Anatomy and Neuroscience - Research Publications
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ItemGenome-wide discovery of human splicing branchpoints(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2015-02)During the splicing reaction, the 5' intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 3' splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.
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ItemImproved definition of the mouse transcriptome via targeted RNA sequencing(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2016-05)Targeted RNA sequencing (CaptureSeq) uses oligonucleotide probes to capture RNAs for sequencing, providing enriched read coverage, accurate measurement of gene expression, and quantitative expression data. We applied CaptureSeq to refine transcript annotations in the current murine GRCm38 assembly. More than 23,000 regions corresponding to putative or annotated long noncoding RNAs (lncRNAs) and 154,281 known splicing junction sites were selected for targeted sequencing across five mouse tissues and three brain subregions. The results illustrate that the mouse transcriptome is considerably more complex than previously thought. We assemble more complete transcript isoforms than GENCODE, expand transcript boundaries, and connect interspersed islands of mapped reads. We describe a novel filtering pipeline that identifies previously unannotated but high-quality transcript isoforms. In this set, 911 GENCODE neighboring genes are condensed into 400 expanded gene models. Additionally, 594 GENCODE lncRNAs acquire an open reading frame (ORF) when their structure is extended with CaptureSeq. Finally, we validate our observations using current FANTOM and Mouse ENCODE resources.
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ItemElectrophysiological characterization of spontaneous recovery in deep dorsal horn interneurons after incomplete spinal cord injury(ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015-09)In the weeks and months following an incomplete spinal cord injury (SCI) significant spontaneous recovery of function occurs in the absence of any applied therapeutic intervention. The anatomical correlates of this spontaneous plasticity are well characterized, however, the functional changes that occur in spinal cord interneurons after injury are poorly understood. Here we use a T10 hemisection model of SCI in adult mice (9-10 wks old) combined with whole-cell patch clamp electrophysiology and a horizontal spinal cord slice preparation to examine changes in intrinsic membrane and synaptic properties of deep dorsal horn (DDH) interneurons. We made these measurements during short-term (4 wks) and long-term (10 wks) spontaneous recovery after SCI. Several important intrinsic membrane properties are altered in the short-term, but recover to values resembling those of uninjured controls in the longer term. AP discharge patterns are reorganized at both short-term and long-term recovery time points. This is matched by reorganization in the expression of voltage-activated potassium and calcium subthreshold-currents that shape AP discharge. Excitatory synaptic inputs onto DDH interneurons are significantly restructured in long-term SCI mice. Plots of sEPSC peak amplitude vs. rise times suggest considerable dendritic expansion or synaptic reorganization occurs especially during long-term recovery from SCI. Connectivity between descending dorsal column pathways and DDH interneurons is reduced in the short-term, but amplified in long-term recovery. Our results suggest considerable plasticity in both intrinsic and synaptic mechanisms occurs spontaneously in DDH interneurons following SCI and takes a minimum of 10 wks after the initial injury to stabilize.
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ItemDeveloping a spinal cord injury research strategy using a structured process of evidence review and stakeholder dialogue. Part III: outcomes(NATURE PUBLISHING GROUP, 2015-10)STUDY DESIGN: Focus Group. OBJECTIVES: To develop a unified, regional spinal cord injury (SCI) research strategy for Australia and New Zealand. SETTING: Australia. METHODS: A 1-day structured stakeholder dialogue was convened in 2013 in Melbourne, Australia, by the National Trauma Research Institute in collaboration with the SCI Network of Australia and New Zealand. Twenty-three experts participated, representing local and international research, clinical, consumer, advocacy, government policy and funding perspectives. Preparatory work synthesised evidence and articulated draft principles and options as a starting point for discussion. RESULTS: A regional SCI research strategy was proposed, whose objectives can be summarised under four themes. (1) Collaborative networks and strategic partnerships to increase efficiency, reduce duplication, build capacity and optimise research funding. (2) Research priority setting and coordination to manage competing studies. (3) Mechanisms for greater consumer engagement in research. (4) Resources and infrastructure to further develop SCI data registries, evaluate research translation and assess alignment of research strategy with stakeholder interests. These are consistent with contemporary international SCI research strategy development activities. CONCLUSION: This first step in a regional SCI research strategy has articulated objectives for further development by the wider SCI research community. The initiative has also reinforced the importance of coordinated, collective action in optimising outcomes following SCI.
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ItemGait recovery following spinal cord injury in mice: Limited effect of treadmill training(TAYLOR & FRANCIS LTD, 2016)BACKGROUND: Several studies in rodents with complete spinal cord transections have demonstrated that treadmill training improves stepping movements. However, results from studies in incomplete spinal cord injured animals have been conflicting and questions regarding the training dosage after injury remain unresolved. OBJECTIVES: To assess the effects of treadmill-training regimen (20 minutes daily, 5 days a week) for 3, 6 or 9 weeks on the recovery of locomotion in hemisected SCI mice. METHODS: A randomized and blinded controlled experimental trial used a mouse model of incomplete spinal cord injury (SCI). After a left hemisection at T10, adult male mice were randomized to trained or untrained groups. The trained group commenced treadmill training one week after surgery and continued for 3, 6 or 9 weeks. Quantitative kinematic gait analysis was used to assess the spatiotemporal characteristics of the left hindlimb prior to injury and at 1, 4, 7 and 10 weeks post-injury. RESULTS: One week after injury there was no movement of the left hindlimb and some animals dragged their foot. Treadmill training led to significant improvements in step duration, but had limited effect on the hindlimb movement pattern. Locomotor improvements in trained animals were most evident at the hip and knee joints whereas recovery of ankle movement was limited, even after 9 weeks of treadmill training. CONCLUSION: These results demonstrate that treadmill training may lead to only modest improvement in recovery of hindlimb movement after incomplete spinal cord injury in mice.
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ItemAAV-Mediated CRISPR/Cas Gene Editing of Retinal Cells In Vivo(ASSOC RESEARCH VISION OPHTHALMOLOGY INC, 2016-06)PURPOSE: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) has recently been adapted to enable efficient editing of the mammalian genome, opening novel avenues for therapeutic intervention of inherited diseases. In seeking to disrupt yellow fluorescent protein (YFP) in a Thy1-YFP transgenic mouse, we assessed the feasibility of utilizing the adeno-associated virus 2 (AAV2) to deliver CRISPR/Cas for gene modification of retinal cells in vivo. METHODS: Single guide RNA (sgRNA) plasmids were designed to target YFP, and after in vitro validation, selected guides were cloned into a dual AAV system. One AAV2 construct was used to deliver Streptococcus pyogenes Cas9 (SpCas9), and the other delivered sgRNA against YFP or LacZ (control) in the presence of mCherry. Five weeks after intravitreal injection, retinal function was determined using electroretinography, and CRISPR/Cas-mediated gene modifications were quantified in retinal flat mounts. RESULTS: Adeno-associated virus 2-mediated in vivo delivery of SpCas9 with sgRNA targeting YFP significantly reduced the number of YFP fluorescent cells of the inner retina of our transgenic mouse model. Overall, we found an 84.0% (95% confidence interval [CI]: 81.8-86.9) reduction of YFP-positive cells in YFP-sgRNA-infected retinal cells compared to eyes treated with LacZ-sgRNA. Electroretinography profiling found no significant alteration in retinal function following AAV2-mediated delivery of CRISPR/Cas components compared to contralateral untreated eyes. CONCLUSIONS: Thy1-YFP transgenic mice were used as a rapid quantifiable means to assess the efficacy of CRISPR/Cas-based retinal gene modification in vivo. We demonstrate that genomic modification of cells in the adult retina can be readily achieved by viral-mediated delivery of CRISPR/Cas.
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ItemRoles and post-translational regulation of cardiac class IIa histone deacetylase isoforms(WILEY, 2015-04-15)Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal-responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro-hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post-translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross-regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms.
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ItemAssessing structural and functional responses of murine hearts to acute and sustained β-adrenergic stimulation in vivo(ELSEVIER SCIENCE INC, 2016)INTRODUCTION: Given the importance of β-adrenoceptor signalling in regulating cardiac structure and function, robust protocols are required to assess potential alterations in such regulation in murine models in vivo. METHODS: Echocardiography was performed in naïve and stressed (isoprenaline; 30μg/g/day s.c. for up to 14days) mice, in the absence or presence of acute β-adrenergic stimulation (dobutamine 0.75μg/g, i.p.). Controls received saline infusion and/or injection. Hearts were additionally analysed gravimetrically, histologically and biochemically. RESULTS: In naïve mice, acute β-adrenoceptor stimulation with dobutamine increased heart rate, left ventricular (LV) fractional shortening (LVFS), ejection fraction (LVEF) and wall thickness and decreased LV diameter (p<0.05). In stressed mice, dobutamine failed to induce further inotropic and chronotropic responses. Furthermore, following dobutamine injection, these mice exhibited lower LVEF and LVFS at identical heart rates, relative to corresponding controls. Sustained isoprenaline infusion induced LV hypertrophy (increased heart weight, heart weight/body weight ratio, heart weight/tibia length ratio and LV wall thickness (p<0.05)) by 3days, with little further change at 14days. In contrast, increases in LVEF and LVFS were seen only at 14days (p<0.05). DISCUSSION: We describe protocols for and illustrative data from the assessment of murine cardiac responses to acute and sustained β-adrenergic stimulation in vivo, which would be of value in determining the impact of genetic or pharmacological interventions on such responses. Additionally, our data indicate that acute dobutamine stimulation unmasks early signs of LV dysfunction in the remodelled heart, even at a stage when basal function is enhanced.
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ItemExploring Motivation and Barriers to Physical Activity among Active and Inactive Australian Adults.(MDPI AG, 2017-06-28)Physical inactivity is a major global public health issue associated with a range of chronic disease outcomes. As such, the underlying motivation and barriers to whether or not an individual engages in physical activity is of critical public health importance. This study examines the National Heart Foundation of Australia Heart Week Survey conducted in March 2015. A total of 894 (40% female) Australian adults aged 25⁻54 years completed the survey, including items relating to motivation and barriers to being physically active. The most frequently selected responses regarding motivation for physical activity among those categorised as active (n = 696) were; to lose or maintain weight (36.6%, 95% CI 33.1⁻40.3), avoid or manage health condition (17.8%, 95% CI 15.1⁻20.8), and improve appearance (12.8%, 95% CI 10.5⁻15.5). Some gender differences were found with a greater proportion of females (43.8%, 95% CI 38.0⁻49.8) reporting lose or maintain weight as their main motivation for being physically active compared to males (31.9%, 95% CI 27.7⁻36.6). Among those categorised as inactive (n = 198), lack of time (50.0%, 95% CI 43.0⁻56.8) was the most frequently reported barrier to physical activity. While empirical studies seek to understand the correlates and determinants of physical activity, it is critical that beliefs and perceptions enabling and prohibiting engagement are identified in order to optimise physical activity promotion in the community.
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