Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin

Huntington\u27s disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that approximately 50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

TLR and complement activation ensures efficient clearance of infection. Previous studies documented synergism between TLRs and the receptor for the pro-inflammatory complement peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. We demonstrate a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identify an underlying mechanistic target. Pre-exposure of PBMCs and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4 signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR upregulation or modulation of C5a-induced Ca2+ mobilization. Rather, TLRs targeted another C5a receptor, C5L2 (acting as a negative modulator of C5aR), by reducing C5L2 activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defense at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation

Delivering a multi-functional and resilient urban forest

Tree planting is widely advocated and applied in urban areas, with large-scaleprojects underway in cities globally. Numerous potential benefits are used to justify these planting campaigns. However, reports of poor tree survival raise questions about the ability of such projects to deliver on their promises over the long-term. Each potential benefit requires different supporting conditions—relating not only to the type and placement of the tree, but also to the broader urban system within which it is embedded. This set of supportingconditions may not always be mutually compatible and may not persist for the lifetime of the tree. Here, we demonstrate a systems-based approach that makes these dependencies, synergies, and tensions more explicit, allowing them to be used to test the decadal-scale resilience of urban street trees. Our analysis highlights social, environmental, and economic assumptions that are implicit within planting projects; notably that high levels of maintenance and public support for urban street trees will persist throughout their natural lifespan, andthat the surrounding built form will remain largely unchanged. Whilst the vulnerability of each benefit may be highly context specific, we identify approaches that address some typical weaknesses, making a functional, resilient, urban forest more attainable.

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

The role of ADAM 12 in determining skeletal muscle development and intramuscular fat deposition in cattle

© 2011 Dr. Chantal ColesThe research undertaken throughout this PhD thesis aimed to better understand the cellular mechanisms that influence development of skeletal muscle and intramuscular fat in beef cattle. In particular, I investigated the role of the extracellular matrix protease disintegrin and metalloprotease protein 12 (ADAM 12) in regulating muscle development and intramuscular fat deposition. These experiments sought to determine whether ADAM12 can be used as a potential modulator to manipulate these processes to increase muscling and intramuscular fat (IMF) deposition in cattle. My intital investigation sought to determine if variation in ADAM 12 expression in skeletal muscle was associated with different muscle phenotypes observed in cattle of various breeds. Primary skeletal muscle cells were isolated from Angus (high muscling), Hereford (moderate muscling) and Wagyu cross (X) Angus (low muscling) and grown in culture. The myogenic potential was found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro. The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight of cattle. ADAM 12 gene expression was not found to be related to in vitro proliferation rates, suggesting that its influence on muscling was negligible, or that its expression in in vitro culture conditions was not reflective of its role in development of muscle tissue in vivo. The role of ADAM 12 during fat development was investigated using an in vitro model of adipogenesis, 3T3-L1 cells. ADAM 12 gene expression was knocked down over a 13-day developmental timecourse using Stealth™ RNAi. DNA, RNA and protein samples were collected to closely analyse the effect of ADAM 12 knockdown on proliferation and differentiation of 3T3-L1 cells. ADAM 12 gene knockdown was found to reduce the proliferation and differentiation of 3T3-L1 cells. However, in ADAM 12 knockdown cells enzyme markers of adipogenesis G3PDH (early adipogenic marker- glucose metabolism) and FAS (late adipogenic marker- lipid filling) were found to be greater. This was consistent with the phenotypic observation and morphometric analysis that the ADAM 12 gene knockdown treated cells displayed hypertrophy, with fewer lipids of greater size. Microarray analysis found the pathway most affected by ADAM 12 gene knockdown to be regulation of insulin-like growth factor by insulin‐like growth factor binding proteins which is thought to be responsible for the effect found on proliferation. Adipocytokines such as adiponectin and adipsin were also found to be significantly down‐regulated in ADAM 12 gene knockdown cells. Absence of these adipocytokines, in particular adiponectin, have previously been shown to cause hypertrophy of mature adipocytes due to alterations in insulin‐sensitivity and fatty acid oxidation. Thus ADAM 12 appears to be an important regulator through all stages of fat development (proliferation, differentiation and energy metabolism). Following in vitro experimentation, the role of ADAM 12 in skeletal muscle and intramuscular fat development in vivo was investigated. The m. longissimus dorsi and m. semitendinosus (LD and ST) muscles from five Angus and Brahman cattle were collected for histology and ADAM 12 gene expression analysis. Muscle fibre typing was used to determine if ADAM 12 expression patterns related to differences in skeletal muscle traits, such as muscling and IMF deposition, which are often influenced by proportions of the different muscle fibres. Consistent with other findings, the ST muscle was found to contain a higher proportion of glycolytic myofibres than the LD muscle which contained a greater proportion of oxidative myofibres. ADAM 12 gene expression was also found to be greater in the LD compared with the ST muscle. Fluorescent immunohistochemical (FIHC) staining for ADAM 12 and image analysis found that the relative amount of ADAM 12 distribution to also be greater in the LD muscle. Regression analysis found a positive relationship for the distribution of ADAM 12 against the proportion of type I myofibres (r2 = 0.86, p < 0.05) and a negative relationship for the distribution of ADAM 12 against type IIX myofibres (r2 = 0.82, p < 0.05). The gene expression and regression analysis suggest ADAM 12 is type I myofibre specific in cattle. This was further confirmed by FIHC staining of ADAM 12 with slow myosin heavy chain I (slow MHC-I) with which ADAM 12 co-localised. The type I myofibre specificity of ADAM 12 may be important in the regulation and maintenance slow myofibres in the LD and of IMF deposition, to which proportion or number of type I myofibres have been related to previously. Lastly, the expression profiles of ADAM 12 was investigated in skeletal muscle across a developmental time-course in two cattle breeds with extreme muscle phenotypes, Piedmontese X Hereford (high muscling) and Wagyu X Hereford (high marbling) cattle. Muscle biopsies (from LD muscle) were collected at 3, 12 and 25 months of age. PiedmonteseX cattle contained a greater proportion type IIX, type IIAX and type IIC myofibres compared with WagyuX cattle which had a higher proportion of the more oxidative type I and type IIA myofibres. Gene and protein expression of ADAM 12 was not different between breeds of cattle. However, ADAM 12 gene expression at 3-months of age was found to be positively related to feedlot average daily weight gain (kg/d) (r2 = 0.62, p < 0.05), liveweight at 25-months of age (kg) (r2 = 0.67, p < 0.05), hot carcass weight (kg) (r2 = 0.81, p < 0.05), retail beef yield (kg) (r2 = 0.65, p < 0.05) and eye muscle area (cm2 ) (r2 = 0.77, p < 0.05). These results suggest ADAM 12 is a candidate to be used to predict muscling and beef yield in cattle from a very young age. Analysis of ADAM 12 protein and gene expression found that expression of ADAM 12 in skeletal muscle was elevated at 3-months of age during pre-weaning muscle growth, with reduced expression at 12-months of age. Expression of ADAM 12 in skeletal muscle was upregulated again at 25-months of age, possibly related to its role in IMF deposition and marbling during this stage of growth in cattle. Thus ADAM 12 proves to be an important regulator during muscle development and IMF deposition in cattle and is a candidate to predict beef yield from a very young age. Findings from this PhD thesis show that ADAM 12 may be an important regulator of muscle and marbling development in cattle. The specific association of ADAM 12 with type I myofibres, and its role in signalling adipogenesis as outlined by this PhD thesis and work by others also suggest it is a candidate to increase marbling in cattle. Greater muscling and marbling by modulation of ADAM 12 may improve the quality and value of beef and efficiency of beef production (by increasing beef yield) to enhance competitiveness of Australia’s beef export