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Pulmonary fibrosis is a progressive scarring disorder of the lung with dismal prognosis and no curative therapy. Clusterin, an extracellular chaperone and regulator of cell functions, is reduced in bronchoalveolar lavage fluid of patients with pulmonary fibrosis. However, its distribution and role in normal and fibrotic human lung are incompletely characterized. Immunohistochemical localization of clusterin revealed strong staining associated with fibroblasts in control lung and morphologically normal areas of fibrotic lung but weak or undetectable staining in fibrotic regions and particularly fibroblastic foci. Clusterin also co-localized with elastin in vessel walls and additionally with amorphous elastin deposits in fibrotic lung. Analysis of primary lung fibroblast isolates in vitro confirmed the down-regulation of clusterin expression in fibrotic compared with control lung fibroblasts and further demonstrated that TGF-β1 is capable of down-regulating fibroblast clusterin expression. shRNA-mediated down-regulation of clusterin did not affect TGF-β1-induced fibroblast-myofibroblast differentiation but inhibited fibroblast proliferative responses and sensitized to apoptosis. Down-regulation of clusterin in fibrotic lung fibroblasts at least partly due to increased TGF-β1 may therefore represent an appropriate but insufficient response to limit fibroproliferation. Reduced expression of clusterin in the lung may also limit its extracellular chaperoning activity contributing to dysregulated deposition of extracellular matrix proteins.

The present study demonstrates that hypermethylation and silencing of chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), a transcriptional regulator of cyclooxygenase-2 (COX-2), is a major contributor to failure of fibroblasts to up-regulate COX-2 in pulmonary fibrosis. DNA methyltransferase (DNMT) inhibition reduces c8orf4 methylation, restores COX-2 expression and normalizes fibroblast function., Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2′-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.

A response to the issues raised by the English GM coexistence consultation.

Overall Aim To develop a robust system for identifying, testing, multiplying and marketing cereal varieties, lines, mixtures and populations best suited to organic production in different parts of the country. Objectives 1. Develop a participatory research and development methodology for UK organic farmers using variety trialling and the management of seed-borne disease as examples. 2. Collect information on the range of cereal varieties currently grown by organic farmers to help identify the major priorities and constraints among the varieties available. 3. Establish a pilot programme of cereal variety trials with organic farmers on organic farms using the methodology developed by Objective 1. 4. To obtain information on which seed-borne diseases, including ergot, may cause problems in the organic seed production chain of wheat, barley, oats and triticale, and to examine any relationship between organic husbandry conditions (seed rate, sowing date, rotation etc.) and incidence/severity of disease. 5. Determine whether cultivars with good potential for organic production are resistant to one or more of the seed-borne disease problems. 6. Working with farmers (Objective 1), review and identify a range of organically acceptable seed treatments and processes, considering both chemical and physical methods, and test these under organic conditions to determine efficacy. 7. Formulate a code of best practice for the production of certified organic seed, and for the processing of seed on organic farms. 8. To evaluate the participatory research and development approach throughout the entire research process and produce guidelines and materials for best practice. Data will be collected throughout the duration of the project. Objective 1. A literature review was undertaken and an agenda for future research set out. Questions to be addressed included: Have we identified the research that farmers and other stakeholders want? What roles do farmers and other stakeholders play? How do we carry out the testing, adaptation and development of options? How can the effective forms of participatory research (if there are any) be ‘mainstreamed’ into other agricultural research? Existing systems of farmer involvement in research were also examined through interviews with farmers, agri-businesses and scientists. It was found that almost all farmers were carrying out some kind of research on their farm. This may be using scientific methods or using a more holistic approach with multiple criteria. Farmers may set hypotheses explicitly before starting the experiment or they may use gut feelings and be experimenting without acknowledging it. They also often changed treatments during the experiment. It was concluded that the best results are more likely to come when topics are addressed by combining farmers’ own research with research on farms controlled and managed by scientists. From discussions with various farmer groups and the previous experience of Elm Farm Organic Research Centre researchers, it was decided that: the project should focus on winter wheat; and the basic experimental protocol must be simple, be able to be undertaken by the farmer with their own machinery and within the farmers’ time constraints. A protocol was established and reviewed each year at annual post harvest review meetings. Objective 2. Planting data was collected from the members of the Organic Arable Marketing Group in the first year of the project. Hereward and Claire were the most popular winter wheat varieties grown, and this was confirmed by a survey of the farmers involved in this project. A major concern of farmers was achieving milling quality specifications (especially protein concentration). Objective 3. Plots of three bread making winter wheat varieties (Hereward, Solstice and Xi19) and a mixture (1:1:1) of the varieties were grown at up to 19 UK farms in two seasons (2003/04 and 2004/05). Measurements were taken of growth habit, yield and grain quality. Grain yields in both seasons showed significant site by variety interactions, although the variation among sites was greater than among varieties in both instances. Wheat grown at Western sites was significantly shorter and higher-yielding than that grown at Eastern sites in 2003/04 but significantly taller in 2004/05. As with grain yield, greater variation among sites than varieties was found in the Hagberg Falling Number and protein concentration results in both seasons. The results from the two years of trials illustrate the variability of organic systems and the difficulty in selecting a single wheat variety suitable for organic farms. Garlic oil was used as a seed treatment for Hereward on two of the sites in the second year of trials. However, the treatment had no effect on establishment of the variety, and yields were variable. Benchmarking data was collected from 24 farms. Exsept appeared to be the highest yielding variety and yields varied with soil type (silts>clays>sands). More data would be needed to give an accurate picture of organic yields across the country. Objective 4. A total of 676 samples were tested between 2002 and 2005. Treatment thresholds for wheat seed have recently been extensively investigated and revised, producing a safe level below which untreated seed could be sown. Results showed that most samples had higher health status than the conventional treatment thresholds. However, there were occasional problems, most notably in the case of bunt on wheat, where very high levels of infection were seen, and the seed would have been unsuitable for further multiplication as seed, or for ware production. It was not possible to relate these occurrences consistently with any particular farm practice, with the possible exception of one site where minimum tillage was used, and the crop was always a second wheat. Ergot (Claviceps purpurea) was present at high levels (e.g. over 50 pieces per kg of seed) in several samples, but ergot infestation has also been increasing in frequency and severity in conventional production recently. Microdochium nivale sometimes reached high levels on wheat seed in seasons favourable to the disease, but similar levels were also seen in conventional samples received for testing at NIAB. Objective 5. Tests on appropriate varieties were carried out in 2 years. Of the wheat varieties: Hereward and Solstice appeared to show ‘resistance’ to bunt although the nature of the resistance is not known; Exsept, was consistently more resistant to Microdochium in the ear than other varieties; and Claire, Deben and Nijinsky appeared to be more resistant to loose smut than other varieties. There has been little effort to breed for seed borne disease resistance but these results indicate that it should be possible to introduce resistance, especially for diseases like loose smut. Objective 6. Seed treatment trials were carried out in 2004 and 2005 and comprised examples of biological, micronutrient and physical treatments. None of the treatments used in 2004 significantly improved establishment when wheat seed had a high level (30%) of Microdochium nivale seedling blight, and none significantly increased final yield. In 2005, one of the biological treatments tested (from Crompton Ltd) did significantly improve plant establishment, though effects on yield were non-significant. Both biological products (Cerall and the Crompton product) suppressed bunt in 2005, as did Radiate (ammonium and zinc ammonium complex), though the latter had no significant effect in 2004. The hot air treatment also reduced bunt in 2005, though the effect was less marked in 2004. Seed cleaning was also investigated as a means of improving establishment in Microdochium infected wheat. Though establishment counts and early spring counts were improved slightly in the cleaned seed compared to uncleaned, effects were not significant, and final yields were not improved. Incidence of disease in the cleaned versus uncleaned seed (% infection on agar plates) was similar, indicating that the process, although removing light and shrivelled seed, did not selectively remove infected seed. Objective 7. The code of best practice for seed production concentrated on bunt since this was the most prevalent problem found in this project. Guidelines included: always test untreated ‘mother’ seed; seed destined for further multiplication should have as close to 0 bunt spores/seed as possible; seed for crop production should have no more than 1 bunt spore/seed; grow farm-saved seed as first wheat; and sow wheat early to minimize any chances of infection. Objective 8. The participatory process was assessed three times using interviews with farmers and researchers involved in the project. It was evident that farmer participatory research was more complicated, time-consuming and expensive than expected. Key issues identified included: • Acknowledging and addressing the training needs of farmers and researchers at the outset of a project • Building a team of people (farmers and researchers) who understand each others’ background, and are able to work together towards an agreed set of goals. • Appreciating the commitment farmers must make on top of their existing workloads to engage in this sort of activity, and doing what is possible to facilitate this. • Identifying appropriate people to act as boundary spanners and draw all stakeholders together in dialogue. • Within this framework, identifying research goals that can be realistically met by all concerned. • A short leaflet and a longer document were produced for farmers/researchers setting out what’s involved in participatory research and the pros and cons of participating. Conclusions. The experimental aspect of this project has highlighted the large variation among organic systems and the problems in recommending a single variety to organic farmers. However, the work has shown that there are few problems in the health of the seed used in organic systems. This is particularly important since none of the potential organic seed treatments tested had a positive effect on yield. The main aspect of this work has been a learning experience in aspects of farmer participatory research in a UK context. Differing views have meant that natural and social scientists have written different parts of the discussion. Researchers have experienced difficulties in engaging farmers and managing their expectations, the challenges of working in multidisciplinary teams spread over different institutions and the extra time needed to build and maintain relationships. Farmer participatory research does have an important role to play in producing both relevant and rigorous results for farmers and funders. It has to be managed appropriately, and has to be recognised that processes are very different to a typical research project.

Farmer participation was essential in developing a uniquely useful set of wheat variety trials data on a wide range of organic farms over two years. Although the trials were successful, it became clear that some of the participating farmers felt there were some limitations in the process. These included a lack of ownership in the project and a concern for more researcher help. It was clear that a greater time in-vestment was needed at the start of the project to help with farmer understanding and ownership. De-spite the negative comments, farmers appreciated their involvement, particularly in contrasting their own views and information with that from the wider scene. Farmer participation is essential for systems-level research and this project helped to develop a small core of trained farmers and researchers.

During 2005 Defra commissioned a study to identify and analyse issues and aspirations that organic stakeholders felt should be addressed by publicly funded organic food and farming research in the UK. How this was undertaken is presented in this paper. A series of 12 workshops were undertaken with stakeholders throughout the UK. Nearly 300 stakeholders attended the workshops. These workshops used participatory approaches to identify and record the most important issues and aspirations from those attending. The use of a highly participatory style was greatly appreciated by stakeholders. In most cases the interaction between stakeholders worked well and resulted in lively discussions. The workshops have served to open up a useful dialogue between groups of stakeholders who do not normally communicate directly. They have produced a significant number of interesting and challenging issues and aspirations.

A participatory research methodology was used to compare the performance of UK wheat varieties under organic conditions. Plots of three breadmaking winter wheat varieties (Hereward, Solstice and Xi19) and a mixture (1:1:1) of the varieties were grown at 19 UK farms in two seasons (2003/04 and 2004/05). Meas-urements were taken of growth habit, yield and grain quality. Grain yields in both seasons showed significant site by variety interactions, although the variation among sites was greater than among varieties in both instances. Wheat grown at Western sites was significantly shorter and higher-yielding than that grown at Eastern sites in 2003/04 but significantly taller in 2004/05. As with grain yield, greater variation among site than variety was found in the Hagberg Falling Number and protein concentra-tion results in both seasons. The results from the two years of trials illustrate the variability of organic systems and the difficulty in selecting a single variety suitable for organic farms.

Mutations in the NRF2-KEAP1 pathway are common in non-small cell lung cancer (NSCLC) and confer broad-spectrum therapeutic resistance, leading to poor outcomes. The cystine/glutamate antiporter, system x c - , is one of the >200 cytoprotective proteins controlled by NRF2, which can be non-invasively imaged by ( S )-4-(3- 18 F-fluoropropyl)-l-glutamate ([ 18 F]FSPG) positron emission tomography (PET). Through genetic and pharmacologic manipulation, we show that [ 18 F]FSPG provides a sensitive and specific marker of NRF2 activation in advanced preclinical models of NSCLC. We validate imaging readouts with metabolomic measurements of system x c - activity and their coupling to intracellular glutathione concentration. A redox gene signature was measured in patients from the TRACERx 421 cohort, suggesting an opportunity for patient stratification prior to imaging. Furthermore, we reveal that system x c - is a metabolic vulnerability that can be therapeutically targeted for sustained tumour growth suppression in aggressive NSCLC. Our results establish [ 18 F]FSPG as predictive marker of therapy resistance in NSCLC and provide the basis for the clinical evaluation of both imaging and therapeutic agents that target this important antioxidant pathway.