The genomic landscape of familial glioma.

Glioma is a rare brain tumor with a poor prognosis. Familial glioma is a subset of glioma with a strong genetic predisposition that accounts for approximately 5% of glioma cases. We performed whole-genome sequencing on an exploratory cohort of 203 individuals from 189 families with a history of familial glioma and an additional validation cohort of 122 individuals from 115 families. We found significant enrichment of rare deleterious variants of seven genes in both cohorts, and the most significantly enriched gene was HERC2 (P = 0.0006). Furthermore, we identified rare noncoding variants in both cohorts that were predicted to affect transcription factor binding sites or cause cryptic splicing. Last, we selected a subset of discovered genes for validation by CRISPR knockdown screening and found that DMBT1, HP1BP3, and ZCH7B3 have profound impacts on proliferation. This study performs comprehensive surveillance of the genomic landscape of familial glioma.

Genome sequencing reveals underdiagnosis of primary ciliary dyskinesia in bronchiectasis.

BACKGROUND: Bronchiectasis can result from infectious, genetic, immunological and allergic causes. 60-80% of cases are idiopathic, but a well-recognised genetic cause is the motile ciliopathy, primary ciliary dyskinesia (PCD). Diagnosis of PCD has management implications including addressing comorbidities, implementing genetic and fertility counselling and future access to PCD-specific treatments. Diagnostic testing can be complex; however, PCD genetic testing is moving rapidly from research into clinical diagnostics and would confirm the cause of bronchiectasis. METHODS: This observational study used genetic data from severe bronchiectasis patients recruited to the UK 100,000 Genomes Project and patients referred for gene panel testing within a tertiary respiratory hospital. Patients referred for genetic testing due to clinical suspicion of PCD were excluded from both analyses. Data were accessed from the British Thoracic Society audit, to investigate whether motile ciliopathies are underdiagnosed in people with bronchiectasis in the UK. RESULTS: Pathogenic or likely pathogenic variants were identified in motile ciliopathy genes in 17 (12%) out of 142 individuals by whole-genome sequencing. Similarly, in a single centre with access to pathological diagnostic facilities, 5-10% of patients received a PCD diagnosis by gene panel, often linked to normal/inconclusive nasal nitric oxide and cilia functional test results. In 4898 audited patients with bronchiectasis, <2% were tested for PCD and <1% received genetic testing. CONCLUSIONS: PCD is underdiagnosed as a cause of bronchiectasis. Increased uptake of genetic testing may help to identify bronchiectasis due to motile ciliopathies and ensure appropriate management.

Magseed localisation of non-palpable breast lesions: experience from a single centre.

AIM: To prospectively analyse patients undergoing magnetic seed (Magseed) localisation (MSL) to evaluate the outcome, and to retrospectively compare re-excision rates for MSL with previous wire-guided localisation (WGL) to assess the hypothesis that the introduction of MSL may lead to a lower re-excision rate. MATERIALS AND METHODS: MSL commenced at University Hospital Crosshouse in December 2017. No other changes were made to radiological or surgical practice during this time. Data were collected prospectively on all patients undergoing MSL between December 2017 and December 2019, in a single breast unit. Data were gathered retrospectively on patients who had undergone localised breast procedures between January 2016 and December 2019 for comparison of re-excision rates. RESULTS: Two hundred and fifty-five patients underwent MSL surgery between December 2017 and December 2019. Of those, 98% (n=250) patients underwent successful MSL at the first attempt. The Magseed was identified intraoperatively in 100% patients and surgical excision was performed. The re-excision rate reduced from 18.9% in 2016/2017, to 11.6% in 2018/2019 (p=0.098). CONCLUSION: In conclusion, Magseed localisation has proved to be a safe and effective way of localising breast lesions, with the advantage of high accuracy. The reduction in re-excision rates at University Hospital Crosshouse with the introduction of Magseed® localisation is a potential benefit, which requires further study.

Norwich COVID-19 testing initiative pilot: evaluating the feasibility of asymptomatic testing on a university campus.

BACKGROUND: There is a high prevalence of COVID-19 in university-age students, who are returning to campuses. There is little evidence regarding the feasibility of universal, asymptomatic testing to help control outbreaks in this population. This study aimed to pilot mass COVID-19 testing on a university research park, to assess the feasibility and acceptability of scaling up testing to all staff and students. METHODS: This was a cross-sectional feasibility study on a university research park in the East of England. All staff and students (5625) were eligible to participate. All participants were offered four PCR swabs, which they self-administered over two weeks. Outcome measures included uptake, drop-out rate, positivity rates, participant acceptability measures, laboratory processing measures, data collection and management measures. RESULTS: 798 (76%) of 1053 who registered provided at least one swab; 687 (86%) provided all four; 792 (99%) of 798 who submitted at least one swab had all negative results and 6 participants had one inconclusive result. There were no positive results. 458 (57%) of 798 participants responded to a post-testing survey, demonstrating a mean acceptability score of 4.51/5, with five being the most positive. CONCLUSIONS: Repeated self-testing for COVID-19 using PCR is feasible and acceptable to a university population.

Identification of pathological complete response after neoadjuvant chemotherapy for breast cancer: comparison of greyscale ultrasound, shear wave elastography, and MRI.

AIM: To assess the value of post-treatment shear-wave elastography (SWE) parameters (maximum stiffness [Emax], mean stiffness [Emean], and standard deviation [SD]) compared to greyscale ultrasonography (US) and magnetic resonance imaging (MRI) in identifying pathological complete response (pCR) to neoadjuvant chemotherapy (NACT) in breast cancer. MATERIALS AND METHODS: In a prospective cohort study, 80 patients receiving NACT for breast cancer underwent baseline and post-treatment US, SWE, and MRI examinations. Four SWE images in two orthogonal planes were obtained. Maximum greyscale US diameter and maximum diameter of lesion enhancement on MRI were measured. Percentage reductions between baseline and post-treatment scans were calculated for MRI and greyscale US diameter, and Emean, Emax, and SD. The percentage reduction in Emean and US diameter were also analysed as a combination. Analysis was undertaken using receiver operating characteristic (ROC) curves and the chi-squared test. RESULTS: pCR occurred in 21 of 80 (26%) women. The area under the ROC curve (AUC) for pCR of percentage reductions in Emean, Emax, SD, and greyscale US diameter were 0.89, 0.85, 0.75, and 0.86, respectively. The combination of percentage reductions in Emean and greyscale ultrasound diameter yielded an AUC of 0.92, which is similar to the AUC for MRI of 0.96 (p=0.28). CONCLUSIONS: SWE combined with greyscale US shows promise for end-of-treatment identification of response to NACT in women with breast cancer, with accuracies similar to breast MRI. This technique could be used to inform surgical decision-making after NACT.

Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

The "Lollypop" technique for polyethylene exchange in total ankle replacement.

UNLABELLED: We describe a simple technique for the removal of the polyethylene meniscus bearing surface in patients undergoing re-operation and meniscus bearing exchange following a previous total ankle replacement. LEVEL OF EVIDENCE: Level V.

MicroRNA profiling of peripheral nerve sheath tumours identifies miR-29c as a tumour suppressor gene involved in tumour progression.

BACKGROUND: Neurofibromatosis type 1 is one of the most common familial diseases, the hallmark of which is the development of multiple neurofibromas. These are benign nerve sheath tumours, which can transform into malignant peripheral nerve sheath tumours (MPNST). METHODS: The aim of this study was to identify differentially expressed microRNA (miRNA) in neurofibromas and MPNST obtained from patients with neurofibromatosis type 1 using microarray analysis. Differential expression was validated by reverse transcription quantitative-PCR, and functional studies were performed after transfection of miRNA oligonucleotide mimics into MPNST cells. RESULTS: Sixteen miRNA were significantly differentially expressed in MPNST compared with NF, and of these fourteen were downregulated in MPNST: these included miR-30e*, miR-29c*, miR-29c, miR-340*, miR-30c, miR-139-5p, miR-195, miR-151-5p, miR-342-5p, miR-146a, miR-150, miR-223, let-7 a and let-7 g with a false discovery rate of q=8.48E-03 for the least significant miRNA. In contrast, miR-210 and miR-339-5p were upregulated in MPNST compared with neurofibromas. Prediction softwares/algorithms identified a list of genes targeted by miR-29c including extracellular matrix genes and matrix metalloproteinase (MMP)-2, all of which are reported to be involved in cell migration and invasion. Functional studies in a MPNST cell line, sNF96.2, using a mimic of the mature miR-29c showed reduced invasion, whereas there was no change in proliferation. Zymography of the manipulated cells showed that MMP2 activity was also reduced when miR-29c expression was forced in sNF96.2. CONCLUSION: We provide evidence that reduction of miR-29c has a pivotal role in the progression of nerve sheath tumours and results by increasing the invasive/migratory properties of nerve sheath tumours.

Evidence-based guideline recommendations on the use of positron emission tomography imaging in head and neck cancer.

AIMS: To provide evidence-based practice guideline recommendations on the use of fluoro-2-deoxy-D-glucose positron emission tomography (PET) for diagnosis, staging and assessing treatment response, restaging or recurrence of head and neck cancer. MATERIALS AND METHODS: A systematic review by Facey et al. (Health Technology Assessment 2007;11(44):iii-iv, xi-267) was used as the evidence base for recommendation development. As the review was limited to August 2005, the evidence base was updated to July 2011 using the same search strategies for MEDLINE and EMBASE used in the original review. The authors of the current systematic review drafted recommendations, which were reviewed, adapted and accepted by consensus by the Ontario provincial Head and Neck Disease Site Group and a special meeting of clinical experts. RESULTS: The results of the Facey et al. review for head and neck cancer included five other systematic reviews and 31 primary studies. The 2005 to 2011 update search included four additional systematic reviews and 53 primary studies. Recommendations were developed based on this evidence and accepted by consensus. CONCLUSIONS: PET is recommended in the M and bilateral nodal staging of all patients with head and neck squamous cell carcinoma where conventional imaging is equivocal, or where treatment may be significantly modified. PET is recommended in all patients after conventional imaging and in addition to, or prior to, diagnostic panendoscopy where the primary site is unknown. PET is recommended for the staging and assessment of recurrence of patients with nasopharyngeal carcinoma if conventional imaging is equivocal. PET is recommended for restaging patients who are being considered for major salvage treatment, including neck dissection.

Activin B is produced early in antral follicular development and suppresses thecal androgen production.

Little is known about the role of activin B during folliculogenesis. This study investigated the expression levels of activin/inhibin subunits (ßA, ßB, and α), steroid enzyme, and gonadotrophin receptors in theca (TC) and granulosa cells (GC) by QPCR and activin A and B and inhibin A protein levels in follicular fluid (FF) of developing sheep follicles during estrus and anestrus. The effect of activin B on androgen production from primary TC cultures in vitro was also assessed. During folliculogenesis, in anestrus and estrus, FF activin B concentrations and thecal and GC activin ßB mRNA levels decreased as follicle diameter increased from 1-3 to >6  mm regardless of estrogenic status. Estrogenic preovulatory follicles had reduced concentrations of FF activins B and A, and TC and GCs expressed higher levels of activin ßA mRNA at 3-4  mm, and TCs more inhibin α mRNA at >4  mm stages of development compared with nonestrogenic follicles. Activin B decreased androstenedione production from primary TCs in vitro, an effect blocked by inhibin A. Thus, sheep follicles 1-3  mm in diameter contained high FF levels of activin B, which decreased as the follicle size increased, and, like activin A, suppressed thecal androgen production in vitro, an effect blocked by inhibin. Furthermore, the theca of large estrogenic follicles expressed high levels of inhibin α and activin ßA mRNA suggesting local thecal derived inhibin A production. This would inhibit the negative effects of thecal activins B and A ensuring maximum androgen production for enhanced estradiol production by the preovulatory follicle(s).

Risk management in clinical practice. Part 11. Oral surgery.

Oral surgery is often an unpleasant experience for a patient and if managed inadequately can be a cause for complaint or a claim in negligence. A practitioner can reduce their risk of complaints, claims or even regulatory body investigations by following some straightforward risk management strategies. Effective communication skills deployed throughout the interaction with the patient, especially during the consent process, are a pre-requisite, as is a proper understanding of the law on consent. An honest reflection by the practitioner on their competence to carry out a procedure, considering their skills, the equipment and support available will result in fewer medico-legal cases. In this article, each stage of the patient's journey is discussed and risk management advice offered for a range of procedures that are regularly encountered in general dental practice.

Activated macrophages utilize glycolytic ATP to maintain mitochondrial membrane potential and prevent apoptotic cell death.

We have previously analysed the bioenergetic consequences of activating J774.A1 macrophages (MΦ) with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) and found that there is a nitric oxide (NO)-dependent mitochondrial impairment and stabilization of hypoxia-inducible factor (HIF)-1α, which synergize to activate glycolysis and generate large quantities of ATP. We now show, using tetramethylrhodamine methyl ester (TMRM) fluorescence and time-lapse confocal microscopy, that these cells maintain a high mitochondrial membrane potential (ΔΨ(m)) despite the complete inhibition of respiration. The maintenance of high ΔΨ(m) is due to the use of a significant proportion of glycolytically generated ATP as a defence mechanism against cell death. This is achieved by the reverse functioning of F(o)F(1)-ATP synthase and adenine nucleotide translocase (ANT). Treatment of activated MΦ with inhibitors of either of these enzymes, but not with inhibitors of the respiratory chain complexes I to IV, led to a collapse in ΔΨ(m) and to an immediate increase in intracellular [ATP], due to the prevention of ATP hydrolysis by the F(o)F(1)-ATP synthase. This collapse in ΔΨ(m) was followed by translocation of Bax from cytosol to the mitochondria, release of cytochrome c into the cytosol, activation of caspases 3 and 9 and subsequent apoptotic cell death. Our results indicate that during inflammatory activation 'glycolytically competent cells' such as MΦ use significant amounts of the glycolytically generated ATP to maintain ΔΨ(m) and thereby prevent apoptosis.

Effect of probiotic treatment in broiler chicks on intestinal macrophage numbers and phagocytosis of Salmonella enteritidis by abdominal exudate cells.

Previous data have indicated that a Lactobacillus-based probiotic culture (FM-B11) is efficacious in reducing Salmonella Enteritidis colonization within 24 h when administered within 1 h of challenge. We hypothesized that the innate immune system, specifically macrophages, may play a role in the observed reduction of Salmonella Enteritidis colonization with probiotic treatment. Day-of-hatch chicks were challenged with Salmonella Enteritidis and then treated with the probiotic culture 1 h later. Three other treatment groups were not treated (negative control), challenged only, or treated with probiotic only. In all experiments, probiotic treatment on the day of hatch reduced (P < 0.05) cecal Salmonella Enteritidis recovery as compared with the control treatment. In experiments 1 and 2, immunohistochemistry was used to evaluate the presence of macrophages (KUL01+) in the ileum and cecum of 7 to 10 chicks per group at 24 h posttreatment. In experiment 1, the number of macrophages observed per 10,000 microm(2) in the ileum of Salmonella Enteritidis-challenged chicks was higher (P < 0.05) than that of nonchallenged chicks (4.87 +/- 0.31 vs. 3.05 +/- 0.19). In the cecum, there were more (P < 0.05) macrophages per 10,000 microm(2) in chicks receiving probiotic treatment without challenge than in negative control chicks (5.32 +/- 0.41 vs. 3.66 +/- 0.35). However, in experiment 2 we found no differences among treatments in the numbers of macrophages for both the ileum and cecum. Experiments 3 and 4 were performed to evaluate the ability of Sephadex-elicited abdominal exudate cells (AEC) from chicks to phagocytose Salmonella Enteritidis in vitro. Abdominal exudate cells were isolated from the abdominal cavity, maintained in tissue culture plates overnight, and then assayed for phagocytic activity by coincubating with Salmonella Enteritidis. In experiment 3, more (P < 0.05) Salmonella Enteritidis was recovered from AEC derived from probiotic-treated chicks than in any other treatment. However, in experiment 4, all treatments resulted in similar levels of elicited AEC, and phagocytosis of Salmonella Enteritidis was at low levels in all groups. Although not conclusive, the modest differences detected in experiments 1 and 3, and the fact that those differences were not repeatedly detectable, suggest that these macrophage-related changes were not solely responsible for the reductions of Salmonella Enteritidis following probiotic treatment.

In vitro testing of Advanced JAX Bone Void Filler System: species differences in the response of bone marrow stromal cells to beta tri-calcium phosphate and carboxymethylcellulose gel.

The Advanced JAX Bone Void Filler System (AJBVFS) is a novel bone graft material manufactured by Smith and Nephew Orthopaedics Ltd. and comprises beta tri-calcium phosphate granules with carboxymethylcellulose (CMC) gel as a handling agent. This study investigated the potential, in vitro, of the AJBVFS to function as a delivery system for cell therapy to enhance healing of bone defects. The attachment of rabbit bone marrow stromal cells (rbBMSCs), human BMSCs (hBMSCs) and human bone-derived cells (hBDCs) to JAX granules and the effect of CMC gel on cell proliferation and differentiation were investigated. There were slight species differences in the number and morphology of cells attached on the JAX granules with less rbBMSC attachment than human. All cells tolerated the presence of CMC gel and a reduction in cell number was only seen after longer exposure to higher gel concentrations. Low concentrations of CMC gel enhanced proliferation, alkaline phosphatase (ALP) expression and ALP activity in human cells but had no effect on rbBMSC. This study suggests that AJBVFS is an appropriate scaffold for the delivery of osteogenic cells and the addition of CMC gel as a handling agent promotes osteogenic proliferation and differentiation and is therefore likely to encourage bone healing.

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells.

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.

Brachyury, a crucial regulator of notochordal development, is a novel biomarker for chordomas.

Chordomas are malignant tumours that occur along the spine and are thought to derive from notochordal remnants. There is significant morphological variability between and within chordomas, with some showing prominent areas of chondroid differentiation. Our microarray data from a broad range of connective tissue neoplasms indicate that, at the transcriptional level, chordomas resemble cartilaginous neoplasms. Here we show that chordomas express many genes known to be involved in cartilage development, but they also uniquely express genes distinguishing them from chondroid neoplasms. The brachyury transcription factor, known to be involved in notochordal development, is only expressed by chordomas. Using a polyclonal antibody, we show that brachyury is expressed in the embryonic notochord and in all 53 chordomas analysed, labelling both chondroid and chordoid areas of these tumours. In contrast, the protein was not detected in over 300 neoplasms, including 163 chondroid tumours. Brachyury was not detected in the nucleus pulposus, arguing against the hypothesis that this tissue derives directly from the notochord. These data provide compelling evidence that chordomas derive from notochord and demonstrate that brachyury is a specific marker for the notochord and notochord-derived tumours.

Total ankle replacement. Early experiences with STAR prosthesis.

Early designs of Total Ankle Replacement (TAR) had a high failure rate. More recent experience with the 3-piece, meniscal bearing, total ankle replacement has been more promising. We report a review of the early results of our first 22 prostheses in 20 patients undergoing Scandinavian Total Ankle Replacement (STAR) in Northern Ireland. There was a mean follow-up time of 26 months. Seventeen patients are pain-free at the ankle joint during normal daily activities. Two of the early cases have required revision surgery due to technical errors. Other complications have included malleolar fractures, poor wound healing and postoperative stiffness. These early results show high levels of patient satisfaction, and we are encouraged to continue with total ankle arthroplasty. There is a steep initial learning curve and use of TAR should be restricted to foot and ankle surgeons.