Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1.

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project.Cancer Research UK, Medical Research CouncilThis is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/srep3241

Epigenetic re-wiring of breast cancer by pharmacological targeting of C-terminal binding protein

The C-terminal binding protein (CtBP) is an NADH-dependent dimeric family of nuclear proteins that scaffold interactions between transcriptional regulators and chromatin-modifying complexes. Its association with poor survival in several cancers implicates CtBP as a promising target for pharmacological intervention. We employed computer-assisted drug design to search for CtBP inhibitors, using quantitative structure-activity relationship (QSAR) modeling and docking. Functional screening of these drugs identified 4 compounds with low toxicity and high water solubility. Micro molar concentrations of these CtBP inhibitors produces significant de-repression of epigenetically silenced pro-epithelial genes, preferentially in the triple-negative breast cancer cell line MDA-MB-231. This epigenetic reprogramming occurs through eviction of CtBP from gene promoters; disrupted recruitment of chromatin-modifying protein complexes containing LSD1, and HDAC1; and re-wiring of activating histone marks at targeted genes. In functional assays, CtBP inhibition disrupts CtBP dimerization, decreases cell migration, abolishes cellular invasion, and improves DNA repair. Combinatorial use of CtBP inhibitors with the LSD1 inhibitor pargyline has synergistic influence. Finally, integrated correlation of gene expression in breast cancer patients with nuclear levels of CtBP1 and LSD1, reveals new potential therapeutic vulnerabilities. These findings implicate a broad role for this class of compounds in strategies for epigenetically targeted therapeutic intervention

General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multicentre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients’ (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16–22) and failed intubation in 1 in 312 (95%CI 1 in 169–667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)

Authentication and characterisation of a new oesophageal adenocarcinoma cell line: MFD-1

New biological tools are required to understand the functional significance of genetic events revealed by whole genome sequencing (WGS) studies in oesophageal adenocarcinoma (OAC). The MFD-1 cell line was isolated from a 55-year-old male with OAC without recombinant-DNA transformation. Somatic genetic variations from MFD-1, tumour, normal oesophagus, and leucocytes were analysed with SNP6. WGS was performed in tumour and leucocytes. RNAseq was performed in MFD-1, and two classic OAC cell lines FLO1 and OE33. Transposase-accessible chromatin sequencing (ATAC-seq) was performed in MFD-1, OE33, and non-neoplastic HET1A cells. Functional studies were performed. MFD-1 had a high SNP genotype concordance with matched germline/tumour. Parental tumour and MFD-1 carried four somatically acquired mutations in three recurrent mutated genes in OAC: TP53, ABCB1 and SEMA5A, not present in FLO-1 or OE33. MFD-1 displayed high expression of epithelial and glandular markers and a unique fingerprint of open chromatin. MFD-1 was tumorigenic in SCID mouse and proliferative and invasive in 3D cultures. The clinical utility of whole genome sequencing projects will be delivered using accurate model systems to develop molecular-phenotype therapeutics. We have described the first such system to arise from the oesophageal International Cancer Genome Consortium project

General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multi-centre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients' (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16-22) and failed intubation in 1 in 312 (95%CI 1 in 169-667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)

Could anionic LDL be a ligand for RAGE and TREM2 in addition to LOX-1 and thus exacerbate lung disease and dementia?

We recently highlighted the potential of protein glycation to generate anionic (electronegative) surfaces. We hypothesised that these anionic proteins are perceived by the innate immune system as arising from infection or damaged cell components, producing an inflammatory response within the lung involving the receptor RAGE. We now review two other pathologies linked to the innate immune response, cardiovascular disease and dementia that involve receptors LOX-1 and TREM2 respectively. Remarkable similarities in properties between RAGE, LOX-1 and TREM2 suggest that electronegative LDL may act as a pathogenic anionic ligand for all three receptors and exacerbate lung inflammation and dementia.</p

A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal

Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.<br/

Catalytic and non-catalytic functions of human IIA phospholipase A2

Group IIA phospholipase A2 (PLA2) is a low-molecular-mass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages

CtBPs promote mitotic fidelity through their activities in the cell nucleus

CtBPs form NADH-sensitive chromatin-modifying complexes, which link cellular metabolism to gene transcription. They also function in the cytoplasm to regulate Golgi fissioning; their inhibition can consequently cause a Golgi-dependent checkpoint in G(2). We have recently identified a novel role of CtBPs in the maintenance of mitotic fidelity; inhibition of CtBP synthesis resulting in reduced association of aurora B with mitotic chromatin and aberrant segregation of chromosomes. Here, we demonstrate that it is the interaction of CtBPs with transcriptional regulators and/or chromatin-modifying enzymes in the cell nucleus, rather than their role in Golgi fission, which is critical for the maintenance of mitotic fidelit