G-quadruplex DNA structures in human stem cells and differentiation.

Funder: Herchel Smith FundsThe establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation

Androgen receptor phosphorylation at serine 515 by Cdk1 predicts biochemical relapse in prostate cancer patients

<br>Background:Prostate cancer cell growth is dependent upon androgen receptor (AR) activation, which is regulated by specific kinases. The aim of the current study is to establish if AR phosphorylation by Cdk1 or ERK1/2 is of prognostic significance.</br> <br>Methods: Scansite 2.0 was utilised to predict which AR sites are phosphorylated by Cdk1 and ERK1/2. Immunohistochemistry for these sites was then performed on 90 hormone-naive prostate cancer specimens. The interaction between Cdk1/ERK1/2 and AR phosphorylation was investigated in vitro using LNCaP cells.</br><br>Results:Phosphorylation of AR at serine 515 (pAR(S515)) and PSA at diagnosis were independently associated with decreased time to biochemical relapse. Cdk1 and pCdk1(161), but not ERK1/2, correlated with pAR(S515). High expression of pAR(S515) in patients with a PSA at diagnosis of ≤20 ng ml(-1) was associated with shorter time to biochemical relapse (P=0.019). This translated into a reduction in disease-specific survival (10-year survival, 38.1% vs 100%, P<0.001). In vitro studies demonstrated that treatment with Roscovitine (a Cdk inhibitor) caused a reduction in pCdk1(161) expression, pAR(S515)expression and cellular proliferation.</br> <br>Conclusion: In prostate cancer patients with PSA at diagnosis of ≤20 ng ml(-1), phosphorylation of AR at serine 515 by Cdk1 may be an independent prognostic marker.</br&gt

TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

TIGIT is an inhibitory receptor expressed on lymphocytes and can inhibit T cells by preventing CD226 co-stimulation through interactions in cis or through competition of shared ligands. Whether TIGIT directly delivers cell-intrinsic inhibitory signals in T cells remains unclear. Here we show, by analysing lymphocytes from matched human tumour and peripheral blood samples, that TIGIT and CD226 co-expression is rare on tumour-infiltrating lymphocytes. Using super-resolution microscopy and other techniques, we demonstrate that ligation with CD155 causes TIGIT to reorganise into dense nanoclusters, which coalesce with T cell receptor (TCR)-rich clusters at immune synapses. Functionally, this reduces cytokine secretion in a manner dependent on TIGIT’s intracellular ITT-like signalling motif. Thus, we provide evidence that TIGIT directly inhibits lymphocyte activation, acting independently of CD226, requiring intracellular signalling that is proximal to the TCR. Within the subset of tumours where TIGIT-expressing cells do not commonly co-express CD226, this will likely be the dominant mechanism of action

Landscape of G-quadruplex DNA structural regions in breast cancer.

Response and resistance to anticancer therapies vary due to intertumor and intratumor heterogeneity1. Here, we map differentially enriched G-quadruplex (G4) DNA structure-forming regions (∆G4Rs) in 22 breast cancer patient-derived tumor xenograft (PDTX) models. ∆G4Rs are associated with the promoters of highly amplified genes showing high expression, and with somatic single-nucleotide variants. Differences in ΔG4R landscapes reveal seven transcription factor programs across PDTXs. ∆G4R abundance and locations stratify PDTXs into at least three G4-based subtypes. ∆G4Rs in most PDTXs (14 of 22) were found to associate with more than one breast cancer subtype, which we also call an integrative cluster (IC)2. This suggests the frequent coexistence of multiple breast cancer states within a PDTX model, the majority of which display aggressive triple-negative IC10 gene activity. Short-term cultures of PDTX models with increased ∆G4R levels are more sensitive to small molecules targeting G4 DNA. Thus, G4 landscapes reveal additional IC-related intratumor heterogeneity in PDTX biopsies, improving breast cancer stratification and potentially identifying new treatment strategies.The Caldas and Balasubramanian laboratories are supported by core funding from Cancer Research UK (C14303/A17197). The Balasubramanian laboratory is supported by Program grant funding from Cancer Research UK (C9681/A18618 and C9681/A29214) and a Wellcome Trust Investigator Award (209441/z/17/z). Prior to the revision of this study work by Dr. Robert Hänsel-Hertsch was supported by the Balasubramanian group, afterwards additionally supported by core funding of the Center for Molecular Medicine Cologne (CMMC)

Distinct Mechanisms for Induction and Tolerance Regulate the Immediate Early Genes Encoding Interleukin 1β and Tumor Necrosis Factor α

Interleukin-1β and Tumor Necrosis Factor α play related, but distinct, roles in immunity and disease. Our study revealed major mechanistic distinctions in the Toll-like receptor (TLR) signaling-dependent induction for the rapidly expressed genes (IL1B and TNF) coding for these two cytokines. Prior to induction, TNF exhibited pre-bound TATA Binding Protein (TBP) and paused RNA Polymerase II (Pol II), hallmarks of poised immediate-early (IE) genes. In contrast, unstimulated IL1B displayed very low levels of both TBP and paused Pol II, requiring the lineage-specific Spi-1/PU.1 (Spi1) transcription factor as an anchor for induction-dependent interaction with two TLR-activated transcription factors, C/EBPβ and NF-κB. Activation and DNA binding of these two pre-expressed factors resulted in de novo recruitment of TBP and Pol II to IL1B in concert with a permissive state for elongation mediated by the recruitment of elongation factor P-TEFb. This Spi1-dependent mechanism for IL1B transcription, which is unique for a rapidly-induced/poised IE gene, was more dependent upon P-TEFb than was the case for the TNF gene. Furthermore, the dependence on phosphoinositide 3-kinase for P-TEFb recruitment to IL1B paralleled a greater sensitivity to the metabolic state of the cell and a lower sensitivity to the phenomenon of endotoxin tolerance than was evident for TNF. Such differences in induction mechanisms argue against the prevailing paradigm that all IE genes possess paused Pol II and may further delineate the specific roles played by each of these rapidly expressed immune modulators. © 2013 Adamik et al

Targeting multiple effector pathways in pancreatic ductal adenocarcinoma with a g-quadruplex-binding small molecule

Human pancreatic ductal adenocarcinoma (PDAC) involves the dysregulation of multiple signaling pathways. A novel approach to the treatment of PDAC is described, involving the targeting of cancer genes in PDAC pathways having over-representation of G-quadruplexes, using the trisubstituted naphthalene diimide quadruplex-binding compound 2,7-bis(3-morpholinopropyl)-4-((2-(pyrrolidin-1-yl)ethyl)amino)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (CM03). This compound has been designed by computer modeling, is a potent inhibitor of cell growth in PDAC cell lines, and has anticancer activity in PDAC models, with a superior profile compared to gemcitabine, a commonly used therapy. Whole-transcriptome RNA-seq methodology has been used to analyze the effects of this quadruplex-binding small molecule on global gene expression. This has revealed the down-regulation of a large number of genes, rich in putative quadruplex elements and involved in essential pathways of PDAC survival, metastasis, and drug resistance. The changes produced by CM03 represent a global response to the complexity of human PDAC and may be applicable to other currently hard-to-treat cancers

Experiences Marketing: A Cultural Philosophy for Contemporary Hospitality Marketing Studies

This article explores the landscape of contemporary hospitality marketing. It is argued that the teaching and academic discussions that surround the subject area adopt a predominantly positivistic approach; although important, that does not adequately reflect the nature of the industry or the products offered. Such a metrics-oriented position, although significant in the formulation of marketing strategy, does not reflect the complex experiential, nontangible nature of the hospitality product. This article presents a culturally located philosophy that reflects the multifaceted nature of the industry. The philosophy is underpinned by three precepts that draw from a multidisciplinary theoretical framework to create a more subject-specific approach to marketing, that when woven with traditional approaches can create a more effective and informed contemporary approach

The effectiveness of interventions to change six health behaviours: a review of reviews

Background: Several World Health Organisation reports over recent years have highlighted the high incidence of chronic diseases such as diabetes, coronary heart disease and cancer. Contributory factors include unhealthy diets, alcohol and tobacco use and sedentary lifestyles. This paper reports the findings of a review of reviews of behavioural change interventions to reduce unhealthy behaviours or promote healthy behaviours. We included six different health-related behaviours in the review: healthy eating, physical exercise, smoking, alcohol misuse, sexual risk taking (in young people) and illicit drug use. We excluded reviews which focussed on pharmacological treatments or those which required intensive treatments (e. g. for drug or alcohol dependency). Methods: The Cochrane Library, Database of Abstracts of Reviews of Effectiveness (DARE) and several Ovid databases were searched for systematic reviews of interventions for the six behaviours (updated search 2008). Two reviewers applied the inclusion criteria, extracted data and assessed the quality of the reviews. The results were discussed in a narrative synthesis. Results: We included 103 reviews published between 1995 and 2008. The focus of interventions varied, but those targeting specific individuals were generally designed to change an existing behaviour (e. g. cigarette smoking, alcohol misuse), whilst those aimed at the general population or groups such as school children were designed to promote positive behaviours (e. g. healthy eating). Almost 50% (n = 48) of the reviews focussed on smoking (either prevention or cessation). Interventions that were most effective across a range of health behaviours included physician advice or individual counselling, and workplace- and school-based activities. Mass media campaigns and legislative interventions also showed small to moderate effects in changing health behaviours. Generally, the evidence related to short-term effects rather than sustained/longer-term impact and there was a relative lack of evidence on how best to address inequalities. Conclusions: Despite limitations of the review of reviews approach, it is encouraging that there are interventions that are effective in achieving behavioural change. Further emphasis in both primary studies and secondary analysis (e.g. systematic reviews) should be placed on assessing the differential effectiveness of interventions across different population subgroups to ensure that health inequalities are addressed.</p

Glucose Availability and AMP-Activated Protein Kinase Link Energy Metabolism and Innate Immunity in the Bovine Endometrium

Defences against the bacteria that usually infect the endometrium of postpartum cattle are impaired when there is metabolic energy stress, leading to endometritis and infertility. The endometrial response to bacteria depends on innate immunity, with recognition of pathogen-associated molecular patterns stimulating inflammation, characterised by secretion of interleukin (IL)-1β, IL-6 and IL-8. How metabolic stress impacts tissue responses to pathogens is unclear, but integration of energy metabolism and innate immunity means that stressing one system might affect the other. Here we tested the hypothesis that homeostatic pathways integrate energy metabolism and innate immunity in bovine endometrial tissue. Glucose deprivation reduced the secretion of IL-1β, IL-6 and IL-8 from ex vivo organ cultures of bovine endometrium challenged with the pathogen-associated molecular patterns lipopolysaccharide and bacterial lipopeptide. Endometrial inflammatory responses to lipopolysaccharide were also reduced by small molecules that activate or inhibit the intracellular sensor of energy, AMP-activated protein kinase (AMPK). However, inhibition of mammalian target of rapamycin, which is a more global metabolic sensor than AMPK, had little effect on inflammation. Similarly, endometrial inflammatory responses to lipopolysaccharide were not affected by insulin-like growth factor-1, which is an endocrine regulator of metabolism. Interestingly, the inflammatory responses to lipopolysaccharide increased endometrial glucose consumption and induced the Warburg effect, which could exacerbate deficits in glucose availability in the tissue. In conclusion, metabolic energy stress perturbed inflammatory responses to pathogen-associated molecular patterns in bovine endometrial tissue, and the most fundamental regulators of cellular energy, glucose availability and AMPK, had the greatest impact on innate immunity

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl(D139V), that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl(D139V) homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO). Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region