A personal reflection upon navigating into a senior academic role.

There are 22,795 University professors in the UK, where 6,340 are women and only 40 are Black women, whilst Asian women are a few more in number. This clearly demonstrates the uncommon narrative of the under-representation of Black minority ethnic (BME) academics in higher education (HE) which has been written about in detail. In contrast, it is rare to read reports on initiatives to successfully navigate senior academic posts. In this article, I will describe two initiatives that I have developed and organized to successfully navigate senior BME academic posts, which have impacted my journey. The first initiative was to understand why postdoctoral researchers were "post-docing" for years, having not been successful in making the transition to lecturers. What was hindering the transition? I was one of them, and some of my female peers as well, who incidentally left HE. I was determined not to leave. I again thought about how to tackle it. It is a known fact that hearing successful BME people experiences and journeys and also understanding how they navigated HE can be powerful. In addition, empowering oneself with additional skills including mentoring, networking, applying for positions, not excluding ourselves due to the lack of confidence, and finally, the importance of having a work-life balance is important as health is wealth. I used this to put together the BME Early Career Researcher (ECR) conference-How to Stay in Academia. After 6 years, it is still going strong. In this article, I will share the impact made over the years which will include testimonies and promotions, including my recent promotion to an associate professor. The second initiative was to understand the barriers and challenges of senior lecturers being promoted to readers and professors. Having successfully transitioned to a lecturer, being overlooked for promotion was now an issue. The project was conducted in 2016/17 at KCL as part of the action plans that needed to be delivered having been a recipient of the Bronze Race Equality Charter Mark. I was provided with a cohort of 51 names of BME staff from different disciplines and was directed to see how I would engage them to hear their experiences. My first concern was that the staff would have engaged in previous initiatives with little or no benefits to them; however, this did not deter me. I thought of the best approach which commenced with a phone interview and then followed up with a focus group, ending with an informal conversation with the Principal of the University. Within 6 months, a BME male was promoted to professor. After a year, both genders were promoted to associate professors (readers) and professors, and to date, I am aware of at least 10 promotions. In both examples, I will demonstrate the support from our allies, some of whom are senior leaders that have openly supported us in our journey. This article will demonstrate a slight shift in the narrative, but a lot more needs to be done, and I am convinced the time is right to start pushing for more. This special issue is an example.

In vitro osteoinductive potential of porous monetite for bone tissue engineering.

Tissue engineering-based bone grafts are emerging as a viable alternative treatment modality to repair and regenerate tissues damaged as a result of disease or injury. The choice of the biomaterial component is a critical determinant of the success of the graft or scaffold; essentially, it must induce and allow native tissue integration, and most importantly mimic the hierarchical structure of the native bone. Calcium phosphate bioceramics are widely used in orthopaedics and dentistry applications due to their similarity to bone mineral and their ability to induce a favourable biological response. One such material is monetite, which is biocompatible, osteoconductive and has the ability to be resorbed under physiological conditions. The osteoinductive properties of monetite in vivo are known; however, little is known of the direct effect on osteoinduction of human mesenchymal stem cells in vitro. In this study, we evaluated the potential of monetite to induce and sustain human mesenchymal stem cells towards osteogenic differentiation. Human mesenchymal stem cells were seeded on the monetite scaffold in the absence of differentiating factors for up to 28 days. The gene expression profile of bone-specific markers in cells on monetite scaffold was compared to the control material hydroxyapatite. At day 14, we observed a marked increase in alkaline phosphatase, osteocalcin and osteonectin expressions. This study provides evidence of a suitable material that has potential properties to be used as a tissue engineering scaffold.

Analysis of giant cell tumour of bone cells for Noonan syndrome/cherubism-related mutations.

BACKGROUND: Giant cell tumour of bone (GCTB) is an osteolytic tumour which contains numerous osteoclast-like giant cells and a proliferation of mononuclear stromal cells (MSC). Giant cell-rich osteolytic lesions can also develop in the jaw bones in Noonan syndrome, a cherubism-like developmental abnormality that is transmitted in an autosomal dominant fashion, often because of mutation in the PTPN11 or BRAF genes. METHODS: We screened GCTBs for mutations in PTPN11 and BRAF to determine whether GCTBs develop through alterations of genes involved in Noonan syndrome. MSC were isolated from 10 GCTBs. RESULTS: Chromosome banding analysis of these cells revealed telomeric associations (tas) in 7 of the 10 cases. Thus, the cultured cells expressed a cytogenetic abnormality typically found in short-term cultures from GCTBs. Sequencing of DNA extracted from the seven GCTB-derived MSC cultures displaying tas did not identify any mutation in PTPN11 or in exons 9-15 of BRAF. CONCLUSION: Our findings indicate that the molecular pathways involved in GCTB development are different from those causing Noonan syndrome. The method for isolating and culturing GCTB stromal cells described in this study generated a population of MSC that contained tas, indicating that it is useful for obtaining stromal cells from GCTB and other giant cell-rich lesions, such as giant cell reparative granuloma, for genetic and other studies.

P16/p53 expression and telomerase activity in immortalized human dental pulp cells.

INTRODUCTION: Residing within human dental pulp are cells of an ectomesenchymal origin which have the potential to differentiate into odontoblast-like cells. These cells have a limited growth potential owing to the effects of cell senescence. This study examines the effects of immortalizing odontoblast-like cells on cell proliferation and mineralization by comparing transformed dental pulp stem cells (tDPSCs) and non-transformed dental pulp stem cells (nDPSCs). RESULTS: With the exogenous expression of hTERT, tDPSCs maintained a continued expression of odontogenic markers for cell proliferation and mineralization (ALP, COL-1, DMP-1, DSPP, OCN amd OPN)as did nDPScs. Oncoprotein expression was seen in both groups except for a noted absence of p16 in the tDPSCs. nDPSCs also showed lower levels of total ALP and DNA activity in comparison to tDPSCs when assayed as well as low telomerase activity readings. METHODS: Using a retroviral vector, exogenous human telomerase reverse transcriptase (hTERT) was expressed in tDPSCs. Both cell groups were cultured and their telomerase activities is determined using a telomerase quantification assay. Also examined were the expression of genes involved in proliferation and mineralization such as human alkaline phosphatase (ALP), ß-actin, collagen 1 (col-1), core binding factor (cbfa-1), dentin matrix protein (DMP-1), dentin sialophosphoprotein (DSPP), GAPDH, hTERT, osteocalcin (OCN), osteopontin (OPN) as well as oncoproteins involved in senescence (p16, p21 and p53) using RT-PCR. DNA and alkaline phosphatase activity was assayed in both cell groups. CONCLUSIONS: These results indicate maintainance of odontoblast-like differentiation characteristics after retroviral transformation with hTERT and suggest a possible link with a reduced p16 expression.

Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors.

Aberrant DNA methylation (DNAm) was first linked to cancer over 25 yr ago. Since then, many studies have associated hypermethylation of tumor suppressor genes and hypomethylation of oncogenes to the tumorigenic process. However, most of these studies have been limited to the analysis of promoters and CpG islands (CGIs). Recently, new technologies for whole-genome DNAm (methylome) analysis have been developed, enabling unbiased analysis of cancer methylomes. By using MeDIP-seq, we report a sequencing-based comparative methylome analysis of malignant peripheral nerve sheath tumors (MPNSTs), benign neurofibromas, and normal Schwann cells. Analysis of these methylomes revealed a complex landscape of DNAm alterations. In contrast to what has been reported for other tumor types, no significant global hypomethylation was observed in MPNSTs using methylome analysis by MeDIP-seq. However, a highly significant (P < 10(-100)) directional difference in DNAm was found in satellite repeats, suggesting these repeats to be the main target for hypomethylation in MPNSTs. Comparative analysis of the MPNST and Schwann cell methylomes identified 101,466 cancer-associated differentially methylated regions (cDMRs). Analysis showed these cDMRs to be significantly enriched for two satellite repeat types (SATR1 and ARLα) and suggests an association between aberrant DNAm of these sequences and transition from healthy cells to malignant disease. Significant enrichment of hypermethylated cDMRs in CGI shores (P < 10(-60)), non-CGI-associated promoters (P < 10(-4)) and hypomethylated cDMRs in SINE repeats (P < 10(-100)) was also identified. Integration of DNAm and gene expression data showed that the expression pattern of genes associated with CGI shore cDMRs was able to discriminate between disease phenotypes. This study establishes MeDIP-seq as an effective method to analyze cancer methylomes.

Role of the transcription factor T (brachyury) in the pathogenesis of sporadic chordoma: a genetic and functional-based study.

A variety of analyses, including fluorescence in situ hybridization (FISH), quantitative PCR (qPCR) and array CGH (aCGH), have been performed on a series of chordomas from 181 patients. Twelve of 181 (7%) tumours displayed amplification of the T locus and an additional two cases showed focal amplification; 70/181 (39%) tumours were polysomic for chromosome 6, and 8/181 (4.5%) primary tumours showed a minor allelic gain of T as assessed by FISH. No germline alteration of the T locus was identified in non-neoplastic tissue from 40 patients. Copy number gain of T was seen in a similar percentage of sacrococcygeal, mobile spine and base of skull tumours. Knockdown of T in the cell line, U-CH1, which showed polysomy of chromosome 6 involving 6q27, resulted in a marked decrease in cell proliferation and morphological features consistent with a senescence-like phenotype. The U-CH1 cell line was validated as representing chordoma by the generation of xenografts, which showed typical chordoma morphology and immunohistochemistry in the NOD/SCID/interleukin 2 receptor [IL2r]gammanull mouse model. In conclusion, chromosomal aberrations resulting in gain of the T locus are common in sporadic chordomas and expression of this gene is critical for proliferation of chordoma cells in vitro.

The role of epidermal growth factor receptor in chordoma pathogenesis: a potential therapeutic target.

Chordoma, the molecular hallmark of which is T (brachyury), is a rare malignant bone tumour with a high risk of local recurrence and a tumour from which metastatic disease is a common late event. Currently, there is no effective drug therapy for treating chordomas, although there is evidence that some patients respond to the empirical use of epidermal growth factor receptor (EGFR) antagonists. The aim of this study was to determine the role of EGFR in the pathogenesis of chordoma. Paraffin-embedded material from 173 chordomas from 160 patients [sacro-coccygeal (n = 94), skull-based (n = 50), and mobile spine (n = 16)] was analysed by immunohistochemistry and revealed total EGFR expression in 69% of cases analysed. Of 147 informative chordomas analysed by FISH, 38% revealed high-level EGFR polysomy, 4% high-level polysomy with focal amplification, 18% low-level polysomy, and 39% disomy. Phospho-receptor tyrosine kinase array membranes showed EGFR activation in the chordoma cell line U-CH1 and all of the three chordomas analysed. Direct sequencing of EGFR (exons 18-21), KRAS, NRAS, HRAS (exons 2, 3), and BRAF (exons 11, 15) using DNA from 62 chordomas failed to reveal mutations. PTEN expression was absent by immunohistochemistry in 19 of 147 (13%) analysed chordomas, only one of which revealed high-level polysomy of EGFR. The EGFR inhibitor tyrphostin (AG 1478) markedly inhibited proliferation of the chordoma cell line U-CH1 in vitro and diminished EGFR phosphorylation in a dose-dependant manner, a finding supported by inhibition of phosphorylated Erk1/2. p-Akt was suppressed to a much lesser degree in these experiments. There was no reduction of T as assessed by western blotting. These data implicate aberrant EGFR signalling in the pathogenesis of chordoma. This study provides a strategy for patient stratification for treatment with EGFR antagonists.

Familial tumoral calcinosis and hyperostosis-hyperphosphataemia syndrome are different manifestations of the same disease: novel missense mutations in GALNT3.

OBJECTIVE: To report on the biochemistry and clinical and genetic findings of two siblings, the younger sister presenting with recurrent bone pain of the radius and ulna, and medullary sclerosis, and the older brother with soft tissue calcific deposits (tumoral calcinosis) but who later developed bone pain. Both were found to be hyperphosphaturic. MATERIALS AND METHODS: The index family comprised four individuals (father, mother, brother, sister). The affected siblings were the offspring of a non-consanguineous Indian family of Tamil origin. Bidirectional sequencing was performed on the DNA from the index family and on 160 alleles from a population of 80 unrelated unaffected control individuals of Tamil extraction and 72 alleles from individuals of non-Tamil origin. RESULTS: Two symptomatic siblings were found to harbour previously unreported compound heterozygous missense UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-transferase; GALNT3) mutations in exon 4 c.842A>G and exon 5 c.1097T>G. This sequence variation was not detected in the control DNA. This is the first report of siblings exhibiting stigmata of familial tumoral calcinosis and hyperostosis-hyperphosphataemia syndrome with documented evidence of autosomal recessive missense GALNT3 mutations. CONCLUSION: The findings from this family add further evidence to the literature that familial tumoral calcinosis and hyperostosis-hyperphosphataemia syndrome are manifestations of the same disease and highlight the importance of appropriate metabolic and genetic investigations.

Analysis of the fibroblastic growth factor receptor-RAS/RAF/MEK/ERK-ETS2/brachyury signalling pathway in chordomas.

Chordomas are rare primary malignant bone tumours that derive from notochord precursor cells and express brachyury, a molecule involved in notochord development. Little is known about the genetic events responsible for driving the growth of this tumour, but it is well established that brachyury is regulated through fibroblastic growth factor receptors (FGFRs) through RAS/RAF/MEK/ERK and ETS2 in ascidian, Xenopus and zebrafish, although little is known about its regulation in mammals. The aim of this study was to attempt to identify the molecular genetic events that are responsible for the pathogenesis of chordomas with particular focus on the FGFR signalling pathway on the basis of the evidence in the ascidian and Xenopus models that the expression of brachyury requires the activation of this pathway. Immunohistochemistry showed that 47 of 50 chordomas (94%) expressed at least one of the FGFRs, and western blotting showed phosphorylation of fibroblast growth factor receptor substrate 2 alpha (FRS2alpha), an adaptor signalling protein, that links FGFR to the RAS/RAF/MEK/ERK pathway. Screening for mutations in brachyury (all coding exons and promoter), FGFRs 1-4 (previously reported mutations), KRAS (codons 12, 13, 51, 61) and BRAF (exons 11 and 15) failed to show any genetic alterations in 23 chordomas. Fluorescent in situ hybridisation analysis on FGFR4, ETS2 and brachyury failed to show either amplification of these genes, although there was minor allelic gain in brachyury in three tumours, or translocation for ERG and ETS2 loci. The key genetic events responsible for the initiation and progression of chordomas remain to be discovered.

GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma.

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabraud's syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

Mutations in SH3BP2, the cherubism gene, were not detected in central or peripheral giant cell tumours of the jaw.

Giant cell granulomas of the jaw (GCGJ) are non-familial, generally unilateral osteoclast-rich lesions that are histopathologically indistinguishable from cherubism. Cherubism is an autosomal dominant disease that is characterised by bilateral radiolucencies of the jaw, and caused by mutations that occur in SH3BP2 exon 10. The aim of the study was to screen lesional GCGJ tissue for SH3BP2 mutations. Lesional mononuclear stromal or spindle cells were microdissected from paraffin-embedded tissue from GCGJ, and DNA was then extracted and sequenced for SH3BP2 mutations associated with cherubism. No mutations were detected in 26 GCGJ (15 central, 11 peripheral), which indicated that people with GCGJ do not harbour cherubism-related germline SH3BP2 mutations, and that GCGJ do not harbour somatic SH3BP2 mutations. This suggests that cherubism and GCGJ arise on a different genetic background, and therefore detection of SH3BP2 mutations can be a useful means of distinguishing between them.

Detection of beta-catenin mutations in paraffin-embedded sporadic desmoid-type fibromatosis by mutation-specific restriction enzyme digestion (MSRED): an ancillary diagnostic tool.

Desmoid-type fibromatosis is a locally aggressive deep soft tissue tumor. Some cases are associated with adenosis polyposis coli germline mutations whereas others harbor somatic beta-catenin point mutations mainly in exon 3, codons 41 and 45. These mutations result in stabilization of beta-catenin, and activation of the Wnt signaling pathway. The aim of this study was to determine the specificity and sensitivity of these 3 most common beta-catenin mutations in the diagnosis of desmoid-type fibromatosis using paraffin-embedded material. The results were compared with nuclear expression of beta-catenin. Mutation-specific restriction enzyme digestion methodology was employed to detect the 3 mutations. One hundred and thirty-three cases were analyzed, including 76 desmoid-type, and 18 superficial fibromatosis, in addition to a further 39 fibromatosis mimics. A restriction site was present for analysis of the codon 41 mutation. Mismatch primers were designed for the codon 45 mutations. Mutations were detected in 66 cases (87%) of 76 desmoid-type fibromatosis (71 extra-abdominal). Of these, 34 (45%) were in codon 45 (TCT>TTT), 27 (35%) in codon 41 (ACC>GCC), and 5 (7%) in codon 45 (TCT>CCT). No mutations were detected in the other lesions studied. All desmoid-type fibromatosis cases and 72% of the mimics tested showed nuclear positivity for beta-catenin indicating immunohistochemistry is a sensitive but not a specific test for desmoid-type fibromatosis. In contrast, to date, beta-catenin mutations have not been detected in any lesions which mimic desmoid-type fibromatosis. Mutation-specific restriction enzyme digestion, a simple and efficient means of detecting the common beta-catenin mutations in desmoid-type fibromatosis, complements light microscopy in reaching a diagnosis.

Laryngeal abductor muscle reinnervation in a pig model.

OBJECTIVE: To develop a large animal model for studies of laryngeal abductor reinnervation. MATERIAL AND METHODS: Six minipigs underwent unilateral anastomosis of the phrenic nerve-abductor branch of the recurrent laryngeal nerve (RLN). Polyhydroxybutyrate (PHB) conduits were used for repair. At each of 30, 60 and 120 days, 2 animals underwent video laryngeal endoscopy (VLE) and were then killed. VLE was also performed in the 120-day pair at 60 days. Nerve-conduit-nerve-muscle samples were fixed for light and immunofluorescence (pan-neurofilaments, S-100) microscopy. Laryngeal muscles were harvested (myosin heavy chain analysis). RESULTS: VLE showed recovery of abductor function in 1 animal at 60 days and in 1 at 120 days. Haematoxylin-eosin staining demonstrated a complex inflammatory response. Eosinophil recruitment was observed. Stepwise regeneration and reorganization of the distal nerve between 30 and 120 days was observed with pan-NF staining. The mean minimum diameter in the reinnervated posterior crico-arytenoids tended to increase for up to 120 days. CONCLUSIONS: Anastomosis of the phrenic nerve-abductor branch of the RLN with a PHB conduit in a pig can result in functional and histological recovery within 2-4 months and appears to at least sustain abductor muscle fibre morphology. Recovery occurs despite a complex inflammatory response, which may be an essential part of healing rather than inhibitory.

A novel function for the U2AF 65 splicing factor in promoting pre-mRNA 3'-end processing.

Splicing and 3'-end processing (including cleavage and polyadenylation) of vertebrate pre-mRNAs are tightly coupled events that contribute to the extensive molecular network that coordinates gene expression. Sequences within the terminal intron of genes are essential to stimulate pre-mRNA 3'-end processing, although the factors mediating this effect are unknown. Here, we show that the pyrimidine tract of the last splice acceptor site of the human beta-globin gene is necessary to stimulate mRNA 3'-end formation in vivo and binds the U2AF 65 splicing factor. Naturally occurring beta-thalassaemia-causing mutations within the pyrimidine tract reduces both U2AF 65 binding and 3'-end cleavage efficiency. Significantly, a fusion protein containing U2AF 65, when tethered upstream of a cleavage/polyadenylation site, increases 3'-end cleavage efficiency in vitro and in vivo. Therefore, we propose that U2AF 65 promotes 3'-end processing, which contributes to 3'-terminal exon definition.