Prostacyclin synthase expression and epigenetic regulation in nonsmall cell lung cancer

BACKGROUND: Prostacyclin synthase (PGIS) metabolizes prostaglandin H(2), into prostacyclin. This study aimed to determine the expression profile of PGIS in nonsmall cell lung cancer (NSCLC) and examine potential mechanisms involved in PGIS regulation. METHODS: PGIS expression was examined in human NSCLC and matched controls by reverse transcriptase polymerase chain reaction (RT-PCR), Western analysis, and immunohistochemistry. A 204-patient NSCLC tissue microarray was stained for PGIS and cyclooxygenase 2 (COX2) expression. Staining intensity was correlated with clinical parameters. Epigenetic mechanisms underpinning PGIS promoter expression were examined using RT-PCR, methylation-specific PCR, and chromatin immunoprecipitation analysis. RESULTS: PGIS expression was reduced/absent in human NSCLC protein samples (P <.0001), but not mRNA relative to matched controls. PGIS tissue expression was higher in squamous cell carcinoma (P =.004) and in male patients (P <.05). No significant correlation of PGIS or COX2 expression with overall patient survival was observed, although COX2 was prognostic for short-term (2-year) survival (P <.001). PGIS mRNA expression was regulated by DNA CpG methylation and histone acetylation in NSCLC cell lines, with chromatin remodeling taking place directly at the PGIS gene. PGIS mRNA expression was increased by both demethylation agents and histone deacetylase inhibitors. Protein levels were unaffected by demethylation agents, whereas PGIS protein stability was negatively affected by histone deacetylase inhibitors. CONCLUSIONS: PGIS protein expression is reduced in NSCLC, and does not correlate with overall patient survival. PGIS expression is regulated through epigenetic mechanisms. Differences in expression patterns between mRNA and protein levels suggest that PGIS expression and protein stability are regulated post-translationally. PGIS protein stability may have an important therapeutic role in NSCLC. © 2011 American Cancer Society.

Relational regulation theory and the role of social support and organisational fairness for nurses in a general acute context

Aims and objectives. To present a novel approach to nurse stress by exploring the demand–control–support model with organisational justice through the lens of relational regulation theory. Background. Nursing is often stressful due to high demands and dissatisfaction with pay, which impacts the mental well-being and productivity of nurses. Design. A cross-sectional design. Methods. A validated questionnaire was sent to the work addresses of all nursing and midwifery staff in a medium-sized general acute hospital in Australia. A total of 190 nurses and midwives returned completed questionnaires for the analyses. Results. The multiple regression analyses demonstrated that the model applies to the prototypical context of a general acute hospital and that job control, supervisor support and outside work support improve the job satisfaction and mental health of nurses. Conclusions. Most importantly, supervisor support was found to buffer the impact of excessive work demands. Fairness of procedures, distribution of resources and the quality and consistency of information are also beneficial. Relational regulation theory is applied to these findings as a novel way to conceptualise the mechanisms of support and fairness in nursing. Relevance to clinical practice. The importance of nurses’ well-being and job satisfaction is a priority for improving clinical outcomes. Practically, this means nurse managers should be encouraging nurses in the pursuit of diverse relational activities both at work and outside work.

Regulation of EP receptors in non-small cell lung cancer by epigenetic modifications

Background: Cyclooxygenase (COX)-2 is frequently overexpressed in non-small cell lung cancer (NSCLC) and results in increased levels of prostaglandin E2 (PGE 2), an important signalling molecule implicated in tumourigenesis. PGE 2 exerts its effects through the E prostanoid (EP) receptors (EPs1-4). Methods: The expression and epigenetic regulation of the EPs were evaluated in a series of resected fresh frozen NSCLC tumours and cell lines. Results: EP expression was dysregulated in NSCLC being up and downregulated compared to matched control samples. For EPs1, 3 and 4 no discernible pattern emerged. EP2 mRNA however was frequently downregulated, with low levels being observed in 13/20 samples as compared to upregulation in 5/20 samples examined. In NSCLC cell lines DNA CpG methylation was found to be important for the regulation of EP3 expression, the demethylating agent decitabine upregulating expression. Histone acetylation was also found to be a critical regulator of EP expression, with the histone deacteylase inhibitors trichostatin A, phenylbutyrate and suberoylanilide hydroxamic acid inducing increased expression of EPs2-4. Direct chromatin remodelling was demonstrated at the promoters for EPs2-4. Conclusions: These results indicate that EP expression is variably altered from tumour to tumour in NSCLC. EP2 expression appears to be predominantly downregulated and may have an important role in the pathogenesis of the disease. Epigenetic regulation of the EPs may be central to the precise role COX-2 may play in the evolution of individual tumours. © 2009 Elsevier Ltd. All rights reserved.

Transcriptional regulation of IRS5/DOK4 expression in non-small-cell lung cancer cells

The insulin-receptor substrate family plays important roles in cellular growth, signaling, and survival. Two new members of this family have recently been isolated: IRS5/Dok4 and IRS6/Dok5. This study examines the expression of IRS5/DOK4 in a panel of lung cancer cell lines and tumor specimens. The results demonstrate that expression of IRS5/DOK4 is frequently altered with both elevated and decreased expression in non-small-cell lung cancer (NSCLC) tumor specimens. The altered expression of IRS5/DOK4 observed in tumor samples is not due to aberrant methylation. In vitro cell culture studies demonstrate that treatment of NSCLC cell lines with the histone deacetylase inhibitor trichostatin A (TSA) upregulates IRS5/DOK4. This finding indicates that expression is regulated epigenetically at the level of chromatin remodeling. Chromatin immunoprecipitation experiments confirm that the IRS5/DOK4 promoter has enhanced histone hyperacetylation following treatments with TSA. Finally, hypoxia was demonstrated to downregulate IRS5/DOK4 expression. This expression was restored by TSA. The clinical relevance of altered IRS5/DOK4 expression in NSCLC requires fur ther evaluation.

bcl-2 and c-erbB-2 proteins are involved in the regulation of VEGF and of thymidine phosphorylase angiogenic activity in non-small-cell lung cancer

Tumour angiogenesis has been recently recognised as one of the most important prognostic factors in lung cancer. Although a variety of angiogenic factors have been identified, the angiogenesis process remains poorly understood. Bcl-2, c-erbB-2 and p53 are well-known oncogenes involved in non- small-cell lung cancer pathogenesis. A direct correlation of thymidine phosphorylase (TP) and of vascular endothelial growth factor (VEGF) with intratumoural angiogenesis has been reported. In the present study we investigated the possible regulatory role if bcl-2, c-erB-2 proteins in angiogenesis and in VEGF and TP expression in non-small-cell lung cancer. Two hundred sixteen specimens from T1,2-NO, 1 staged patients treated with surgery alone were immunohistochemically examined. Bcl-2 and c-erbB-2 were significantly inversely related to each other (P = 0.04) and both were inversely associated with microvessel density (P < 0.02). High TP and VEGF reactivity was statistically related to loss of bcl-2 expression (P < 0.01). A significant co-expression of c-erbB-2 with TP was noted (P = 0.01). However, TP expression was related to high angiogenesis only in cases with absence of c-erB-2 expression (P < 0.0001). c-erbB-2 expression in poorly vascularised tumours was linked with poor outcome (P = 0.03). The present study provides strong evidence that the bcl-2 gene has a suppressive function over genes involved in both angiogenesis (VEGF and TP) and cell migration (c- erbB-2) in NSCLC. TP and c-erbB-2 proteins are significantly, and often simultaneously, expressed in bcl-2 negative cases. However, expression of the c-erbB-2 abolishes the TP-related angiogenic activity. Whether this is a result of a direct activity of the c-erbB-2 protein or a consequence of a c- erbB-2-related immune response remains to be further investigated.

Epigenetic regulation of glucose transporters in non-small cell lung cancer

Due to their inherently hypoxic environment, cancer cells often resort to glycolysis, or the anaerobic breakdown of glucose to form ATP to provide for their energy needs, known as the Warburg effect. At the same time, overexpression of the insulin receptor in non-small cell lung cancer (NSCLC) is associated with an increased risk of metastasis and decreased survival. The uptake of glucose into cells is carried out via glucose transporters or GLUTs. Of these, GLUT-4 is essential for insulin-stimulated glucose uptake. Following treatment with the epigenetic targeting agents histone deacetylase inhibitors (HDACi), GLUT-3 and GLUT-4 expression were found to be induced in NSCLC cell lines, with minimal responses in transformed normal human bronchial epithelial cells (HBECs). Similar results for GLUT-4 were observed in cells derived from liver, muscle, kidney and pre-adipocytes. Bioinformatic analysis of the promoter for GLUT-4 indicates that it may also be regulated by several chromatin binding factors or complexes including CTCF, SP1 and SMYD3. Chromatin immunoprecipitation studies demonstrate that the promoter for GLUT-4 is dynamically remodeled in response to HDACi. Overall, these results may have value within the clinical setting as (a) it may be possible to use this to enhance fluorodeoxyglucose (18F) positron emission tomography (FDG-PET) imaging sensitivity; (b) it may be possible to target NSCLC through the use of HDACi and insulin mediated uptake of the metabolic targeting drugs such as 2-deoxyglucose (2-DG); or (c) enhance or sensitize NSCLC to chemotherapy. © 2011 by the authors; licensee MDPI, Basel, Switzerland.

EGFR expression as a predictor of survival for first-line chemotherapy plus cetuximab in patients with advanced non-small-cell lung cancer : analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 First-Line ErbituX in lung cancer (FLEX) study showed that the addition of cetuximab to first-line chemotherapy significantly improved overall survival compared with chemotherapy alone (hazard ratio [HR] 0·871, 95% CI 0·762-0·996; p=0·044) in patients with advanced non-small-cell lung cancer (NSCLC). To define patients benefiting most from cetuximab, we studied the association of tumour EGFR expression level with clinical outcome in FLEX study patients. Methods: We used prospectively collected tumour EGFR expression data to generate an immunohistochemistry score for FLEX study patients on a continuous scale of 0-300. We used response data to select an outcome-based discriminatory threshold immunohistochemistry score for EGFR expression of 200. Treatment outcome was analysed in patients with low (immunohistochemistry score <200) and high (≥200) tumour EGFR expression. The primary endpoint in the FLEX study was overall survival. We analysed patients from the FLEX intention-to-treat (ITT) population. The FLEX study is registered with ClinicalTrials.gov, number NCT00148798. Findings: Tumour EGFR immunohistochemistry data were available for 1121 of 1125 (99·6%) patients from the FLEX study ITT population. High EGFR expression was scored for 345 (31%) evaluable patients and low for 776 (69%) patients. For patients in the high EGFR expression group, overall survival was longer in the chemotherapy plus cetuximab group than in the chemotherapy alone group (median 12·0 months [95% CI 10·2-15·2] vs 9·6 months [7·6-10·6]; HR 0·73, 0·58-0·93; p=0·011), with no meaningful increase in side-effects. We recorded no corresponding survival benefit for patients in the low EGFR expression group (median 9·8 months [8·9-12·2] vs 10·3 months [9·2-11·5]; HR 0·99, 0·84-1·16; p=0·88). A treatment interaction test assessing the difference in the HRs for overall survival between the EGFR expression groups suggested a predictive value for EGFR expression (p=0·044). Interpretation: High EGFR expression is a tumour biomarker that can predict survival benefit from the addition of cetuximab to first-line chemotherapy in patients with advanced NSCLC. Assessment of EGFR expression could offer a personalised treatment approach in this setting. Funding: Merck KGaA. © 2012 Elsevier Ltd.

Monitoring and benchmarking population diet quality globally : a step-wise approach

INFORMAS (International Network for Food and Obesity/non-communicable diseases Research, Monitoring and Action Support) aims to monitor and benchmark the healthiness of food environments globally. In order to assess the impact of food environments on population diets, it is necessary to monitor population diet quality between countries and over time. This paper reviews existing data sources suitable for monitoring population diet quality, and assesses their strengths and limitations. A step-wise framework is then proposed for monitoring population diet quality. Food balance sheets (FBaS), household budget and expenditure surveys (HBES) and food intake surveys are all suitable methods for assessing population diet quality. In the proposed ‘minimal’ approach, national trends of food and energy availability can be explored using FBaS. In the ‘expanded’ and ‘optimal’ approaches, the dietary share of ultra-processed products is measured as an indicator of energy-dense, nutrient-poor diets using HBES and food intake surveys, respectively. In addition, it is proposed that pre-defined diet quality indices are used to score diets, and some of those have been designed for application within all three monitoring approaches. However, in order to enhance the value of global efforts to monitor diet quality, data collection methods and diet quality indicators need further development work.

Coexpression of epidermal growth factor receptor with related factors is associated with a poor prognosis in non-small-cell lung cancer

The epidermal growth factor receptor (EGFR) is commonly expressed in non-small-cell lung cancer (NSCLC) and promotes a host of mechanisms involved in tumorigenesis. However, EGFR expression does not reliably predict prognosis or response to EGFR-targeted therapies. The data from two previous studies of a series of 181 consecutive surgically resected stage I-IIIA NSCLC patients who had survived in excess of 60 days were explored. Of these patients, tissue was available for evaluation of EGFR in 179 patients, carbonic anhydrase (CA) IX in 177 patients and matrix metalloproteinase-9 (MMP-9) in 169 patients. We have previously reported an association between EGFR expression and MMP-9 expression. We have also reported that MMP-9 (P=0.001) and perinuclear (p)CA IX (P=0.03) but not EGFR expression were associated with a poor prognosis. Perinuclear CA IX expression was also associated with EGFR expression (P<0.001). Multivariate analysis demonstrated that coexpression of MMP-9 with EGFR conferred a worse prognosis than the expression of MMP-9 alone (P<0.001) and coexpression of EGFR and pCA IX conferred a worse prognosis than pCA IX alone (P=0.05). A model was then developed where the study population was divided into three groups: group 1 had expression of EGFR without coexpression of MMP-9 or pCA IX (number=21); group 2 had no expression of EGFR (number=75); and group 3 had coexpression of EGFR with pCA IX or MMP-9 or both (number=70). Group 3 had a worse prognosis than either groups 1 or 2 (P=0.0003 and 0.027, respectively) and group 1 had a better prognosis than group 2 (P=0.036). These data identify two cohorts of EGFR-positive patients with diametrically opposite prognoses. The group expressing either EGFR and or both MMP-9 and pCA IX may identify a group of patients with activated EGFR, which is of clinical relevance with the advent of EGFR-targeted therapies. © 2004 Cancer Research UK.

Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18:1 to elongation reactions beginning with 18:1 as substrate. These V2-based leaf assays produced ~50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays. © 2012 Naim et al.

Regulation of dormancy in barley by blue light and after-ripening : effects on abscisic acid and gibberellin metabolism

White light strongly promotes dormancy in freshly harvested cereal grains, whereas dark and after-ripening have the opposite effect. We have analyzed the interaction of light and after-ripening on abscisic acid (ABA) and gibberellin (GA) metabolism genes and dormancy in barley (Hordeum vulgare ‘Betzes’). Analysis of gene expression in imbibed barley grains shows that different ABA metabolism genes are targeted by white light and after-ripening. Of the genes examined, white light promotes the expression of an ABA biosynthetic gene, HvNCED1, in embryos. Consistent with this result, enzyme-linked immunosorbent assays show that dormant grains imbibed under white light have higher embryo ABA content than grains imbibed in the dark. After-ripening has no effect on expression of ABA biosynthesis genes, but promotes expression of an ABA catabolism gene (HvABA8′OH1), a GA biosynthetic gene (HvGA3ox2), and a GA catabolic gene (HvGA2ox3) following imbibition. Blue light mimics the effects of white light on germination, ABA levels, and expression of GA and ABA metabolism genes. Red and far-red light have no effect on germination, ABA levels, or HvNCED1. RNA interference experiments in transgenic barley plants support a role of HvABA8′OH1 in dormancy release. Reduced HvABA8′OH1 expression in transgenic HvABA8′OH1 RNAi grains results in higher levels of ABA and increased dormancy compared to nontransgenic grains.

Can water security be achieved in the urban environment through the implementation of the Neo Institutional theory government regulation and legislation?

The research seeks to address the current global water crisis and the built environments effect on the increasing demand for sustainability and water security. The fundamental question in determining the correct approach for water security in the built environment is whether government regulation and legislation could provide the framework for sustainable development and the conscious shift providing that change is the only perceivable option, there is no alternative. This article will attempt to analyse the value of the neo institutional theory as a method for directing individuals and companies to conform to water saving techniques. As is highlighted throughout the article, it will be investigated whether an incentive verse punishment approach to government legislations and regulations would provide the framework required to ensure water security within the built environment. Individuals and companies make certain choices or perform certain actions not because they fear punishment or attempt to conform; neither do they do so because an action is appropriate or feels some sort of social obligation. Instead, the cognitive element of neo institutionalism suggests that individuals make certain choices because they can conceive no alternative. The research seeks to identify whether sustainability and water security can become integrated into all aspects of design and architecture through the perception that 'there is no alternative.' This report seeks to address the omission of water security in the built environment by reporting on a series of investigations, interviews, literature reviews, exemplars and statistics relating to the built environment and the potential for increased water security. The results and analysis support the conclusions that through the support of government and local council, sustainability in the built environment could be achieved and become common practice for developments. Highlighted is the approach required for water management systems integration into the built environment and how these can be developed and maintained effectively between cities, states, countries and cultures.

Mouse estrous cycle regulation of vaginal versus uterine cytokines, chemokines, α-/β-defensins and TLRs

This study investigates the cyclic changes in innate immunity in the female reproductive tract (FRT) of mice during the estrous cycle. By examining uterine and vaginal tissues and secretions we show that innate immunity varies with the stage of the estrous cycle and site in the FRT. Secretions from the uterine lumen contained cytokines and chemokines that were significantly higher at proestrus and estrus relative to that measured at diestrus. In contrast, analysis of vaginal secretions indicated that only IL-1β and CXCL1/mouse KC changed during the cycle, with highest levels measured at diestrus and estrus. In contrast, vaginal α-defensin 2 and β-defensins 1-4 mRNA levels peaked at proestrus and estrus and are expressed 1-4 logs greater than that seen in the uterus. These studies further indicate that TLR5 and TLR12 in the uterus, and TLR1, TLR2, TLR5 and TLR13 in the vagina varies with stage of the estrous cycle, with some peaking at proestrus/estrus and others at diestrus. Overall, these studies indicate that innate immune parameters in the uterus and vagina are separate and discrete, and regulated precisely during the estrous cycle.

Sex hormone regulation of innate immunity in the female reproductive tract : the role of epithelial cells in balancing reproductive potential with protection against sexually transmitted pathogens

The immune system in the female reproductive tract (FRT) does not mount an attack against HIV or other sexually transmitted infections (STI) with a single endogenously produced microbicide or with a single arm of the immune system. Instead, the body deploys dozens of innate antimicrobials to the secretions of the female reproductive tract. Working together, these antimicrobials along with mucosal antibodies attack many different viral, bacterial and fungal targets. Within the FRT, the unique challenges of protection against sexually transmitted pathogens coupled with the need to sustain the development of an allogeneic fetus have evolved in such a way that sex hormones precisely regulate immune function to accomplish both tasks. The studies presented in this review demonstrate that estradiol and progesterone secreted during the menstrual cycle act both directly and indirectly on epithelial cells and other immune cells in the reproductive tract to modify immune function in a way that is unique to specific sites throughout the FRT. As presented in this review, studies from our laboratory and others demonstrate that the innate immune response is under hormonal control, varies with the stage of the menstrual cycle, and as such is suppressed at mid-cycle to optimize conditions for successful fertilization and pregnancy. In doing so, a window of STI vulnerability is created during which potential pathogens including HIV enter the reproductive tract to infect host targets.