Chromatin remodeling and genome stability

A report on the 12th Tenovus Scotland Symposium 'Stability and Regulation of Genes and Genomes', Glasgow UK, 6-7 April 2006

Chromatin insulator elements: establishing barriers to set heterochromatin boundaries

Epigenomic profiling has revealed that substantial portions of genomes in higher eukaryotes are organized into extensive domains of transcriptionally repressive chromatin. The boundaries of repressive chromatin domains can be fixed by DNA elements known as barrier insulators, to both shield neighboring gene expression and to maintain the integrity of chromosomal silencing. Here, we examine the current progress in identifying vertebrate barrier elements and their binding factors. We overview the design of the reporter assays used to define enhancer-blocking and barrier insulators. We look at the mechanisms vertebrate barrier proteins, such as USF1 and VEZF1, employ to counteract Polycomb- and heterochromatin-associated repression. We also undertake a critical analysis of whether CTCF could also act as a barrier protein. There is good evidence that barrier elements in vertebrates can form repressive chromatin domain boundaries. Future studies will determine whether barriers are frequently used to define repressive domain boundaries in vertebrates

Validity of two new brief instruments to estimate vegetable intake in adults

Cost effective population-based monitoring tools are needed for nutritional surveillance and interventions. The aim was to evaluate the relative validity of two new brief instruments (three item: VEG3 and five item: VEG5) for estimating usual total vegetable intake in comparison to a 7-day dietary record (7DDR). Sixty-four Australian adult volunteers aged 30 to 69 years (30 males, mean age ± SD 56.3 ± 9.2 years and 34 female mean age ± SD 55.3 ± 10.0 years). Pearson correlations between 7DDR and VEG3 and VEG5 were modest, at 0.50 and 0.56, respectively. VEG3 significantly (p < 0.001) underestimated mean vegetable intake compared to 7DDR measures (2.9 ± 1.3 vs. 3.6 ± 1.6 serves/day, respectively), whereas mean vegetable intake assessed by VEG5 did not differ from 7DDR measures (3.3 ± 1.5 vs. 3.6 ± 1.6 serves/day). VEG5 was also able to correctly identify 95%, 88% and 75% of those subjects not consuming five, four and three serves/day of vegetables according to their 7DDR classification. VEG5, but not VEG3, can estimate usual total vegetable intake of population groups and had superior performance to VEG3 in identifying those not meeting different levels of vegetable intake. VEG5, a brief instrument, shows measurement characteristics useful for population-based monitoring and intervention targeting

Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. &lt;p/&gt;Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. &lt;p/&gt;Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. &lt;p/&gt;Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters

Protecting a transgene expression from the HAC-based vector by different chromatin insulators

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00018-013-1362-9) contains supplementary material, which is available to authorized users

The Understated Ampersand

Donna Barkess very kindly came down from her Northumbrian castle to explain to us how the ampersand – 27th letter in our alphabet – has taken over her life, and why the ampersand is so much part of our lives; arguably the most expressive and versatile of characters, evolved from the Latin et, meaning ‘and’. It is steeped in history. Donna has explored the ‘&’ and its history over two millennia; how the symbol has been pushed and pulled around artistically by typographers, calligraphers, designers and artists and is everywhere, from company logos to our computer or phone keyboards. It is also an important character in computer coding. The ampersand is one of the oldest alphabetic abbreviations; by its very nature, history and definition, it is inviting and inclusive. Its versatility is astonishing. Whatever the designing of a new font inflicts upon it, the ampersand holds its own. Still recognisable after flourishes are added, or elements of its anatomy pruned back. Looking at the ampersand within any font reveals so much about the character of the typeface. Donna recently curated a successful exhibition to celebrate the beauty and versatility of the Ampersand and garnered much fascinating information about this outwardly innocuous and ubiquitous character. The exhibition explored the history and evolution of the ampersand, exploring how well it has stood the test of time. As well as taking the viewer through the intriguing life and times of the ampersand, the exhibition featured the work of many local and international artists and designers, who submitted pieces of artwork to celebrate the beauty and versatility of the ampersand. Donna is a graphic designer, illustrator, typographer and lecturer. She currently works at the University of Sunderland part-time, and spends the rest of the week working as Creative Director within a design consultancy. Her links to industry are strong, so she is able to draw upon her professional practice and use this a teaching tool. Predominantly lecturing in graphic design, Donna has taught across other programmes, such as advertising and illustration. Her main area of research during the past decade has been linked to sustainable design practice and the role of the contemporary designer. Currently researching ‘creative protest culture’ that considers the benefits and implications of creative protest in both theory and practice; aiming to demonstrate that creative and unconventional approaches to protest can be more effective than ‘traditional’ methods, thus examining the role of designers and creatives within a political context. More information can be found at: http://narcmagazine.com/news-and-per-say-and-sunderland-university

Curating Expertise: An Anthropological Approach

This thesis is an exploration of the expertise of UK-based contemporary art curators using an anthropological approach. By engaging with anthropology’s methodological and ethnographic promise, this thesis explores the lived experiences of UK-based professional contemporary art curators. It will consider their professional knowledge, identity, and practice for the purpose of illuminating their expertise. An extensive ethnographic and anthropological study of contemporary art curators in the UK does not currently exist. By extension, there are very few examples of ethnographic studies of contemporary art curators generally. Art curators do not feature within anthropological studies of professionals or indeed more widely within industry-focused literature, for example. Through taking an ethnographic approach, the thesis sheds light on the everyday lives of contemporary art curators in the UK. Moving beyond a generalised focus on curatorial narratives as ‘discourse’, it aims to reveal a more nuanced version of their professional expertise that. These situated examples will demonstrate the types of contradictions, ethical dilemmas, ideological aspirations, and personal aspects that reside in the everyday lives of UK-based contemporary art curators. Therefore, this thesis will provide novel perspectives on their practices and sociality, with a focus on the professional culture of curators that constructs art. Building on anthropological approaches to expert knowledge and professional culture, this research sheds new light on professional narratives surrounding the curatorial profession. A consideration through the lens of social anthropology of UK-based contemporary art curators in their metamorphic state, provides an ideal opportunity by which to consider the impacts of professional narratives, and to make a case for a different way of thinking, predicated on a consideration of curatorial expertise based upon ethnographic insights into their everyday lives

A question of design-exhibition

Sunderland Universities A Question of Design which explores the relationship between design and the environment. THE EXHIBITION EXPLORES THE ISSUES RELATED TO SUSTAINABILITY IN GRAPHIC DESIG