were collected in the form of satisfaction scores, VAS for pain, the VAS foot and ankle and survivorship data. RESULTS: The results of the original study demonstrated that pain relief was greater following arthrodesis at 2 years. At 15 years, patients with an arthrodesis experienced less pain and were more satisfied compared to those with an arthroplasty. No functional differences were seen between these 2 groups. There were more revisions in the arthroplasty group. CONCLUSION: Despite the hope of better function, less pain, and greater satisfaction from MTPJ replacement, this was not found in our patient population. The long-term results of our study showed that arthrodesis outperformed arthroplasty. If an arthroplasty failed, then salvage was likely to be technically difficult, with significant potential for complications. LEVEL OF EVIDENCE: Level I, randomized controlled trial.

Differential regulation of blood flow-induced neovascularization and mural cell recruitment by vascular endothelial growth factor and angiopoietin signalling.

KEY POINTS: Combining nitric oxide (NO)-mediated increased blood flow with angiopoietin-1-Tie2 receptor signalling induces arteriolargenesis - the formation of arterioles from capillaries - in a model of physiological angiogenesis. This NO-Tie-mediated arteriolargenesis requires endogenous vascular endothelial growth factor (VEGF) signalling. Inhibition of VEGF signalling increases pericyte coverage in microvessels. Together these findings indicate that generation of functional neovasculature requires close titration of NO-Tie2 signalling and localized VEGF induction, suggesting that the use of exogenous VEGF expression as a therapeutic for neovascularization may not be successful. ABSTRACT: Signalling through vascular endothelial growth factor (VEGF) receptors and the tyrosine kinase with IgG and EGF domains-2 (Tie2) receptor by angiopoietins is required in combination with blood flow for the formation of a functional vascular network. We tested the hypothesis that VEGF and angiopoietin-1 (Ang1) contribute differentially to neovascularization induced by nitric oxide (NO)-mediated vasodilatation, by comparing the phenotype of new microvessels in the mesentery during induction of vascular remodelling by over-expression of endothelial nitric oxide synthase in the fat pad of the adult rat mesentery during inhibition of angiopoietin signalling with soluble Tie2 (sTie2) and VEGF signalling with soluble Fms-like tyrosine kinase receptor-1 (sFlt1). We found that NO-mediated angiogenesis was blocked by inhibition of VEGF with sFlt1 (from 881 ± 98% increase in functional vessel area to 279 ± 72%) and by inhibition of angiopoietin with sTie2 (to 337 ± 67%). Exogenous angiopoietin-1 was required to induce arteriolargenesis (8.6 ± 1.3% of vessels with recruitment of vascular smooth muscle cells; VSMCs) in the presence of enhanced flow. sTie2 and sFlt1 both inhibited VSMC recruitment (both 0%), and VEGF inhibition increased pericyte recruitment to newly formed vessels (from 27 ± 2 to 54 ± 3% pericyte ensheathment). We demonstrate that a fine balance of VEGF and angiopoietin signalling is required for the formation of a functional vascular network. Endogenous VEGF signalling prevents excess neovessel pericyte coverage, and is required for VSMC recruitment during increased nitric oxide-mediated vasodilatation and angiopoietin signalling (NO-Tie-mediated arteriogenesis). Therapeutic vascular remodelling paradigms may therefore require treatments that modulate blood flow to utilize endogenous VEGF, in combination with exogenous Ang1, for effective neovascularization.

Cloche is a bHLH-PAS transcription factor that drives haemato-vascular specification.

Vascular and haematopoietic cells organize into specialized tissues during early embryogenesis to supply essential nutrients to all organs and thus play critical roles in development and disease. At the top of the haemato-vascular specification cascade lies cloche, a gene that when mutated in zebrafish leads to the striking phenotype of loss of most endothelial and haematopoietic cells and a significant increase in cardiomyocyte numbers. Although this mutant has been analysed extensively to investigate mesoderm diversification and differentiation and continues to be broadly used as a unique avascular model, the isolation of the cloche gene has been challenging due to its telomeric location. Here we used a deletion allele of cloche to identify several new cloche candidate genes within this genomic region, and systematically genome-edited each candidate. Through this comprehensive interrogation, we succeeded in isolating the cloche gene and discovered that it encodes a PAS-domain-containing bHLH transcription factor, and that it is expressed in a highly specific spatiotemporal pattern starting during late gastrulation. Gain-of-function experiments show that it can potently induce endothelial gene expression. Epistasis experiments reveal that it functions upstream of etv2 and tal1, the earliest expressed endothelial and haematopoietic transcription factor genes identified to date. A mammalian cloche orthologue can also rescue blood vessel formation in zebrafish cloche mutants, indicating a highly conserved role in vertebrate vasculogenesis and haematopoiesis. The identification of this master regulator of endothelial and haematopoietic fate enhances our understanding of early mesoderm diversification and may lead to improved protocols for the generation of endothelial and haematopoietic cells in vivo and in vitro.

Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish.

Skeletal muscles provide metazoans with the ability to feed, reproduce and avoid predators. In humans, a heterogeneous group of genetic diseases, termed muscular dystrophies (MD), lead to skeletal muscle dysfunction. Mutations in the gene encoding Caveolin-3, a principal component of the membrane micro-domains known as caveolae, cause defects in muscle maintenance and function; however it remains unclear how caveolae dysfunction underlies MD pathology. The Cavin family of caveolar proteins can form membrane remodeling oligomers and thus may also impact skeletal muscle function. Changes in the distribution and function of Cavin4/Murc, which is predominantly expressed in striated muscles, have been reported to alter caveolae structure through interaction with Caveolin-3. Here, we report the generation and phenotypic analysis of murcb mutant zebrafish, which display impaired swimming capacity, skeletal muscle fibrosis and T-tubule abnormalities during development. To understand the mechanistic importance of Murc loss of function, we assessed Caveolin-1 and 3 localization and found it to be abnormal. We further identified an in vivo function for Murc in Erk signaling. These data link Murc with developmental defects in T-tubule formation and progressive muscle dysfunction, thereby providing a new candidate for the etiology of muscular dystrophy.

A Cross-sectional Study of Current Doctors' Performance in a Modified Version of a Medical School Admission Aptitude Test: The UKCAT.

The 2-hour long United Kingdom Clinical Aptitude Test (UKCAT) is used by many universities in the United Kingdom as part of their selection process for undergraduate medical and dentistry degrees. We aimed to compare the performance of senior doctors in primary and secondary care and across a range of specialties, in a modified version of the medical school entrance examination-the mUKCAT. Lay people were also included in the study. Despite its widespread use, this is the first study that examines the performance of senior clinicians in the UKCAT.The study used a prospective cross-sectional design. It used mock questions from the UKCAT website to generate an mUKCAT that was anticipated to take 15 minutes to complete. In all, 167 doctors at consultant, general practitioner (GP), or specialty trainee grade and 26 lay people took part.The overall mean mUKCAT score of all participants was 2486 (69.1%). Of the total cohort, 126 (65.3%) scored above our designated threshold of 2368 and were deemed to have passed the mUKCAT. Excluding lay people, 113 (67.7%) of the 167 doctors scored above that threshold. Medical specialty was associated with overall score (P = 0.003), with anesthetists/intensive care physicians scoring highest (n = 20, mean score 2660) and GPs scoring lowest (n = 38, mean score 2302). Academics outperformed nonacademics (mean score of academics, n = 44 vs nonacademics, n = 123: 2750 vs 2406; P < 0.001). Those clinicians in senior management positions scored lower than those in "standard" roles (mean score of senior management, n = 31 vs standard roles, n = 136: 2332 vs 2534, mean difference 202, 95% confidence interval 67-337, P = 0.004).In the situational judgement section, there was no evidence that specialty was associated with score (P = 0.15). Academics exhibited greater situational judgement than their nonacademic colleagues (academics vs nonacademics: 69.8 vs 63.6%; P = 0.01).The majority of senior clinicians passed our mUKCAT. Academics and anesthetists were found to be the best performers, with GPs and those in senior management positions performing the worst.

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/ß-catenin pathway during brain angiogenesis.

Despite the critical role of endothelial Wnt/ß-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/ß-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/ß-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/ß-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function.

An unusual complication after shoulder hemiarthroplasty.

Hemiarthroplasty of the shoulder can be a safe and an effective treatment for pain in patients with rheumatoid arthritis. Many complications have been previously described in the literature; the most common of which are dislocation, loosening, periprosthetic fractures, and infection. We report a patient who presented with a discharging sinus over the tip of the acromium which was created by the displacement of the prosthesis and erosion of the AC joint and distal clavicle. The erosion of the distal clavicle and AC joint caused the remaining proximal clavicle to become mobile and displaced posteriorly; this spike of clavicle was then able to penetrate the trapezius muscle and eventually the skin causing an aseptic sinus. This was successfully treated with the exploration and excision of the distal 2 cm of the clavicle.