Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

<p>Abstract</p> <p>Background</p> <p>Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds.</p> <p>Methods</p> <p>Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25.</p> <p>Results</p> <p>It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer.</p> <p>Conclusion</p> <p>This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.</p

The effects of beta-blockers on dobutamine-atropine stress echocardiography: early protocol versus standard protocol

BACKGROUND: To study the effects of Beta-blockers during Dobutamine Stress Echocardiography (DSE) comparing the hemodynamic benefits of an early administration of atropine in patients taking or not Beta-blockers. METHODS: One hundred and twenty-one patients were submitted to dobutamine stress echocardiography for the investigation of myocardial ischemia. The administration of atropine was randomized into two groups: A or B (early protocol when atropine was administered at 10 and 20 mcg/kg/min of dobutamine, respectively) and C (standard protocol with atropine at 40 mcg/kg/min of dobutamine). Analysis of the effects of Beta-blockers was done regarding the behavior pattern of heart rate and blood pressure, test time, number of conclusive and inconclusive (negative sub-maximum test) results, total doses of atropine and dobutamine, and general complications. RESULTS: Beta-blocked patients who received early atropine (Group A&B) had a significantly lower double product (p = 0.008), a higher mean test time (p = 0.010) and required a higher dose of atropine (p = 0.0005) when compared to the patients in this group who were not Beta-blocked. The same findings occurred in the standard protocol (Group C), however the early administration of atropine reduced test time both in the presence and absence of this therapy (p = 0.0001). The patients with Beta-blockers in Group A&B had a lower rate of inconclusive tests (26%) compared to those in Group C (40%). Complications were similar in both groups. CONCLUSION: The chronotropic response during dobutamine stress echocardiography was significantly reduced with the use of Beta-blockers. The early administration of atropine optimized the hemodynamic response, reduced test time in patients with or without Beta-blockers and reduced the number of inconclusive tests in the early protocol

Sterile neutrino portal to Dark Matter I: the U(1) B−L case

In this paper we explore the possibility that the sterile neutrino and Dark Matter sectors in the Universe have a common origin. We study the consequences of this assumption in the simple case of coupling the dark sector to the Standard Model via a global U(1)B−L, broken down spontaneously by a dark scalar. This dark scalar provides masses to the dark fermions and communicates with the Higgs via a Higgs portal coupling. We find an interesting interplay between Dark Matter annihilation to dark scalars — the CP-even that mixes with the Higgs and the CP-odd which becomes a Goldstone boson, the Majoron — and heavy neutrinos, as well as collider probes via the coupling to the Higgs. Moreover, Dark Matter annihilation into sterile neutrinos and its subsequent decay to gauge bosons and quarks, charged leptons or neutrinos lead to indirect detection signatures which are close to current bounds on the gamma ray flux from the galactic center and dwarf galaxies

Altered adipocyte differentiation and unbalanced autophagy in type 2 Familial Partial Lipodystrophy: an in vitro and in vivo study of adipose tissue browning

Type-2 Familial Partial Lipodystrophy is caused by LMNA mutations. Patients gradually lose subcutaneous fat from the limbs, while they accumulate adipose tissue in the face and neck. Several studies have demonstrated that autophagy is involved in the regulation of adipocyte differentiation and the maintenance of the balance between white and brown adipose tissue. We identified deregulation of autophagy in laminopathic preadipocytes before induction of differentiation. Moreover, in differentiating white adipocyte precursors, we observed impairment of large lipid droplet formation, altered regulation of adipose tissue genes, and expression of the brown adipose tissue marker UCP1. Conversely, in lipodystrophic brown adipocyte precursors induced to differentiate, we noticed activation of autophagy, formation of enlarged lipid droplets typical of white adipocytes, and dysregulation of brown adipose tissue genes. In agreement with these in vitro results indicating conversion of FPLD2 brown preadipocytes toward the white lineage, adipose tissue from FPLD2 patient neck, an area of brown adipogenesis, showed a white phenotype reminiscent of its brown origin. Moreover, in vivo morpho-functional evaluation of fat depots in the neck area of three FPLD2 patients by PET/CT analysis with cold stimulation showed the absence of brown adipose tissue activity. These findings highlight a new pathogenetic mechanism leading to improper fat distribution in lamin A-linked lipodystrophies and show that both impaired white adipocyte turnover and failure of adipose tissue browning contribute to disease.We thank FPLD2 patients for donating biological samples. We thank the Italian Network for Laminopathies and the European Consortium of Lipodystrophies (ECLip) for support and helpful discussion. We thank Aurelio Valmori for the technical support. The studies were supported by Rizzoli Orthopedic Institute “5 per mille” 2014 project to MC, AIProSaB project 2016 and Fondazione Del Monte di Bologna e Ravenna grant 2015–2016 “New pharmacological approaches in bone laminopathies based on the use of antibodies neutralizing TGF beta 2” to GL. GL is also supported by PRIN MIUR project 2015FBNB5Y.S

The neurogenic effects of exogenous neuropeptide Y: early molecular events and long-lasting effects in the hippocampus of trimethyltin-treated rats.

Modulation of endogenous neurogenesis is regarded as a promising challenge in neuroprotection. In the rat model of hippocampal neurodegeneration obtained by Trimethyltin (TMT) administration (8 mg/kg), characterised by selective pyramidal cell loss, enhanced neurogenesis, seizures and cognitive impairment, we previously demonstrated a proliferative role of exogenous neuropeptide Y (NPY), on dentate progenitors in the early phases of neurodegeneration. To investigate the functional integration of newly-born neurons, here we studied in adult rats the long-term effects of intracerebroventricular administration of NPY (2 \ub5g/2 \ub5l, 4 days after TMT-treatment), which plays an adjuvant role in neurodegeneration and epilepsy. Our results indicate that 30 days after NPY administration the number of new neurons was still higher in TMT+NPY-treated rats than in control+saline group. As a functional correlate of the integration of new neurons into the hippocampal network, long-term potentiation recorded in Dentate Gyrus (DG) in the absence of GABAA receptor blockade was higher in the TMT+NPY-treated group than in all other groups. Furthermore, qPCR analysis of Kruppel-like factor 9, a transcription factor essential for late-phase maturation of neurons in the DG, and of the cyclin-dependent kinase 5, critically involved in the maturation and dendrite extension of newly-born neurons, revealed a significant up-regulation of both genes in TMT+NPY-treated rats compared with all other groups. To explore the early molecular events activated by NPY administration, the Sonic Hedgehog (Shh) signalling pathway, which participates in the maintenance of the neurogenic hippocampal niche, was evaluated by qPCR 1, 3 and 5 days after NPY-treatment. An early significant up-regulation of Shh expression was detected in TMT+NPY-treated rats compared with all other groups, associated with a modulation of downstream genes. Our data indicate that the neurogenic effect of NPY administration during TMT-induced neurodegeneration involves early Shh pathway activation and results in a functional integration of newly-generated neurons into the local circuit

Phenomenology of Dark Matter from A4 Flavor Symmetry

We investigate a model in which Dark Matter is stabilized by means of a Z2 parity that results from the same non-abelian discrete flavor symmetry which accounts for the observed pattern of neutrino mixing. In our A4 example the standard model is extended by three extra Higgs doublets and the Z2 parity emerges as a remnant of the spontaneous breaking of A4 after electroweak symmetry breaking. We perform an analysis of the parameter space of the model consistent with electroweak precision tests, collider searches and perturbativity. We determine the regions compatible with the observed relic dark matter density and we present prospects for detection in direct as well as indirect Dark Matter search experiments.Comment: 15 pages, 4 figures.v2: minor modifications, matches version accepted for publication in JHE

Binding of Pramipexole to Extrastriatal Dopamine D2/D3 Receptors in the Human Brain: A Positron Emission Tomography Study Using 11C-FLB 457

The purpose of this study was to determine the binding sites of pramipexole in extrastriatal dopaminergic regions because its antidepressive effects have been speculated to occur by activating the dopamine D2 receptor subfamily in extrastriatal areas. Dynamic positron emission tomography (PET) scanning using 11C-FLB 457 for quantification of D2/D3 receptor subtype was performed on 15 healthy volunteers. Each subject underwent two PET scans before and after receiving a single dose of pramipexole (0, 0.125, or 0.25 mg). The study demonstrated that pramipexole significantly binds to D2/D3 receptors in the prefrontal cortex, amygdala, and medial and lateral thalamus at a dose of 0.25 mg. These regions have been indicated to have some relation to depression and may be part of the target sites where pramipexole exerts its antidepressive effects

Altered Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in the Exceptionally Long-Lived Naked Mole-Rat

The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life