A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Blood and immune development in human fetal bone marrow and Down syndrome.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).

The learning curves of a validated virtual reality hip arthroscopy simulator.

INTRODUCTION: Decreases in trainees' working hours, coupled with evidence of worse outcomes when hip arthroscopies are performed by inexperienced surgeons, mandate an additional means of training. Though virtual reality simulation has been adopted by other surgical specialities, its slow uptake in arthroscopic training is due to a lack of evidence as to its benefits. These benefits can be demonstrated through learning curves associated with simulator training-with practice reflecting increases in validated performance metrics. METHODS: Twenty-five medical students with no previous experience of hip arthroscopy completed seven weekly simulated arthroscopies of a healthy virtual hip joint using a 70° arthroscope in the supine position. Twelve targets were visualised within the central compartment, six via the anterior portal, three via the anterolateral portal and three via the posterolateral portal. Task duration, number of collisions (bone and soft-tissue), and distance travelled by arthroscope were measured by the simulator for every session of each student. RESULTS: Learning curves were demonstrated by the students, with improvements in time taken, number of collisions (bone and soft-tissue), collision length and efficiency of movement (all p < 0.01). Improvements in time taken, efficiency of movement and number of collisions with soft-tissue were first seen in session 3 and improvements in all other parameters were seen in session 4. No differences were found after session 5 for time taken and length of soft-tissue collision. No differences in number of collisions (bone and soft-tissue), length of collisions with bone, and efficiency of movement were found after session 6. CONCLUSIONS: The results of this study demonstrate learning curves for a hip arthroscopy simulator, with significant improvements seen after three sessions. All performance metrics were found to improved, demonstrating sufficient visuo-haptic consistency within the virtual environment, enabling individuals to develop basic arthroscopic skills.

The use of an electronic health record system reduces errors in the National Hip Fracture Database.

AIM: to compare the validity of data submitted from a UK level 1 trauma centre to the National Hip Fracture Database (NHFD) before and after the introduction of an electronic health record system (EHRS). PATIENTS AND METHODS: a total of 3224 records were reviewed from July 2009 to July 2017. 2,133 were submitted between July 2009 and October 2014 and 1,091 between October 2014 and July 2017, representing data submitted before and after the introduction of the EHRS, respectively. Data submitted to the NHFD were scrutinised against locally held data. RESULTS: use of an EHRS was associated with significant reductions in NHFD errors. The operation coding error rate fell significantly from 23.2% (494/2133) to 7.6% (83/1091); P < 0.001. Prior to EHRS introduction, of the 109 deaths recorded in the NHFD, 64 (59%) were incorrect. In the EHRS dataset, all the 112 recorded deaths were correct (P < 0.001). There was no significant difference in the error rate for fracture coding. In the EHRS dataset, after controlling for sample month, entries utilising an operation note template with mandatory fields relevant to NHFD data were more likely to be error free than those not using the template (OR 2.69; 95% CI 1.92-3.78). CONCLUSION: this study highlights a potential benefit of EHR systems, which offer automated data collection for auditing purposes. However, errors in data submitted to the NHFD remain, particularly in cases where an NHFD-specific operation note template is not used. Clinician engagement with new technologies is vital to avoid human error and ensure database integrity.

Virtual reality hip arthroscopy simulator demonstrates sufficient face validity.

PURPOSE: To test the face validity of the hip diagnostics module of a virtual reality hip arthroscopy simulator. METHODS: A total of 25 orthopaedic surgeons, 7 faculty members and 18 orthopaedic residents, performed diagnostic supine hip arthroscopies of a healthy virtual reality hip joint using a 70° arthroscope. Twelve specific targets were visualised within the central compartment; six via the anterior portal, three via the anterolateral portal and three via the posterolateral portal. This task was immediately followed by a questionnaire regarding the realism and training capability of the system. This consisted of seven questions addressing the verisimilitude of the simulator and five questions addressing the training environment of the simulator. Each question consisted of a statement stem and 10-point Likert scale. Following similar work in surgical simulators, a rating of 7 or above was considered an acceptable level of realism. RESULTS: The diagnostic hip arthroscopy module was found to have an acceptable level of realism in all domains apart from the tactile feedback received from the soft tissue. 23 out of 25 participants (92%) felt the simulator provided a non-threatening learning environment and 22 participants (88%) stated they enjoyed using the simulator. It was most frequently agreed that the level of trainees who would benefit most from the simulator were registrars and fellows (22 participants; 88%). Additionally, 21 of the participants (84%) agreed that this would be a beneficial training modality for foundation and core trainees, and 20 participants (80%) agreed that his would be beneficial for consultants. CONCLUSIONS: This VR hip arthroscopy simulator was demonstrated to have a sufficient level of realism, thus establishing its face validity. These results suggest this simulator has sufficient realism for use in the acquisition of basic arthroscopic skills and supports its use in orthopaedics surgical training. LEVEL OF EVIDENCE: I.

Close encounters of the blurred kind.

We present a visually arresting scout image obtained during a CT head scan of an elderly patient for assessment of new onset confusion. The patient moved during the scout image acquisition resulting in distortion of the cranial vault that never the less remained largely in focus.

Current concepts and future perspectives in computer-assisted navigated total knee replacement.

BACKGROUND: Total knee replacements (TKR) aim to restore stability of the tibiofemoral and patella-femoral joints and provide relief of pain and improved quality of life for the patient. In recent years, computer-assisted navigation systems have been developed with the aim of reducing human error in joint alignment and improving patient outcomes. METHODS: We examined the current body of evidence surrounding the use of navigation systems and discussed their current and future role in TKR. RESULTS: The current body of evidence shows that the use of computer navigation systems for TKR significantly reduces outliers in the mechanical axis and coronal prosthetic position. Also, navigation systems offer an objective assessment of soft tissue balancing that had previously not been available. Although these benefits represent a technical superiority to conventional TKR techniques, there is limited evidence to show long-term clinical benefit with the use of navigation systems, with only a small number of studies showing improvement in outcome scores at short-term follow-up. Because of the increased costs and operative time associated with their use as well as the emergence of more affordable and patient-specific technologies, it is unlikely for navigation systems to become more widely used in the near future. CONCLUSIONS: Whilst this technology helps surgeons to achieve improved component positioning, it is important to consider the clinical and functional implications, as well as the added costs and potential learning curve associated with adopting new technology.

What Is the Risk Posed to the Lateral Femoral Cutaneous Nerve During the Use of the Anterior Portal of Supine Hip Arthroscopy and the Minimally Invasive Anterior Approach for Total Hip Arthroplasty?

PURPOSE: To determine: (1) What is the proximity of the lateral femoral cutaneous nerve (LFCN) to the anterior portal (AP) used in supine hip arthroscopy? (2) What is the proximity of the LCFN to the incision in the minimally invasive anterior approach (MIAA) for total hip arthroplasty? (3) What effect does lateralizing the AP have on the likelihood of nerve injury? (4) What branching patterns are observable in the LFCN? METHODS: Forty-five hemipelves were dissected. The LFCN was identified and its path dissected. The positions of the nerve in relation to the AP and the MIAA incision were measured. RESULTS: The AP intersected with 38% of nerves. In the remainder, the LFCN was located 5.7 ± 4.5 mm from the portal's edge. In addition, 44% of nerves crossed the incision of the MIAA. Of those that did not, the average minimum distance from the incision was 14.4 ± 7.0 mm. We found a significant reduction in risk if the AP is moved medially by 5 mm or laterally by 15 mm (P = .0054 and P = .0002). The LFCN showed considerable variation with 4 branching variants. CONCLUSIONS: These results show that the LFCN is at high risk during supine hip arthroscopy and the MIAA, emphasizing the need for meticulous dissection. We suggest that relocation of the AP 5 mm medially or 15 mm laterally will reduce the risk to the LFCN. CLINICAL RELEVANCE: These findings should aid surgeons in minimizing the risk to the LCFN during hip arthroscopy and the minimally invasive anterior approach to the hip.

Do Patients Taking Warfarin Experience Delays to Theatre, Longer Hospital Stay, and Poorer Survival After Hip Fracture?

BACKGROUND: Patients sustaining a fractured neck of the femur are typically of advanced age with multiple comorbidities. As a consequence, the proportion of these patients receiving warfarin therapy is approximately 10%. There are currently few studies investigating outcomes in this subset of patients. QUESTIONS/PURPOSES: The purpose of this study was to assess the association between warfarin therapy and time to surgery, length of hospital stay, and survival in patients sustaining a fractured neck of the femur. METHODS: Data for 2036 patients admitted to our center between July 2009 and July 2014 with a fractured neck of the femur were extracted from the National Hip Fracture Database. Fifty-seven patients received no surgical treatment and were excluded from analysis. Multivariable ordinary least squares regression was performed to test the association between warfarin treatment on time to surgery and length of stay, and Cox proportional hazards to test followup survival. Variables included in the regression model were age, sex, American Society of Anesthesiologists (ASA) score, admission Abbreviated Mental Test Score (AMTS), fracture type, operation type, and premorbid Work Ability Index (WAI). One hundred fifty-two of 1979 surgically treated patients (8%) were receiving warfarin therapy at the time of admission. RESULTS: After controlling for age, sex, ASA score, AMTS, fracture type, operation type, and WAI, we found that patients taking warfarin were less likely to go to surgery by 36 hours (odds ratio [OR], 0.20; 95% CI, 0.14-0.30), and less likely to go to surgery by 48 hours (OR, 0.17; 95% CI, 0.11-0.24). Patients taking warfarin had a longer length of stay (median, 15 days; interquartile range [IQR], 12-22 days) compared with patients not taking warfarin (median, 13 days; IQR, 9-20 days; p < 0.001). Survival analysis to June 2015 showed a higher mortality for patients taking warfarin (12-month survival, 66% vs 76%; hazard ratio, 1.57; 95% CI, 1.21-2.04; p < 0.001). CONCLUSIONS: After controlling for multiple prognostic factors such as age, ASA score, AMTS, and WAI, warfarin therapy at the time of injury is associated with increased time to surgery, length of stay, and decreased survival. This study highlights the need to view warfarin therapy as a 'red flag' in patients presenting with a fractured neck of the femur. Preoperatively, prompt warfarin reversal together with adequate investigation and optimization of the patient should ensure timely, safe surgery. Early involvement of the anesthesia team should ensure an appropriate level of postoperative care for these patients. LEVEL OF EVIDENCE: Level III, therapeutic study.

Testing the Construct Validity of a Virtual Reality Hip Arthroscopy Simulator.

PURPOSE: To test the construct validity of the hip diagnostics module of a virtual reality hip arthroscopy simulator. METHODS: Nineteen orthopaedic surgeons performed a simulated arthroscopic examination of a healthy hip joint using a 70° arthroscope in the supine position. Surgeons were categorized as either expert (those who had performed 250 hip arthroscopies or more) or novice (those who had performed fewer than this). Twenty-one specific targets were visualized within the central and peripheral compartments; 9 via the anterior portal, 9 via the anterolateral portal, and 3 via the posterolateral portal. This was immediately followed by a task testing basic probe examination of the joint in which a series of 8 targets were probed via the anterolateral portal. During the tasks, the surgeon's performance was evaluated by the simulator using a set of predefined metrics including task duration, number of soft tissue and bone collisions, and distance travelled by instruments. No repeat attempts at the tasks were permitted. Construct validity was then evaluated by comparing novice and expert group performance metrics over the 2 tasks using the Mann-Whitney test, with a P value of less than .05 considered significant. RESULTS: On the visualization task, the expert group outperformed the novice group on time taken (P = .0003), number of collisions with soft tissue (P = .001), number of collisions with bone (P = .002), and distance travelled by the arthroscope (P = .02). On the probe examination, the 2 groups differed only in the time taken to complete the task (P = .025) with no significant difference in other metrics. CONCLUSIONS: Increased experience in hip arthroscopy was reflected by significantly better performance on the virtual reality simulator across 2 tasks, supporting its construct validity. CLINICAL RELEVANCE: This study validates a virtual reality hip arthroscopy simulator and supports its potential for developing basic arthroscopic skills. LEVEL OF EVIDENCE: Level III.

Adult-onset hypophosphatasia diagnosed following bilateral atypical femoral fractures in a 55-year-old woman.

We report the case of a 55-year-old woman who presented to the emergency department having woken from sleep with right sided thigh swelling. Pelvic radiographs revealed bilateral atypical subtrochanteric femoral fractures (ASFFs). In the two years leading up to this admission, the patient had experienced gradually increasing pain and weakness in her legs which had resulted in a decrease in her mobility from fully mobile to bed-bound. During this time a neurologist had organised a magnetic-resonance imaging (MRI) scan of the brain and spine which was normal. There was no history of bisphosphonate (BP) use. Historical and admission blood tests revealed a persistently low serum alkaline phosphatase (ALP), with all other results within normal limits. The patient was treated with intramedullary nailing of both femurs and histological analysis of bone reamings were characteristic of hypophosphatasia (HPP). The patient was independently mobilising with a walking frame on discharge. Subsequent genetic testing revealed bi-allelic pathogenic variants in the TNSALP gene: c.526G>A, p.(Ala176Thr) and c.1171C>T, p.(Arg391Cys). HPP is an inborn error in metabolism caused by mutation in the gene coding for tissue non-specific alkaline phosphatase (TNSALP), resulting in a decrease in serum ALP concentrations. The age at which it presents which can vary from childhood to middle age, with symptoms ranging from perinatal death to late-onset osteomalacia. In those patients who survive to adulthood, there is a predisposition to fractures, including ASFFs. Treatment with asfotase alfa (a bone-targeted, recombinant human TNSALP) has been approved for perinatal, infantile and paediatric-onset hypophosphatasia. This case emphasises the importance of viewing persistent low ALP as a 'red flag' in patients presenting with musculoskeletal symptoms. Timely diagnosis and treatment of HPP can reduce the risk of serious complications, such as those experienced by this patient.

Neuropsychiatric Manifestations of Colloid Cysts: a review of the literature.

Colloid cysts account for approximately 2% of primary brain tumours and the majority of cases are identified in the fourth and fifth decade. They are small, gelatinous neoplasms lined by a single layer of mucin-secreting columnar epithelium that are thought to arise from errors in folding of the primitive neuroepithelium. They develop in the rostral aspect of the third ventricle in the foramen of Monro in 99% of cases and despite their benign histology carry a poor prognosis, with a mortality greater than 10% in symptomatic cases. The location of colloid cysts within the ventricular system results in obstruction of the foramen of Monro as the cyst grows, disrupting the circulation of cerebrospinal fluid (CSF) and causing hydrocephalus. This is the mechanism behind the most common presenting symptoms of postural headache, nausea and vomiting - a clinical picture synonymous with hydrocephalus and intracranial pathology. In addition to these classical neurological symptoms, there is a high prevalence of psychiatric symptoms in the patient population, with symptoms ranging from anterograde amnesia to gustatory hallucination. These symptoms can occur with or without the presence of hydrocephalus, and are thought to be secondary to compression of connecting pathways between the mesocortices and subcortical limbic regions. These symptoms have been shown to be comparative in frequency to the classical symptoms, yet are rarely the reason for referral to a neurological or neurosurgical service for investigation.

Anti-NMDA Encephalitis in the Acute Setting.

BACKGROUND: Encephalitis associated with antibodies targeted against the N-methyl-D-aspartate (NMDA) receptor is increasingly recognised as a major cause of an acute presentation of organic psychosis. Misdiagnosis and subsequent inappropriate referral to psychiatric services is common and avoidable. This review focuses on addressing this issue in the acute setting. METHODS: The authors present a review of existing literature relating to the pathophysiology and presentation of anti-NMDA receptor encephalitis, prior to proposing a management pathway avoiding delays to treatment incurred through misdiagnosis or inappropriate referral. CONCLUSIONS: Acute care physicians should have a low threshold for suspecting anti-NMDA receptor encephalitis in any patient presenting with acute psychosis in the context of non-specific coryzal and constitutional symptoms in whom infective causes have been excluded. The presence of pleocytosis and reduced protein in routine CSF analysis should further raise suspicion, and samples should be sent for immunohistochemical testing. Availability and efficiency of this testing is currently suboptimal.

Human skeletal muscle aging atlas.

Skeletal muscle aging is a key contributor to age-related frailty and sarcopenia with substantial implications for global health. Here we profiled 90,902 single cells and 92,259 single nuclei from 17 donors to map the aging process in the adult human intercostal muscle, identifying cellular changes in each muscle compartment. We found that distinct subsets of muscle stem cells exhibit decreased ribosome biogenesis genes and increased CCL2 expression, causing different aging phenotypes. Our atlas also highlights an expansion of nuclei associated with the neuromuscular junction, which may reflect re-innervation, and outlines how the loss of fast-twitch myofibers is mitigated through regeneration and upregulation of fast-type markers in slow-twitch myofibers with age. Furthermore, we document the function of aging muscle microenvironment in immune cell attraction. Overall, we present a comprehensive human skeletal muscle aging resource ( https://www.muscleageingcellatlas.org/ ) together with an in-house mouse muscle atlas to study common features of muscle aging across species.

Targetable NOTCH1 rearrangements in reninoma.

Reninomas are exceedingly rare renin-secreting kidney tumours that derive from juxtaglomerular cells, specialised smooth muscle cells that reside at the vascular inlet of glomeruli. They are the central component of the juxtaglomerular apparatus which controls systemic blood pressure through the secretion of renin. We assess somatic changes in reninoma and find structural variants that generate canonical activating rearrangements of, NOTCH1 whilst removing its negative regulator, NRARP. Accordingly, in single reninoma nuclei we observe excessive renin and NOTCH1 signalling mRNAs, with a concomitant non-excess of NRARP expression. Re-analysis of previously published reninoma bulk transcriptomes further corroborates our observation of dysregulated Notch pathway signalling in reninoma. Our findings reveal NOTCH1 rearrangements in reninoma, therapeutically targetable through existing NOTCH1 inhibitors, and indicate that unscheduled Notch signalling may be a disease-defining feature of reninoma.

Diagnostic classification of childhood cancer using multiscale transcriptomics.

The causes of pediatric cancers' distinctiveness compared to adult-onset tumors of the same type are not completely clear and not fully explained by their genomes. In this study, we used an optimized multilevel RNA clustering approach to derive molecular definitions for most childhood cancers. Applying this method to 13,313 transcriptomes, we constructed a pediatric cancer atlas to explore age-associated changes. Tumor entities were sometimes unexpectedly grouped due to common lineages, drivers or stemness profiles. Some established entities were divided into subgroups that predicted outcome better than current diagnostic approaches. These definitions account for inter-tumoral and intra-tumoral heterogeneity and have the potential of enabling reproducible, quantifiable diagnostics. As a whole, childhood tumors had more transcriptional diversity than adult tumors, maintaining greater expression flexibility. To apply these insights, we designed an ensemble convolutional neural network classifier. We show that this tool was able to match or clarify the diagnosis for 85% of childhood tumors in a prospective cohort. If further validated, this framework could be extended to derive molecular definitions for all cancer types.

From Cape Town to Cambridge: Orthopaedic trauma in contrasting environments.

AIM: To compare the trauma experience gained by a trainee at a United Kingdom major trauma centre and a secondary level hospital in South Africa. METHODS: A profile of inpatient trauma cases during a five-week period in Addenbrooke's Hospital, Cambridge and Somerset Hospital, Cape Town was created. This was achieved by recording various parameters for each patient admitted including age, gender, injury, mechanism of injury and postal/area code. This, together with details of the departments themselves, allows a comparison of the amount and variety of orthopaedic trauma cases experienced by an individual trainee in each setting. RESULTS: The trauma profiles differed significantly. Patients in Cape Town were younger and more likely to be male. In the young, injury in Cape Town was more likely to occur due to assault or being struck by a vehicle, whilst patients in Cambridge were more likely to be injured whilst in a vehicle or in high energy falls. In older patients, trauma at both centres was almost exclusively due to mechanical falls. In a given age group, injuries at the two centres were similar, however the majority of patients admitted to Addenbrooke's were elderly, resulting in less variation in the overall injury profile. CONCLUSION: The trauma profile of a major trauma centre in the United Kingdom is less varied than that of a South African secondary centre, with significantly fewer cases per surgeon. This suggests a more varied training experience in the developing world with a greater caseload.

Delayed Presentation of Gluteal Compartment Syndrome: The Argument for Fasciotomy.

A male patient in his fifties presented to his local hospital with numbness and weakness of the right leg which left him unable to mobilise. He reported injecting heroin the previous morning. Following an initial diagnosis of acute limb ischaemia the patient was transferred to a tertiary centre where Computed Tomography Angiography was reported as normal. Detailed neurological examination revealed weakness in hip flexion and extension (1/5 on the Medical Research Council scale) with complete paralysis of muscle groups distal to this. Sensation to pinprick and light touch was globally reduced. Blood tests revealed acute kidney injury with raised creatinine kinase and the patient was treated for rhabdomyolysis. Orthopaedic referral was made the following day and a diagnosis of gluteal compartment syndrome (GCS) was made. Emergency fasciotomy was performed 56 hours after the onset of symptoms. There was immediate neurological improvement following decompression and the patient was rehabilitated with complete nerve recovery and function at eight-week follow-up. This is the first documented case of full functional recovery following a delayed presentation of GCS with sciatic nerve palsy. We discuss the arguments for and against fasciotomy in cases of compartment syndrome with significant delay in presentation or diagnosis.