Next-generation muscle-directed gene therapy by in silico vector design

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cisregulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy

Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders

Abstract Background Labrador retrievers are reportedly predisposed to many disorders but accurate prevalence information relating to the general population are lacking. This study aimed to describe demography, mortality and commonly recorded diseases in Labrador retrievers under UK veterinary care. Methods The VetCompass™ programme collects electronic patient record data on dogs attending UK primary-care veterinary practices. Demographic analysis covered all33,320 Labrador retrievers in the VetCompass™ database under veterinary care during 2013 while disorder and mortality data were extracted from a random sample of 2074 (6.2%) of these dogs. Results Of the Labrador retrievers with information available, 15,427 (46.4%) were female and 15,252 (53.6%) were male. Females were more likely to be neutered than males (59.7% versus 54.8%, P <  0.001). The overall mean adult bodyweight was 33.0 kg (SD 6.1). Adult males were heavier (35.2 kg, SD 5.9 kg) than adult females (30.4 kg, SD 5.2 kg) (P <  0.001). The median longevity of Labrador retrievers overall was 12.0 years (IQR 9.9–13.8, range 0.0–16.0). The most common recorded colours were black (44.6%), yellow (27.8%) and liver/chocolate (reported from hereon as chocolate) (23.8%). The median longevity of non-chocolate coloured dogs (n = 139, 12.1 years, IQR 10.2–13.9, range 0.0–16.0) was longer than for chocolate coloured animals (n = 34, 10.7 years, IQR 9.0–12.4, range 3.8–15.5) (P = 0.028). Of a random sample of 2074 (6.2%) Labrador retrievers under care in 2013 that had full disorder data extracted, 1277 (61.6%) had at least one disorder recorded. The total number of dogs who died at any date during the study was 176. The most prevalent disorders recorded were otitis externa (n = 215, prevalence 10.4%, 95% CI: 9.1–11.8), overweight/obesity (183, 8.8%, 95% CI: 7.6–10.1) and degenerative joint disease (115, 5.5%, 95% CI: 4.6–6.6). Overweight/obesity was not statistically significantly associated with neutering in females (8.3% of entire versus 12.5% of neutered, P = 0.065) but was associated with neutering in males (4.1% of entire versus 11.4% of neutered, P < 0.001). The prevalence of otitis externa in black dogs was 12.8%, in yellow dogs it was 17.0% but, in chocolate dogs, it rose to 23.4% (P < 0.001). Similarly, the prevalence of pyo-traumatic dermatitis in black dogs was 1.1%, in yellow dogs it was 1.6% but in chocolate dogs it rose to 4.0% (P = 0.011). Conclusions The current study assists prioritisation of health issues within Labrador retrievers. The most common disorders were overweight/obesity, otitis externa and degenerative joint disease. Males were significantly heavier females. These results can alert prospective owners to potential health issues and inform breed-specific wellness checks

Induced pseudoscalar coupling of the proton weak interaction

The induced pseudoscalar coupling $g_p$ is the least well known of the weak coupling constants of the proton's charged--current interaction. Its size is dictated by chiral symmetry arguments, and its measurement represents an important test of quantum chromodynamics at low energies. During the past decade a large body of new data relevant to the coupling $g_p$ has been accumulated. This data includes measurements of radiative and non radiative muon capture on targets ranging from hydrogen and few--nucleon systems to complex nuclei. Herein the authors review the theoretical underpinnings of $g_p$, the experimental studies of $g_p$, and the procedures and uncertainties in extracting the coupling from data. Current puzzles are highlighted and future opportunities are discussed.Comment: 58 pages, Latex, Revtex4, prepared for Reviews of Modern Physic

Increased Liver Uptake and Reduced Hepatic Stellate Cell Activation with a Cell-Specific Conjugate of the Rho-kinase Inhibitor Y27632

Rho-kinase regulates activation of hepatic stellate cells (HSC) during liver fibrosis, but the ubiquitous presence of this kinase may hinder examination of its exact role and the therapeutic use of inhibitors. We therefore coupled the Rho-kinase inhibitor Y27632 to a drug carrier that binds the mannose-6-phosphate insulin-like growth factor II (M6P/IGFII)-receptor which is upregulated on activated HSC. Y27632 was coupled to mannose-6-phosphate human serum albumin (M6PHSA), and in vitro experiments were performed on primary rat HSC. Biodistribution and effect studies were performed in an acute CCl(4) model in mice. Y27-conjugate remained stable in serum, while drug was efficiently released in liver homogenates. Receptor-blocking studies revealed that it was specifically taken up through the M6P/IGFII-receptor on fibroblasts, and it inhibited expression of fibrotic markers in activated HSC. In vivo, liver drug levels were significantly higher after injection of Y27-conjugate as compared to Y27632, and the conjugate accumulated specifically in HSC. After acute CCl(4)-induced liver injury, Y27-conjugate reduced the local activation of HSC, whereas an equimolar dose of free drug did not. We conclude that specific targeting of a Rho-kinase inhibitor to HSC leads to enhanced accumulation of the drug in HSC, reducing early fibrogenesis in the liver

Feasibility studies of time-like proton electromagnetic form factors at PANDA at FAIR

Simulation results for future measurements of electromagnetic proton form factors at \PANDA (FAIR) within the PandaRoot software framework are reported. The statistical precision with which the proton form factors can be determined is estimated. The signal channel $\bar p p \to e^+ e^-$ is studied on the basis of two different but consistent procedures. The suppression of the main background channel, $\textit{i.e.}$ $\bar p p \to \pi^+ \pi^-$, is studied. Furthermore, the background versus signal efficiency, statistical and systematical uncertainties on the extracted proton form factors are evaluated using two different procedures. The results are consistent with those of a previous simulation study using an older, simplified framework. However, a slightly better precision is achieved in the PandaRoot study in a large range of momentum transfer, assuming the nominal beam conditions and detector performance

Decay Properties of p pi- Systems Produced in Neutron Dissociation at Fermilab Energies

The authors have examined the decay distributions of (p{pi}{sup -}) systems produced in the reaction n + p {yields} (p{pi}{sup -}) + p for neutron momenta between 120 GeV/c and 300 GeV/c. Preliminary analysis of decay moments indicates the presence of large helicity-flip amplitudes even for small (p{pi}{sup -}) mass values, and does not support the hypothesis that the helicity non-flip (p{pi}{sup -}) states are produced peripherally in impact parameter. These results are in approximate agreement with predictions of the Deck mechanism. The experiment was performed at the M-3 neutral beam of Fermilab

Hepatocyte Growth Factor (HGF) Inhibits Collagen I and IV Synthesis in Hepatic Stellate Cells by miRNA-29 Induction

BACKGROUND: In chronic liver disease, hepatic stellate cells (HSC) transdifferentiate into myofibroblasts, promoting extracellular matrix (ECM) synthesis and deposition. Stimulation of HSC by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis due to its impact on myofibroblastic transition and ECM induction. In contrast, hepatocyte growth factor (HGF), exerts antifibrotic activities. Recently, miR-29 has been reported to be involved in ECM synthesis. We therefore studied the influence of HGF and TGF-β on the miR-29 collagen axis in HSC. METHODOLOGY: HSC, isolated from rats, were characterized for HGF and Met receptor expression by Real-Time PCR and Western blotting during culture induced myofibroblastic transition. Then, the levels of TGF-β, HGF, collagen-I and -IV mRNA, in addition to miR-29a and miR-29b were determined after HGF and TGF-β stimulation of HSC or after experimental fibrosis induced by bile-duct obstruction in rats. The interaction of miR-29 with 3'-untranslated mRNA regions (UTR) was analyzed by reporter assays. The repressive effect of miR-29 on collagen synthesis was studied in HSC treated with miR-29-mimicks by Real-Time PCR and immunoblotting. PRINCIPAL FINDINGS: The 3'-UTR of the collagen-1 and -4 subtypes were identified to bind miR-29. Hence, miR-29a/b overexpression in HSC resulted in a marked reduction of collagen-I and -IV synthesis. Conversely, a decrease in miR-29 levels is observed during collagen accumulation upon experimental fibrosis, in vivo, and after TGF-β stimulation of HSC, in vitro. Finally, we show that during myofibroblastic transition and TGF-β exposure the HGF-receptor, Met, is upregulated in HSC. Thus, whereas TGF-β stimulation leads to a reduction in miR-29 expression and de-repression of collagen synthesis, stimulation with HGF was definitely associated with highly elevated miR-29 levels and markedly repressed collagen-I and -IV synthesis. CONCLUSIONS: Upregulation of miRNA-29 by HGF and downregulation by TGF-β take part in the anti- or profibrogenic response of HSC, respectively

Models of care for musculoskeletal health: A cross-sectional qualitative study of Australian stakeholders' perspectives on relevance and standardised evaluation

Background: The prevalence and impact of musculoskeletal conditions are predicted to rapidly escalate in the coming decades. Effective strategies are required to minimise 'evidence-practice', 'burden-policy' and 'burden-service' gaps and optimise health system responsiveness for sustainable, best-practice healthcare. One mechanism by which evidence can be translated into practice and policy is through Models of Care (MoCs), which provide a blueprint for health services planning and delivery. While evidence supports the effectiveness of musculoskeletal MoCs for improving health outcomes and system efficiencies, no standardised national approach to evaluation in terms of their 'readiness' for implementation and 'success' after implementation, is yet available. Further, the value assigned to MoCs by end users is uncertain. This qualitative study aimed to explore end users' views on the relevance of musculoskeletal MoCs to their work and value of a standardised evaluation approach. Methods: A cross-sectional qualitative study was undertaken. Subject matter experts (SMEs) with health, policy and administration and consumer backgrounds were drawn from three Australian states. A semi-structured interview schedule was developed and piloted to explore perceptions about musculoskeletal MoCs including: i) aspects important to their work (or life, for consumers) ii) usefulness of standardised evaluation frameworks to judge 'readiness' and 'success' and iii) challenges associated with standardised evaluation. Verbatim transcripts were analysed by two researchers using a grounded theory approach to derive key themes. Results: Twenty-seven SMEs (n = 19; 70.4 % female) including five (18.5 %) consumers participated in the study. MoCs were perceived as critical for influencing and initiating changes to best-practice healthcare planning and delivery and providing practical guidance on how to implement and evaluate services. A 'readiness' evaluation framework assessing whether critical components across the health system had been considered prior to implementation was strongly supported, while 'success' was perceived as an already familiar evaluation concept. Perceived challenges associated with standardised evaluation included identifying, defining and measuring key 'readiness' and 'success' indicators; impacts of systems and context changes; cost; meaningful stakeholder consultation and developing a widely applicable framework. Conclusions: A standardised evaluation framework that includes a strong focus on 'readiness' is important to ensure successful and sustainable implementation of musculoskeletal MoCs