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Background: Cystic fibrosis transmembrane conductance regulator metabolic syndrome/cystic fibrosis screen-positive, inconclusive diagnosis (CRMS/CFSPID) is a designation given following a positive newborn screen for cystic fibrosis (CF) when CF is not excluded but cannot be confirmed. We describe the long-term clinical outcomes of a CRMS/CFSPID cohort. Methods: A retrospective, single centre study of children with a current or previous diagnosis of CRMS/CFSPID. Study period extended from February 1, 2007 to August 1, 2022. Baseline and longitudinal data were assessed. Results: 30 children were designated as CRMS/CFSPID between 2007 and 2021. At baseline, 13 CFTR variants were identified, of which F508del and R117H 7T/9T were most common (occurring in 25 and 20 children respectively). Initial mean immunoreactive trypsinogen and sweat chloride were 82.8 mmol/L and 34.3 mmol/L respectively. During longitudinal assessment (n = 27), occurring over a mean duration of 8.5 years, five children progressed to CF at a mean age of 9.5 years. All children were pancreatic sufficient except one who reclassified to CF. Four isolated Pseudomonas aeruginosa and 12 isolated Staphylococcus aureus, of which one and two progressed to CF respectively. All recent Z-scores for weight and spirometry were above −2. Initial mean sweat chloride was higher in those who progressed to CF versus those who did not, although this did not reach statistical significance (38.4 mmol/L versus 32.0 mmol/L respectively, p = 0.105). Conclusions: Most children with CRMS/CFSPID remained well with a low progression rate to CF. This supports a less intensive medical surveillance approach. Our results highlight the importance of assessment in a dedicated CRMS/CFSPID clinic during adolescence to detect progression to CF after 6 years of age.

Degenerative cervical myelopathy (DCM) is a common neurological condition, with disease progression that is both variable and difficult to predict. Here, we present a case of DCM in a gentleman in his late 60s with significant radiological disease progression without consequent change in clinical symptoms. The case serves as a reminder of an enduring medical aphorism that clinical history and examination should be prioritized above more complex data, such as imaging investigations. In addition, the case also highlights that guidelines should be contextualized within individual clinical circumstances.

Abstract Background The ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route. Results We present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods. Conclusions Our results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9–4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

The growing importance of advanced endoscopy in the diagnosis and treatment of pancreatic neuroendocrine neoplasms (PanNETs) necessitates a comprehensive understanding of various biochemical markers, genetic testing methods, radiological techniques, and treatment approaches that encompass multiple disciplines within and beyond gastrointestinal oncology. This review aims to highlight key aspects of these topics, with a specific focus on emerging EUS-guided procedures for the management of PanNETs.

Normal function of the C-terminal Eps15 homology domain-containing protein 1 (EHD1) has previously been associated with endocytic vesicle trafficking, shaping of intracellular membranes, and ciliogenesis. We recently identified an autosomal recessive missense mutation c.1192C>T (p.R398W) of EHD1 in patients who had low molecular weight proteinuria (0.7–2.1 g/d) and high-frequency hearing loss. It was already known from Ehd1 knockout mice that inactivation of Ehd1 can lead to male infertility. However, the exact role of the EHD1 protein and its p.R398W mutant during spermatogenesis remained still unclear. Here, we report the testicular phenotype of a knockin mouse model carrying the p.R398W mutation in the EHD1 protein. Male homozygous knockin mice were infertile, whereas the mutation had no effect on female fertility. Testes and epididymes were significantly reduced in size and weight. The testicular epithelium appeared profoundly damaged and had a disorganized architecture. The composition of developing cell types was altered. Malformed acrosomes covered underdeveloped and misshaped sperm heads. In the sperm tail, midpieces were largely missing indicating disturbed assembly of the sperm tail. Defective structures, i.e., nuclei, acrosomes, and sperm tail midpieces, were observed in large vacuoles scattered throughout the epithelium. Interestingly, cilia formation itself did not appear to be affected, as the axoneme and other parts of the sperm tails except the midpieces appeared to be intact. In wildtype mice, EHD1 co-localized with acrosomal granules on round spermatids, suggesting a role of the EHD1 protein during acrosomal development. Wildtype EHD1 also co-localized with the VPS35 component of the retromer complex, whereas the p.R398W mutant did not. The testicular pathologies appeared very early during the first spermatogenic wave in young mice (starting at 14 dpp) and tubular destruction worsened with age. Taken together, EHD1 plays an important and probably multifaceted role in spermatogenesis in mice. Therefore, EHD1 may also be a hitherto underestimated infertility gene in humans.

BackgroundDegenerative cervical myelopathy (DCM) is estimated to affect 2% of the adult population. DCM occurs when degenerative processes cause compression and injure the spinal cord. Surgery to remove the stress caused by the compression of the spinal cord is the mainstay of treatment, with a range of techniques in use. Although various factors are described to inform the selection of these techniques, there needs to be more consensus and limited comparative evidence. ObjectiveThe main objective of this survey was to explore the variation of practice and decision-making, with a focus on laminectomy versus laminectomy and fusion in posterior surgery of the cervical spine. We present the results of a survey conducted among the principal investigators (PIs) of the National Institute for Health and Care Research (NIHR) randomized controlled trial on posterior laminectomy with fixation for degenerative cervical myelopathy (POLYFIX-DCM). MethodsA series of 7 cases were shared with 24 PIs using SurveyMonkey. Each case consisted of a midsagittal T2-weighted magnetic resonance imaging and lateral cervical x-rays in flexion and extension. Surgeons were asked if their preferred approach was anterior or posterior. If posterior, they were asked whether they preferred to instrument and whether they had the equipoise to randomize in the NIHR POLYFIX-DCM trial. Variability in decision-making was then explored using factors reported to inform decision-making, such as alignment, location of compression, number of levels operated, presence of mobile spondylolisthesis, and patient age. ResultsThe majority of PIs (16/30, 53%) completed the survey. Overall, PIs favored a posterior approach (12/16, 75%) with instrumentation (75/112, average 66%) and would randomize (67/112, average 62%) most cases. Factors reported to inform decision-making poorly explained variability in responses in both univariate testing and with a multivariate model (R2=0.1). Only surgeon experience of more than 5 years and orthopedic specialty training background were significant predictors, both associated with an anterior approach (odds ratio [OR] 1.255; P=.02 and OR 1.344; P=.007, respectively) and fusion for posterior procedures (OR 0.628; P

Despite an upregulation in anergic B cells, N-myc downstream-regulated gene 1 (NDRG1) is not required for the tolerogenic downstream responses, reducing risk of immune modulation on targeting of NDRG1 for cancer therapeutics.

Objectives To explore whether a James Lind Alliance Priority Setting Partnership could provide insights on knowledge translation within the field of degenerative cervical myelopathy (DCM).Design Secondary analysis of a James Lind Alliance Priority Setting Partnership process for DCM.Participants and setting DCM stake holders, including spinal surgeons, people with myelopathy and other healthcare professionals, were surveyed internationally. Research suggestions submitted by stakeholders but considered answered were identified. Sampling characteristics of respondents were compared with the overall cohort to identify subgroups underserved by current knowledge translation.Results The survey was completed by 423 individuals from 68 different countries. A total of 22% of participants submitted research suggestions that were considered ‘answered’. There was a significant difference between responses from different stakeholder groups (p

Abstract Background Chromatin states and enhancers associate gene expression, cell identity and disease. Here, we systematically delineate the acute innate immune response to endotoxin in terms of human macrophage enhancer activity and contrast with endotoxin tolerance, profiling the coding and non-coding transcriptome, chromatin accessibility and epigenetic modifications. Results We describe the spectrum of enhancers under acute and tolerance conditions and the regulatory networks between these enhancers and biological processes including gene expression, splicing regulation, transcription factor binding and enhancer RNA signatures. We demonstrate that the vast majority of differentially regulated enhancers on acute stimulation are subject to tolerance and that expression quantitative trait loci, disease-risk variants and eRNAs are enriched in these regulatory regions and related to context-specific gene expression. We find enrichment for context-specific eQTL involving endotoxin response and specific infections and delineate specific differential regions informative for GWAS variants in inflammatory bowel disease and multiple sclerosis, together with a context-specific enhancer involving a bacterial infection eQTL for KLF4. We show enrichment in differential enhancers for tolerance involving transcription factors NFκB-p65, STATs and IRFs and prioritize putative causal genes directly linking genetic variants and disease risk enhancers. We further delineate similarities and differences in epigenetic landscape between stem cell-derived macrophages and primary cells and characterize the context-specific enhancer activities for key innate immune response genes KLF4, SLAMF1 and IL2RA. Conclusions Our study demonstrates the importance of context-specific macrophage enhancers in gene regulation and utility for interpreting disease associations, providing a roadmap to link genetic variants with molecular and cellular functions.

Introduction: Rural populations routinely rank poorly on common health indicators. While it is understood that rural residents face barriers to health care, the exact nature of these barriers remains unclear. To further define these barriers, a qualitative study of primary care physicians practicing in rural communities was performed. Methods: Semistructured interviews were conducted with primary care physicians practicing in rural areas within western Pennsylvania, the third largest rural population within the USA, using purposively sampling. Data were then transcribed, coded, and analyzed by thematic analysis. Results: Three key themes emerged from the analysis addressing barriers to rural health care: (1) cost and insurance, (2) geographic dispersion, and (3) provider shortage and burnout. Providers mentioned strategies that they either employed or thought would be beneficial for their rural communities: (1) subsidize services, (2) establish mobile and satellite clinics (particularly for specialty care), (3) increase utilization of telehealth, (4) improve infrastructure for ancillary patient support (ie social work services), and (5) increase utilization of advanced practice providers. Conclusion: There are numerous barriers to providing rural communities with quality health care. Barriers that are encountered are multidimensional. Patients are unable to obtain the care they need because of cost-related barriers. More providers need to be recruited to rural areas to combat the shortage and burnout. Advanced care-delivery methods such as telehealth, satellite clinics, or advanced practice providers can help bridge the gaps caused by geographic dispersion. Policy efforts should target all these aspects in order to appropriately address rural healthcare needs.

Introduction Degenerative cervical myelopathy (DCM) is a common and disabling condition of symptomatic cervical spinal cord compression secondary to degenerative changes in spinal structures leading to a mechanical stress injury of the spinal cord. RECEDE-Myelopathy aims to test the disease-modulating activity of the phosphodiesterase 3/phosphodiesterase 4 inhibitor Ibudilast as an adjuvant to surgical decompression in DCM.Methods and analysis RECEDE-Myelopathy is a multicentre, double-blind, randomised, placebo-controlled trial. Participants will be randomised to receive either 60–100 mg Ibudilast or placebo starting within 10 weeks prior to surgery and continuing for 24 weeks after surgery for a maximum of 34 weeks. Adults with DCM, who have a modified Japanese Orthopaedic Association (mJOA) score 8–14 inclusive and are scheduled for their first decompressive surgery are eligible for inclusion. The coprimary endpoints are pain measured on a visual analogue scale and physical function measured by the mJOA score at 6 months after surgery. Clinical assessments will be undertaken preoperatively, postoperatively and 3, 6 and 12 months after surgery. We hypothesise that adjuvant therapy with Ibudilast leads to a meaningful and additional improvement in either pain or function, as compared with standard routine care.Study design Clinical trial protocol V.2.2 October 2020.Ethics and dissemination Ethical approval has been obtained from HRA—Wales.The results will be presented at an international and national scientific conferences and in a peer-reviewed journals.Trial registration number ISRCTN Number: ISRCTN16682024.

Background: Spondylodiscitis is a potentially life-threatening infection of the intervertebral disk and adjacent vertebral bodies, with a mortality rate of 2–20%. Given the aging population, the increase in immunosuppression, and intravenous drug use in England, the incidence of spondylodiscitis is postulated to be increasing; however, the exact epidemiological trend in England remains unknown. Objective: The Hospital Episode Statistics (HES) database contains details of all secondary care admissions across NHS hospitals in England. This study aimed to use HES data to characterise the annual activity and longitudinal change of spondylodiscitis in England. Methods: The HES database was interrogated for all cases of spondylodiscitis between 2012 and 2019. Data for the length of stay, waiting time, age-stratified admissions, and ‘Finished Consultant Episodes’ (FCEs), which correspond to a patient's hospital care under a lead clinician, were analysed. Results: In total, 43135 FCEs for spondylodiscitis were identified between 2012 and 2022, of which 97.1% were adults. Overall admissions for spondylodiscitis have risen from 3 per 100,000 population in 2012/13 to 4.4 per 100,000 population in 2020/21. Similarly, FCEs have increased from 5.8 to 10.3 per 100,000 population, in 2012–2013 and 2020/21 respectively. The highest increase in admissions from 2012 to 2021 was recorded for those aged 70–74 (117% increase) and aged 75-59 (133% increase), among those of working age for those aged 60–64 years (91% increase). Conclusion: Population-adjusted admissions for spondylodiscitis in England have risen by 44% between 2012 and 2021. Healthcare policymakers and providers must acknowledge the increasing burden of spondylodiscitis and make spondylodiscitis a research priority.

Summary: Background: Cauda equina syndrome (CES) results from nerve root compression in the lumbosacral spine, usually due to a prolapsed intervertebral disc. Evidence for management of CES is limited by its infrequent occurrence and lack of standardised clinical definitions and outcome measures. Methods: This is a prospective multi-centre observational cohort study of adults with CES in the UK. We assessed presentation, investigation, management, and all Core Outcome Set domains up to one year post-operatively using clinician and participant reporting. Univariable and multivariable associations with the Oswestry Disability Index (ODI) and urinary outcomes were investigated. Findings: In 621 participants with CES, catheterisation for urinary retention was required pre-operatively in 31% (191/615). At discharge, only 13% (78/616) required a catheter. Median time to surgery from symptom onset was 3 days (IQR:1–8) with 32% (175/545) undergoing surgery within 48 h. Earlier surgery was associated with catheterisation (OR:2.2, 95%CI:1.5–3.3) but not with admission ODI or radiological compression. In multivariable analyses catheter requirement at discharge was associated with pre-operative catheterisation (OR:10.6, 95%CI:5.8–20.4) and one-year ODI was associated with presentation ODI (r = 0.3, 95%CI:0.2–0.4), but neither outcome was associated with time to surgery or radiological compression. Additional healthcare services were required by 65% (320/490) during one year follow up. Interpretation: Post-operative functional improvement occurred even in those presenting with urinary retention. There was no association between outcomes and time to surgery in this observational study. Significant healthcare needs remained post-operatively. Funding: DCN Endowment Fund funded study administration. Castor EDC provided database use. No other study funding was received.

None.

Aims: Hydroxyapatite (HA)-coated collars have been shown to reduce aseptic loosening of massive endoprostheses following primary surgery. Limited information exists about their effectiveness in revision surgery. The aim of this study was to radiologically assess osteointegration to HA-coated collars of cemented massive endoprostheses following revision surgery. Methods: Retrospective review of osseointegration frequency, pattern, and timing to a specific HA-coated collar on massive endoprostheses used in revision surgery at our tertiary referral centre between 2010 to 2017 was undertaken. Osseointegration was radiologically classified on cases with a minimum follow-up of six months. Results: In all, 39 patients underwent radiological review at mean 43.5 months; 22/39 (56.4%) showed no osseointegration to the collar. Revision endoprostheses for aseptic loosening were less likely to show osseointegration compared with other indications for revision. Oncological cases with previous or current infection were more likely to show osseointegration to ≥ 1 collar side than those without evidence of prior infection. Conclusion: This seven-year review identified osseointegration of HA-coated collars after revision surgery is less likely (43.6%, 17/39) than after primary surgery. Young patients who undergo revision surgery following initial oncological indication may benefit the most from this collar design. Use in revision oncological cases with a history of infection may be beneficial. HA-coated collars showed limited benefit for patients undergoing revision for failed arthroplasty with history of infection.

Introduction Degenerative cervical myelopathy (DCM) is the most common cause of spinal cord impairment. Unfortunately, the condition remains poorly recognised and underdiagnosed. To better identify patients, screening tests that target individuals at high risk would be helpful. One group in particular known to have a high prevalence of DCM consists of patients with lumbar degenerative disease (LDD), with the combined presentation referred to as tandem stenosis. Given that LDD is one of the most common presentations in neurosurgical practice and primary care, it is the objective of the proposed study to administer a screening test to these patients as well as those with risk factors or symptoms which raise the suspicion of underlying DCM.Methods and analysis A screening test based on clinical signs/symptoms and known risk factors of DCM was designed. Screening will be performed in neurosurgical consultations for patients with LDD or those with any suspicion of myelopathy. Points are attributed based on the presence of signs/symptoms of DCM (eg, Hoffmann sign, hyper-reflexia) and for comorbidities that predispose or are frequently associated with cervical myelopathy (eg, rheumatoid arthritis, carpal tunnel syndrome). Patients with ≥3 points undergo cervical MRI examination. Patients with positive MRIs will be consulted and receive assessment via modified Japanese Orthopedic Association and Neck Disability Index scores, and subsequent clinical management will be based on practice guidelines. An exploratory multivariate analysis of the effectiveness and efficiency of this proposed screening test will be evaluated after positively screening 50 patients for DCM.Ethics and dissemination This study has received research ethics approval from the Swiss Association of Research Ethics Committees (ID: 2020-02785). The results of this study will be disseminated in a journal targeting physicians commonly encountering patients with LDD.

BackgroundDegenerative cervical myelopathy (DCM) is a common, disabling condition of symptomatic cervical spinal cord compression that requires significant research advances to improve patient outcomes. A James Lind Alliance Partnership recently identified the top research priorities for DCM. To effectively address these priorities, appropriate funding of DCM research is essential. ObjectiveThe aim of this paper is to review current funding in DCM research and highlight future research funding opportunities. MethodsA systematic search of Web of Science for “cervical AND myelopathy” was conducted. Papers exclusively studying DCM with declared funding and published between January 1, 1995, and March 21, 2020, were considered eligible. Funding sources were classified by country of origin and organization type. A grant search was also conducted using Dimensions.ai (Digital Science Ltd). ResultsA total of 621 papers were included, with 300 unique funding bodies. The top funders were AO Spine (n=87); National Institutes of Health, USA (n=63); and National Natural Science Foundation, China (n=63). Funding sources in the USA (n=242) supported the most DCM research, followed by China (n=209) and Japan (n=116). Funding in the USA was primarily provided by corporate or nonprofit organizations (146/242, 60.3%), while in China, the majority of funding was from institutions (208/209, 99.5%). Dimensions.ai gives an estimate for the total declared grant funding awards for DCM-specific research. Data here showed 180 grants awarded specifically for DCM research, with a total value of US $45.6 million since 1996. ConclusionsDCM funding appears to be predominantly from the USA, China, and Japan, aligning with areas of high DCM research activity and underpinning the importance of funding to increasing research capacity. The existing funding sources differ from medical research in general, representing opportunities for future investment in DCM.

Abstract Background Genome editing in mice using either classical approaches like homologous recombination or CRISPR/Cas9 has been reported to harbor off target effects (insertion/deletion, frame shifts or gene segment duplications) that lead to mutations not only in close proximity to the target site but also outside. Only the genomes of few engineered mouse strains have been sequenced. Since the role of the ether-lipid cleaving enzyme alkylglycerol monooxygenase (AGMO) in physiology and pathophysiology remains enigmatic, we created a knockout mouse model for AGMO using EUCOMM stem cells but unforeseen genotyping issues that did not agree with Mendelian distribution and enzyme activity data prompted an in-depth genomic validation of the mouse model. Results We report a gene segment tandem duplication event that occurred during the generation of an Agmo knockout-first allele by homologous recombination. Only low homology was seen between the breakpoints. While a single copy of the recombinant 18 kb cassette was integrated correctly around exon 2 of the Agmo gene, whole genome nanopore sequencing revealed a 94 kb duplication in the Agmo locus that contains Agmo wild-type exons 1–3. The duplication fooled genotyping by routine PCR, but could be resolved using qPCR-based genotyping, targeted locus amplification sequencing and nanopore sequencing. Despite this event, this Agmo knockout mouse model lacks AGMO enzyme activity and can therefore be used to study its physiological role. Conclusions A duplication event occurred at the exact locus of the homologous recombination and was not detected by conventional quality control filters such as FISH or long-range PCR over the recombination sites. Nanopore sequencing provides a cost convenient method to detect such underrated off-target effects, suggesting its use for additional quality assessment of gene editing in mice and also other model organisms.

Background Binding of Slit ligands to their Robo receptors regulates signaling pathways that are important for heart development. Genetic variants in ROBO1and ROBO4 have been linked to congenital heart defects in humans. These defects are recapitulated in mouse models with ubiquitous deletions of the Slit ligands or Robo receptors and include additional heart defects not currently linked to SLIT or ROBO mutations in humans. Given the broad expression patterns of these genes, the question remains open which tissue‐specific ligand‐receptor interactions are important for the correct development of different cardiac structures. Methods and Results We used tissue‐specific knockout mouse models of Robo1/Robo2, Robo4, Slit2 andSlit3 and scored cardiac developmental defects in perinatal mice. Knockout of Robo2 in either the whole heart, endocardium and its derivatives, or the neural crest in ubiquitous Robo1 knockout background resulted in ventricular septal defects. Neural crest‐specific removal of Robo2 in Robo1 knockouts showed fully penetrant bicuspid aortic valves (BAV). Endocardial knock‐out of either Slit2or Robo4 caused low penetrant BAV. In contrast, endocardial knockout of Slit3 using a newly generated line resulted in fully penetrant BAV, while removal from smooth muscle cells also resulted in BAV. Caval vein and diaphragm defects observed in ubiquitous Slit3 mutants were recapitulated in the tissue‐specific knockouts. Conclusions Our data will help understand defects observed in patients with variants in ROBO1 and ROBO4. The results strongly indicate interaction between endocardial Slit3and neural crest Robo2 in the development of BAV, highlighting the need for further studies of this connection.

BackgroundDegenerative cervical myelopathy (DCM) is a chronic neurological condition estimated to affect 1 in 50 adults. Due to its diverse impact, trajectory and management options, patient-centred care and shared decision making are essential. In this scoping review, we aim to explore whether information needs in DCM are currently being met in available DCM educational resources. This forms part of a larger Myelopathy.org project to promote shared decision making in DCM.MethodsA search was completed encompassing MEDLINE, Embase and grey literature. Resources relevant to DCM were compiled for analysis. Resources were grouped into 5 information types: scientific literature, videos, organisations, health education websites and patient information leaflets. Resources were then further arranged into a hierarchical framework of domains and subdomains, formed through inductive analysis. Frequency statistics were employed to capture relative popularity as a surrogate marker of potential significance.ResultsOf 2674 resources, 150 information resources addressing DCM were identified: 115 scientific literature resources, 28 videos, 5 resources from health organisations and 2 resources from health education websites. Surgical management was the domain with the largest number of resources (66.7%, 100/150). The domain with the second largest number of resources was clinical presentation and natural history (28.7%, 43/150). Most resources (83.3%, 125/150) were designed for professionals. A minority (11.3% 17/150) were written for a lay audience or for a combined audience (3.3%, 5/150).ConclusionEducational resources for DCM are largely directed at professionals and focus on surgical management. This is at odds with the needs of stakeholders in a lifelong condition that is often managed without surgery, highlighting an unmet educational need.

Pathogenic variants in ACTN2, coding for alpha-actinin 2, are known to be rare causes of Hypertrophic Cardiomyopathy. However, little is known about the underlying disease mechanisms. Adult heterozygous mice carrying the Actn2 p.Met228Thr variant were phenotyped by echocardiography. For homozygous mice, viable E15.5 embryonic hearts were analysed by High Resolution Episcopic Microscopy and wholemount staining, complemented by unbiased proteomics, qPCR and Western blotting. Heterozygous Actn2 p.Met228Thr mice have no overt phenotype. Only mature males show molecular parameters indicative of cardiomyopathy. By contrast, the variant is embryonically lethal in the homozygous setting and E15.5 hearts show multiple morphological abnormalities. Molecular analyses, including unbiased proteomics, identified quantitative abnormalities in sarcomeric parameters, cell-cycle defects and mitochondrial dysfunction. The mutant alpha-actinin protein is found to be destabilised, associated with increased activity of the ubiquitin-proteasomal system. This missense variant in alpha-actinin renders the protein less stable. In response, the ubiquitin-proteasomal system is activated; a mechanism that has been implicated in cardiomyopathies previously. In parallel, a lack of functional alpha-actinin is thought to cause energetic defects through mitochondrial dysfunction. This seems, together with cell-cycle defects, the likely cause of the death of the embryos. The defects also have wide-ranging morphological consequences.

The morbidity associated with pancreatectomies limits surgical options for high-risk patients with pancreatic neoplasms that warrant resection. Endoscopic ultrasound-guided radiofrequency ablation (EUS-RFA) offers a minimally invasive and potentially definitive means to treat pancreatic neuroendocrine tumors and precancerous pancreatic cystic lesions. In addition, EUS-RFA may play a role in the treatment and palliation of non-surgical cases of pancreatic adenocarcinoma. The efficacy of RFA appears to be further enhanced by systemic immunomodulatory effects. Here, we review current studies on the developing role of EUS-RFA in these pancreatic pathologies.

Objectives To explore the frequency and nature of complaints and compliments reported to Patient Advice and Liaison (PALS) in individuals undergoing surgery for a chronic subdural haematoma (cSDH).Design A retrospective study of PALS user interactions.Subjects Individuals undergoing treatment for cSDH between 2014 and 2019.Methods PALS referrals from patients with cSDH between 2014 and 2019 were identified. Case records were reviewed and data on the frequency, nature and factors leading up to the complaint were extracted and coded according to Healthcare Complaints Analysis Tool (HCAT).Results Out of 531 patients identified, 25 (5%) had a PALS interaction, of which 15 (3%) were complaints and 10 (2%) were compliments. HCAT coding showed 8/15 (53%) of complaints were relationship problems, 6/15 (33%) a management problem and 1/15 (7%) other. Of the relationship problems, 6 (75%) were classed as problems with communication and 2 (25%) as a problem with listening. Of the compliments, 9/10 (90%) related to good clinical quality and 1/10 (10%) to staff–patient relationship. Patients were more likely to register a compliment than family members, who in turn were more likely to register a complaint (p

During meiotic recombination, genetic information is transferred or exchanged between parental chromosome copies. Using a large hybrid mouse pedigree, the authors generated high-resolution maps of these transfer/exchange events and discovered new properties governing their processing and resolution.

This paper presents an investigation into the experiences and perceptions of volunteers and community managers of an Australian voluntary-sector organization that supports young help-seeking people. The process focused specifically on the design of a chat tool, a rudimentary version of which was conceptualized and tested during a trial completed prior to this study. The process explored the motivations and experiences of these volunteers using a codesign approach, which led to the development of specific features of the chat tool that were tailored to the nature of their work and organization, as well as the sector-specific ethos. We employed several research methods, which included interviews, focus groups, and participatory design workshops. Thematic analyses were performed on the resultant qualitative data. The methods, motivational themes, and the ensuing design solutions that were implemented are discussed in detail with the aim of encouraging codesign of technology for voluntary-sector organizations.

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of ‘sporadic’ cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.

Understanding how fertilizer application (particularly N, the most used chemical fertilizer worldwide) interacts with soil microbes is important for the development of best management practices that target improved microbial activity to enhance sustainable food production. This study was conducted to determine whether urea N rate and time of application to maize (Zea mays) influenced soil enzyme activity. Enzyme activity was determined by monitoring fluorescein diacetate (FDA) hydrolysis, ß-glucosidase, acid-phosphomonoesterase, and arylsulfatase activities. Experiments were conducted from 2014 through 2016 to compare single (fall or spring applications) and split applications of N at varying N rates under irrigation (Becker) and rainfed conditions (Lamberton and Waseca) in MN, USA. Nitrogen rates varied by location and were based on University of Minnesota guidelines. Soil samples were collected seven times each season. Nitrogen application split into two applications increased FDA activity by 10% compared with fall and spring applied N at Waseca. Fall or spring N application decreased arylsulfatase activity by 19% at Becker and by between 13% and 16% at Lamberton. ß-Glucosidase and acid-phosphomonoesterase activities were unaffected by N application. Sampling time and year had the greatest impact on enzyme activity, but the results varied by location. A negative linear relationship occurred between FDA and ß-glucosidase activity at all three sites. In summary, urea N application had small effects on enzyme activity at the sites studied, suggesting that some form of organic N could be more important than the ammonium provided by urea.

Introduction Integrated care pathways (ICPs) are a pre-defined framework of evidence based, multidisciplinary practice for specific patients. They have the potential to enhance continuity of care, patient safety, patient satisfaction, efficiency gains, teamwork and staff education. In order to inform the development of neurosurgical ICPs in the future, we performed a systematic review to aggregate examples of neurosurgical ICP, to consider their impact and design features that may be associated with their success. Methods Electronic databases MEDLINE, EMBASE, and CENTRAL were searched for relevant literature published from date of inception to July 2020. Primary studies reporting details of neurosurgical ICPs, across all pathologies and age groups were eligible for inclusion. Patient outcomes in each case were also recorded. Results Twenty-four studies were included in our final dataset, from the United States, United Kingdom, Italy, China, Korea, France, Netherlands and Switzerland, and a number of sub-specialties. 3 for cerebrospinal fluid diversion, 1 functional, 2 neurovascular, 1 neuro-oncology, 2 paediatric, 2 skull base, 10 spine, 1 for trauma, 2 miscellaneous (other craniotomies). All were single centre studies with no regional or national examples. Thirteen were cohort studies while 11 were case series which lacked a control group. Effectiveness was typically evaluated using hospital or professional performance metrics, such as length of stay (n = 11, 45.8%) or adverse events (n = 17, 70.8%) including readmission, surgical complications and mortality. Patient reported outcomes, including satisfaction, were evaluated infrequently (n = 3, 12.5%). All studies reported a positive impact. No study reported how the design of the ICP was informed by published literature or other methods Conclusions ICPs have been successfully developed across numerous neurosurgical sub-specialities. However, there is often a lack of clarity over their design and weaknesses in their evaluation, including an underrepresentation of the patient’s perspective.

Introduction Degenerative cervical myelopathy (DCM) is a common, disabling and progressive neurological condition triggered by chronic compression of the cervical spinal cord by surrounding degenerative changes. Early diagnosis and specialist management are essential to reduce disability, yet time to diagnosis is typically prolonged. Lack of sufficient representation of DCM in undergraduate and postgraduate medical curricula may contribute to the poor recognition of DCM by non-specialist doctors in clinical practice.In this study, our objective, therefore, is to assess DCM teaching provision in medical schools throughout the UK and to assess the impact of teaching on the DCM knowledge of UK medical students.Methods and analysis A 19-item questionnaire capturing data on medical student demographics, myelopathy teaching and myelopathy knowledge was designed. Ethical approval was granted by the Psychology Research Ethics Committee, University of Cambridge. An online survey was hosted on Myelopathy.org, an international myelopathy charity. Students studying at a UK medical school are eligible for inclusion. The survey is advertised nationally through university social media pages, university email bulletins and the national student network of Myelopathy.org. Advertisements are scheduled monthly over a 12-month recruitment period. Participation is incentivised by entering consenting participants of completed surveys to an Amazon voucher prize draw. Responses are anonymised using participant-chosen unique identifier codes. A participant information sheet followed by an explicit survey question captures participant informed consent. Regular updates on the progress of the study will be published on Myelopathy.org.Ethics and Dissemination Ethical approval for the study was granted by the Psychology Research Ethics Committee, University of Cambridge (PRE.2018.099). The findings of the study described in this protocol, and all other related work, will be submitted for publication in a peer-reviewed journal and will be presented at scientific conferences.

There is a growing use of agent-based model (ABM) simulations to reconstruct past human-environment interactions. ABMs are useful in that they offer scientists the opportunity to model processes and study phenomena and systems that may not be otherwise reproducible or testable. Replication or re-implementation studies of ABMs are, however, infrequently undertaken, and there are few examples within archaeology or other social sciences. This paper documents the process of a successful ABM replication study, as well as two additional modifications to the original model. Results corroborate the findings of the original geoarchaeological model and indicate that episodic geomorphic events significantly affect archaeological deposit formation and the inferences drawn from associated radiocarbon records. One revision of the model further demonstrates that episodic fluvial events can create highly varied radiocarbon distributions. The second modification illustrates that excavation data helps to fill in hiatuses in radiocarbon chronologies on depositional landforms, although there is no effect across landscapes subject to erosion. This successful replication exercise also illustrates the value of Open Access data and analyses in reproducing, testing, and expanding upon archaeological research and theory building.

Timing and rate of nitrogen (N) fertilizer application can influence maize (Zea mays L.) grain yield, N uptake, and nitrogen use efficiency (NUE) parameters, but results have been inconsistent across the upper Midwest. This study compared single (fall and preplant) and split applications of differing N rates for maize under irrigated conditions on loamy sand at Becker, MN and under rainfed conditions on loam and clay loam soils at Lamberton, MN and Waseca, MN, respectively, in 2014 to 2016. Fall and preplant applications of N were applied at recommended and 125% of recommended rates (RN) according to University of Minnesota guidelines. Split-application treatments included a two-way (Sp, applied at 75% and 100% of RN) and a three-way split (TSp applied at 50%, 75%, and 100% of RN), with the total N rate equally split among application times. At Becker, maize grain yield with TSp was 12.6 to 15.7 Mg ha-1 among years and significantly greater than that with fall or preplant treatments. The TSp treatment also improved agronomic efficiency (AE) and recovery efficiency (RE) by an average of 30% over fall or preplant treatments. At Lamberton, maize grain yield, AE and RE did not differ among treatments. However, TSp75 improved AE by 8.3 kg kg-1 while producing comparable yields to fall and preplant treatments. At Waseca, Sp or TSp improved grain yield and AE compared with fall treatments. These results suggest that split applications of N can increase maize grain yield, AE, and RE on irrigated coarse-textured soils and applying N fertilizer near planting or as a split application can improve N management on non-irrigated clay loam soils.

Avulsion fracture of the tibial tuberosity is an infrequent injury in adolescents and an extremely rare occurrence in adults. We describe the case of an 86-year-old gentleman presenting after a fall, sustaining injury to the left knee. Radiographs of the left knee showed avulsion fracture of the tibial tuberosity. The purpose of this study was to present a rare case of tibial tuberosity avulsion fracture in an adult, the treatment performed, and the challenges faced. The case is discussed with the review of the literature.

Priming of the adult mouse heart with Tβ4 activates dormant epicardium-derived cells to aid repair of injured myocardium. Here, Vieiraet al. explain this process and show that Tβ4 binds a chromatin remodeller BRG1 and activates Wt1, the key regulator of epicardial epithelial-to-mesenchymal transformation, by altering the epigenetic landscape of the Wt1 locus.

Abstract Background Cellular-based therapies represent a platform technology within the rapidly expanding field of regenerative medicine and are distinct from conventional therapeutics—offering a unique approach to managing what were once considered untreatable diseases. Despite a significant increase in basic science activity within the cell therapy arena, alongside a growing portfolio of cell therapy trials and promising investment, the translation of cellular-based therapeutics from “bench to bedside” remains challenging, and the number of industry products available for widespread clinical use remains comparatively low. This systematic review identifies unique intrinsic and extrinsic barriers in the cell-based therapy domain. Methods/design Eight electronic databases will be searched, specifically Medline, EMBASE (OvidSP), BIOSIS & Web of Science, Cochrane Library & HEED, EconLit (ProQuest), WHOLIS WHO Library Database, PAIS International (ProQuest), and Scopus. Addition to this gray literature was searched by manually reviewing relevant work. All identified articles will be subjected for review by two authors who will decide whether or not each article passes our inclusion/exclusion criteria. Eligible papers will subsequently be reviewed, and key data extracted into a pre-designed data extraction scorecard. An assessment of the perceived impact of broad commercial barriers to the adoption of cell-based therapies will be conducted. These broad categories will include manufacturing, regulation and intellectual property, reimbursement, clinical trials, clinical adoption, ethics, and business models. This will inform further discussion in the review. There is no PROSPERO registration number. Discussion Through a systematic search and appraisal of available literature, this review will identify key challenges in the commercialization pathway of cellular-based therapeutics and highlights significant barriers impeding successful clinical adoption. This will aid in creating an adaptable, acceptable, and harmonized approach supported by apposite regulatory frameworks and pertinent expertise throughout the respective stages of the adoption cycle to facilitate the adoption of new products and technologies in the industry.

Recent advances have made pluripotent stem cell (PSC)-derived cardiomyocytes an attractive option to model both normal and diseased cardiac function at the single-cell level. However, in vitro differentiation yields heterogeneous populations of cardiomyocytes and other cell types, potentially confounding phenotypic analyses. Fluorescent PSC-derived cardiomyocyte reporter systems allow specific cell lineages to be labelled, facilitating cell isolation for downstream applications including drug testing, disease modelling and cardiac regeneration. In this review, the different genetic strategies used to generate such reporter lines are presented with an emphasis on their relative technical advantages and disadvantages. Next, we explore how the fluorescent reporter lines have provided insights into cardiac development and cardiomyocyte physiology. Finally, we discuss how exciting new approaches using PSC-derived cardiomyocyte reporter lines are contributing to progress in cardiac cell therapy with respect to both graft adaptation and clinical safety.

INTRODUCTION:Degenerative Cervical Myelopathy [DCM] often presents with non-specific symptoms and signs. It progresses insidiously and leads to permanent neurological dysfunction. Decompressive surgery can halt disease progression, however significant delays in diagnosis result in increased disability and limit recovery. The nature of early DCM symptoms is unknown, moreover it has been suggested incomplete examination contributes to missed diagnosis. This study examines how DCM is currently assessed, if assessment differs between stages of healthcare, and whether this influences patient management. STUDY DESIGN:Retrospective cohort study. METHODS:Cervical MRI scans (N = 1123) at a tertiary neurosciences center, over a single year, were screened for patients with DCM (N = 43). Signs, symptoms, and disease severity of DCM were extracted from patient records. Patients were considered at 3 phases of clinical assessment: primary care, secondary care, and surgical assessment. RESULTS:Upper limb paraesthesia and urinary dysfunction were consistently the most and least prevalent symptoms respectively. Differences between assessing clinicians were present in the reporting of: limb pain (p

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply.

The identification of mutated metabolic enzymes in hereditary cancer syndromes has established a direct link between metabolic dysregulation and cancer. Mutations in the Krebs cycle enzyme, fumarate hydratase (FH), predispose affected individuals to leiomyomas, renal cysts, and cancers, though the respective pathogenic roles of mitochondrial and cytosolic FH isoforms remain undefined. On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism. Remarkably, transgenic re-expression of cytosolic FH ameliorated both renal cyst development and urea cycle defects associated with renal-specific FH1 deletion in mice. Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls. Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

Learning about what young children with limited spoken language know about the grammar of their language is extremely challenging. Researchers have traditionally used looking behavior as a measure of language processing and to infer what overt choices children might make. However, these methods are expensive to setup, require specialized training, are time intensive for data analysis and can have considerable dropout rates. For these reasons, we have developed a forced choice task delivered on an iPad based on our eye-tracking studies with English monolinguals (Davies et al., accepted; Davies et al., under review). Using the iPad we investigated 3- and 4-year-olds’ understanding of the English plural in preschool centers. The primary aim of the study was to provide evidence for the usefulness of the iPad as a language research tool. We evaluated the usefulness of the iPad with second language (L2) learning children who have limited L2 language skills. Studies with school aged Chinese-speaking children show below native performance on English inflectional morphology despite 5-6 years of immersion (Jia, 2003; Jia & Fuse, 2007; Paradis et al., 2016). However, it is unclear whether this is specific only to children who speak Chinese as their first language (L1) or if younger preschoolers will also show similar challenges. We tested three groups of preschoolers with different L1s (English, Chinese, and Other languages). L1 Chinese children’s performance was below both English monolinguals and children speaking Other L1 languages, providing evidence that English inflections are specifically challenging for Chinese-speaking children. The results provide further evidence to support previous eye-tracking findings with monolinguals and studies with older bilinguals. The study provides evidence for the usefulness of iPads as research tool for studying language acquisition. Implications for future application of the iPad as a teaching and intervention tool, and limitations for the method, are discussed.

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain.

The principal argument of this paper is that migrant women with secure mobility rights and supportive social networks can avoid or mitigate many trafficking harms. However the paper contends that some actors have conspired to prevent such circumstances so as to pursue diverse political agendas at the expense of migrant women. The paper’s analysis restructures the trafficking contest from organised criminals versus law enforcement agencies to principally a contest between migrant women and those political agents who benefit from the moral panic associated with trafficking. It is then argued that it is these more sophisticated political actors rather than organised criminals and the clients of sex workers are the most important stakeholders in sustaining or exploiting trafficking harm. Therefore, it is concluded that resolving many trafficking harms in the EEA could be achieved by subverting political traffickers through improving migration policy rather than fighting organised crime.

Most retroperitoneal tumors such as renal cell carcinoma have been associated with tumor thrombus extending into the renal vein, inferior vena cava (IVC), and heart. The retroperitoneal metastatic potential of testicular tumors is well known. We report here the first instance of a cardiac murmur prompting diagnosis of metastatic testicular neoplasia in an 18-year-old patient. Chemotherapy was delayed and after successful surgical resection of the ventricular mass, the patient recovered uneventfully. This case underscores the need to pursue abnormal cardiac exams in newly diagnosed testicular cancer patients.

Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing.