A04 The role of splicing factor SRSF6 in incomplete splicing of the HTT transcript

Background Huntington’s disease (HD) is caused by an expanded CAG repeat in exon 1 of the HTT gene. In models of HD, an expanded CAG repeat in HTT causes premature termination of HTT RNA during transcription; this occurs by a process called incomplete splicing. Incompletely spliced HTT (HTTexon1) includes exon 1 of the coding region of HTT, as well as a 5’ region of intron 1, which is non-coding. HTTexon1 encodes a truncated exon 1 HTT protein, which is implicated in HD pathogenesis. Although the precise RNA processing mechanism of Httexon1 is unknown, splicing factor SRSF6 has been shown to co-precipitate with transcripts containing Htt intron 1 in HD mice. Aim To elucidate the role of splicing factor SRSF6 in incomplete splicing of Htt in HD mice. Methods Heterozygous Srsf6 knock-out (KO) mice (Srsf6±) were generated by CRISPR/Cas9. Characterisation of Srsf6± mice was undertaken by quantitative RT-PCR and western blotting. Viability of homozygous Srsf6 KO (Srsf6-/-) mice was examined by inbreeding of Srsf6± mice. To assess the modulation of incomplete splicing by decreasing SRSF6, Srsf6± mice were bred to HD knock in mice (zQ175) and tissues were analysed. Levels of Httexon1 were measured by Quantigene, a gene expression assay. Results Srsf6-/- homozygotes were embryonic lethal, limiting us to the use of Srsf6± mice only. In Srsf6± heterozygotes, Srsf6 mRNA was decreased by 50% in brain and peripheral regions, and SRSF6 protein was decreased by 70% in brain compared to wild type mice. However, heterozygosity for Srsf6 knock out did not modulate the level on incomplete splicing in zQ175 mice. Conclusion Ablation of a single Srsf6 allele did not reduce levels of incomplete splicing in HD mice and therefore, further Srsf6 knock down may be required. Accordingly, mouse embryonic fibroblasts (MEFs) have been generated and will be used to measure Httexon1 levels after further Srsf6 knockdown by RNA interference. This work is supported by the CHDI foundation

Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay

Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by a CAG repeat expansion within exon 1 of the huntingtin (HTT) gene. HTT mRNA contains 67 exons and does not always splice between exon 1 and exon 2 leading to the production of a small polyadenylated HTTexon1 transcript, and the full-length HTT mRNA has three 3′UTR isoforms. We have developed a QuantiGene multiplex panel for the simultaneous detection of all of these mouse Htt transcripts directly from tissue lysates and demonstrate that this can replace the more work-intensive Taqman qPCR assays. We have applied this to the analysis of brain regions from the zQ175 HD mouse model and wild type littermates at two months of age. We show that the incomplete splicing of Htt occurs throughout the brain and confirm that this originates from the mutant and not endogenous Htt allele. Given that HTTexon1 encodes the highly pathogenic exon 1 HTT protein, it is essential that the levels of all Htt transcripts can be monitored when evaluating HTT lowering approaches. Our QuantiGene panel will allow the rapid comparative assessment of all Htt transcripts in cell lysates and mouse tissues without the need to first extract RNA

The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction.

Huntington's disease (HD) is a devastating neurodegenerative disorder, caused by a CAG/polyglutamine repeat expansion, that results in the aggregation of the huntingtin protein, culminating in the deposition of inclusion bodies in HD patient brains. We have previously shown that the heat shock response becomes impaired with disease progression in mouse models of HD. The disruption of this inducible arm of the proteostasis network is likely to exacerbate the pathogenesis of this protein-folding disease. To allow a rapid and more comprehensive analysis of the heat shock response, we have developed, and validated, a 16-plex QuantiGene assay that allows the expression of Hsf1 and nine heat shock genes, to be measured directly, and simultaneously, from mouse tissue. We used this QuantiGene assay to show that, following pharmacological activation in vivo, the heat shock response impairment in tibialis anterior, brain hemispheres and striatum was comparable between zQ175 and R6/2 mice. In contrast, although a heat shock impairment could be detected in R6/2 cortex, this was not apparent in the cortex from zQ175 mice. Whilst the mechanism underlying this impairment remains unknown, our data indicated that it is not caused by a reduction in HSF1 levels, as had been reported

SPRAT: Spectrograph for the Rapid Acquisition of Transients

We describe the development of a low cost, low resolution (R ~ 350), high throughput, long slit spectrograph covering visible (4000-8000) wavelengths. The spectrograph has been developed for fully robotic operation with the Liverpool Telescope (La Palma). The primary aim is to provide rapid spectral classification of faint (V ∼ 20) transient objects detected by projects such as Gaia, iPTF (intermediate Palomar Transient Factory), LOFAR, and a variety of high energy satellites. The design employs a volume phase holographic (VPH) transmission grating as the dispersive element combined with a prism pair (grism) in a linear optical path. One of two peak spectral sensitivities are selectable by rotating the grism. The VPH and prism combination and entrance slit are deployable, and when removed from the beam allow the collimator/camera pair to re-image the target field onto the detector. This mode of operation provides automatic acquisition of the target onto the slit prior to spectrographic observation through World Coordinate System fitting. The selection and characterisation of optical components to maximise photon throughput is described together with performance predictions. © (2014) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

Workgroup Report: Biomonitoring Study Design, Interpretation, and Communication—Lessons Learned and Path Forward

Human biomonitoring investigations have provided data on a wide array of chemicals in blood and urine and in other tissues and fluids such as hair and human milk. These data have prompted questions such as a) What is the relationship between levels of environmental chemicals in humans and external exposures? b) What is the baseline or “background” level against which individual levels should be compared? and c) How can internal levels be used to draw conclusions about individual and/or population health? An interdisciplinary panel was convened for a 1-day workshop in November 2004 with the charge of focusing on three specific aspects of biomonitoring: characteristics of scientifically robust biomonitoring studies, interpretation of human biomonitoring data for potential risks to human health, and communication of results, uncertainties, and limitations of biomonitoring studies. In this report we describe the recommendations of the panel

Liverpool Telescope 2: beginning the design phase

The Liverpool Telescope is a fully robotic 2-metre telescope located at the Observatorio del Roque de los Muchachos on the Canary Island of La Palma. The telescope began routine science operations in 2004, and currently seven simultaneously mounted instruments support a broad science programme, with a focus on transient followup and other time domain topics well suited to the characteristics of robotic observing. Work has begun on a successor facility with the working title ‘Liverpool Telescope 2’. We are entering a new era of time domain astronomy with new discovery facilities across the electromagnetic spectrum, and the next generation of optical survey facilities such as LSST are set to revolutionise the field of transient science in particular. The fully robotic Liverpool Telescope 2 will have a 4-metre aperture and an improved response time, and will be designed to meet the challenges of this new era. Following a conceptual design phase, we are about to begin the detailed design which will lead towards the start of construction in 2018, for first light ∼2022. In this paper we provide an overview of the facility and an update on progress. © (2016) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only

The quality case for information technology in healthcare

BACKGROUND: As described in the Institute of Medicine's Crossing the Quality Chasm report, the quality of health care in the U.S. today leaves much to be desired. DISCUSSION: One major opportunity for improving quality relates to increasing the use of information technology, or IT. Health care organizations currently invest less in IT than in any other information-intensive industry, and not surprisingly current systems are relatively primitive, compared with industries such as banking or aviation. Nonetheless, a number of organizations have demonstrated that quality can be substantially improved in a variety of ways if IT use is increased in ways that improve care. Specifically, computerization of processes that are error-prone and computerized decision support may substantially improve both efficiency and quality, as well as dramatically facilitate quality measurement. This report discusses the current levels of IT and quality in health care, how quality improvement and management are currently done, the evidence that more IT might be helpful, a vision of the future, and the barriers to getting there. SUMMARY: This report suggests that there are five key policy domains that need to be addressed: standards, incentives, security and confidentiality, professional involvement, and research, with financial incentives representing the single most important lever

Investigating and learning lessons from early experiences of implementing ePrescribing systems into NHS hospitals:a questionnaire study

Background: ePrescribing systems have significant potential to improve the safety and efficiency of healthcare, but they need to be carefully selected and implemented to maximise benefits. Implementations in English hospitals are in the early stages and there is a lack of standards guiding the procurement, functional specifications, and expected benefits. We sought to provide an updated overview of the current picture in relation to implementation of ePrescribing systems, explore existing strategies, and identify early lessons learned.Methods: a descriptive questionnaire-based study, which included closed and free text questions and involved both quantitative and qualitative analysis of the data generated.Results: we obtained responses from 85 of 108 NHS staff (78.7% response rate). At least 6% (n = 10) of the 168 English NHS Trusts have already implemented ePrescribing systems, 2% (n = 4) have no plans of implementing, and 34% (n = 55) are planning to implement with intended rapid implementation timelines driven by high expectations surrounding improved safety and efficiency of care. The majority are unclear as to which system to choose, but integration with existing systems and sophisticated decision support functionality are important decisive factors. Participants highlighted the need for increased guidance in relation to implementation strategy, system choice and standards, as well as the need for top-level management support to adequately resource the project. Although some early benefits were reported by hospitals that had already implemented, the hoped for benefits relating to improved efficiency and cost-savings remain elusive due to a lack of system maturity.Conclusions: whilst few have begun implementation, there is considerable interest in ePrescribing systems with ambitious timelines amongst those hospitals that are planning implementations. In order to ensure maximum chances of realising benefits, there is a need for increased guidance in relation to implementation strategy, system choice and standards, as well as increased financial resources to fund local activitie

Size constancy in bat biosonar?

Perception and encoding of object size is an important feature of sensory systems. In the visual system object size is encoded by the visual angle (visual aperture) on the retina, but the aperture depends on the distance of the object. As object distance is not unambiguously encoded in the visual system, higher computational mechanisms are needed. This phenomenon is termed "size constancy". It is assumed to reflect an automatic re-scaling of visual aperture with perceived object distance. Recently, it was found that in echolocating bats, the 'sonar aperture', i.e., the range of angles from which sound is reflected from an object back to the bat, is unambiguously perceived and neurally encoded. Moreover, it is well known that object distance is accurately perceived and explicitly encoded in bat sonar. Here, we addressed size constancy in bat biosonar, recruiting virtual-object techniques. Bats of the species Phyllostomus discolor learned to discriminate two simple virtual objects that only differed in sonar aperture. Upon successful discrimination, test trials were randomly interspersed using virtual objects that differed in both aperture and distance. It was tested whether the bats spontaneously assigned absolute width information to these objects by combining distance and aperture. The results showed that while the isolated perceptual cues encoding object width, aperture, and distance were all perceptually well resolved by the bats, the animals did not assign absolute width information to the test objects. This lack of sonar size constancy may result from the bats relying on different modalities to extract size information at different distances. Alternatively, it is conceivable that familiarity with a behaviorally relevant, conspicuous object is required for sonar size constancy, as it has been argued for visual size constancy. Based on the current data, it appears that size constancy is not necessarily an essential feature of sonar perception in bats