Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms

Huntington’s disease is caused by a CAG / polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington’s disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1 huntingtin protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of huntingtin protein-lowering approaches in preclinical studies and clinical trials. Huntingtin protein bioassays for soluble and aggregated forms of huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 huntingtin protein and full-length huntingtin protein. In addition, they are frequently used to quantify huntingtin protein levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel huntingtin protein bioassays to ensure that all soluble huntingtin protein isoforms could be distinguished. We utilized the zQ175 Huntington’s disease mouse model that has ∼190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyglutamine-length-matched standard, the vast majority of soluble mutant huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant huntingtin protein, soluble exon 1 huntingtin protein, soluble mutant huntingtin protein (excluding the exon 1 huntingtin protein) and total soluble full-length huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare the levels of huntingtin protein isoforms in a wide variety of mouse models of Huntington’s disease and to determine how these change in response to genetic or therapeutic manipulations

Highway to heaven: mammary gland development and differentiation

In recent years, the mammary gland epithelium has been shown to be a mixture of differentiated cell populations in a hierarchical relationship with their stem and progenitor cells. However, the mechanisms that regulate their cellular differentiation processes are still unclear. The identification of genes that govern stem and progenitor cell expansion, or that determine daughter cell fate, will be of crucial interest for understanding breast cancer diversity and, ultimately, improving treatment. Two recent analyses have identified some of the key genes that regulate these processes, lighting up the highway to normal mammary gland development

The impact of libraries as creative spaces

Ready access to information through digital media has challenged the perceived societal roles of public libraries. Since the mid 1990s, libraries have reoriented themselves towards public participation beyond lending and reading. Libraries now offer an increasing range of community-focused creative activities.Library spaces are transforming. In addition to housing archival and loan materials, desks and reading spaces, libraries are becoming even more exible and activity-oriented. Given these transformations, understanding and demonstrating the new contributions public libraries make to their communities is critical.In 2015, the State Library of Queensland (SLQ) commissioned researchers at the Digital Media Research Centre at Queensland University of Technology (QUT) to explore the impact of libraries as creative spaces.The objectives of the Libraries as Creative Spaces project were to:• Investigate the community impact of creative spaces in public libraries• Provide clear evidence of this impact• Articulate the opportunities to further embed creative spaces in public libraries or community spaces.The methods used included:• A contextual review of thinking regarding libraries as creative spaces• A literature review on the evaluation of creative activity in libraries and creative spaces more generally• Interviews and observational eld research at selected Queensland libraries• The development and refinement of an evidence-based Creative Spaces Impact Framework• The development of an associated rich media package (including videos, photos and digital stories) showcasing library-based creative activity across Queensland in 2015.The contextual review highlights the attention being paid to makerspaces as a site of creative activity in public libraries. However, the review also points to the many other ways public libraries are facilitating creative activity. It also demonstrates that while libraries have been formally evaluated for more than a hundred years, only recently has there been a concerted e ort to develop frameworks that can qualitatively assess the social impact on individuals and their communities. Moreover, few of these frameworks directly assess libraries as creative spaces.Following the contextual review, an extended literature review was undertaken for two reasons. Firstly, to develop our understanding of how creative spaces are evaluated (outside the library context). And secondly, to develop our understanding of how libraries are evaluated in more general terms. From this initial evidence base, a preliminary Creative Spaces Impact Framework was developed.The framework was used to interrogate creative activity at ve public library sites across Queensland – Ayr, Cleveland, Helensvale, Roma, and Victoria Point. The framework was revised in conjunction with the eldwork and further feedback from a range of stakeholders. The Creative Spaces Impact Framework allows for the evaluation of libraries as creative spaces through the creative activities they enable. These activities may be formally organised by the library, informally enacted by library clients, or involve a combination of both approaches. The contents of the framework represent a range of impact potentials, some of which will be relevant to certain library creative space activities more than others, if at all

Mass-flowering crops have a greater impact than semi-natural habitat on crop pollinators and pollen deposition

This is the final version. Available from Springer via the DOI in this record. Datasets available in the NERC Environmental Information Data Centre repository https://doi.org/10.5285/6128a4f7-d2ac-43c5-b492-af4c654e89b8.Context: Maximising insect pollination of mass-flowering crops is a widely-discussed approach to sustainable agriculture. Management actions can target landscape-scale semi-natural habitat, cropping patterns or field-scale features, but little is known about their relative effectiveness. Objective: To test how landscape composition (area of mass-flowering crops and semi-natural habitat) and field-scale habitat (margins and hedges) affect pollinator species richness, abundance, and pollen deposition within crop fields. Methods: We surveyed all flower visitors (Diptera, Coleoptera and Hymenoptera) in oilseed rape fields and related them to landscape composition and field features. Flower visitors were classified as bees, non-bee pollinators and brassica specialists. Total pollen deposition by individual taxa was estimated using single visit pollen deposition on stigmas combined with insect abundance. Results: The area of mass-flowering crop had a negative effect on the species richness and abundance of bees in fields, but not other flower visitors. The area of semi-natural habitat in the surrounding landscape had a positive effect on bees, but was not as important as the area of mass-flowering crop. Taxonomic richness and abundance varied significantly between years for non-bee pollinators. Greater cover of mass-flowering crops surrounding fields had a negative effect on pollen deposition, but only when non-bee pollinator numbers were reduced. Conclusions: Management choices that result in landscape homogenisation, such as large areas of mass-flowering crops, may reduce pollination services by reducing the numbers of bees visiting fields. Non-bee insect pollinators may buffer these landscape effects on pollen deposition, and management to support their populations should be considered.Natural Environment Research Counci

Spatial and topological organization of DNA chains induced by gene co-localization

Transcriptional activity has been shown to relate to the organization of chromosomes in the eukaryotic nucleus and in the bacterial nucleoid. In particular, highly transcribed genes, RNA polymerases and transcription factors gather into discrete spatial foci called transcription factories. However, the mechanisms underlying the formation of these foci and the resulting topological order of the chromosome remain to be elucidated. Here we consider a thermodynamic framework based on a worm-like chain model of chromosomes where sparse designated sites along the DNA are able to interact whenever they are spatially close-by. This is motivated by recurrent evidence that there exists physical interactions between genes that operate together. Three important results come out of this simple framework. First, the resulting formation of transcription foci can be viewed as a micro-phase separation of the interacting sites from the rest of the DNA. In this respect, a thermodynamic analysis suggests transcription factors to be appropriate candidates for mediating the physical interactions between genes. Next, numerical simulations of the polymer reveal a rich variety of phases that are associated with different topological orderings, each providing a way to increase the local concentrations of the interacting sites. Finally, the numerical results show that both one-dimensional clustering and periodic location of the binding sites along the DNA, which have been observed in several organisms, make the spatial co-localization of multiple families of genes particularly efficient.Comment: Figures and Supplementary Material freely available on http://dx.doi.org/10.1371/journal.pcbi.100067