Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option

Decision Problems for Nash Equilibria in Stochastic Games

We analyse the computational complexity of finding Nash equilibria in stochastic multiplayer games with $\omega$-regular objectives. While the existence of an equilibrium whose payoff falls into a certain interval may be undecidable, we single out several decidable restrictions of the problem. First, restricting the search space to stationary, or pure stationary, equilibria results in problems that are typically contained in PSPACE and NP, respectively. Second, we show that the existence of an equilibrium with a binary payoff (i.e. an equilibrium where each player either wins or loses with probability 1) is decidable. We also establish that the existence of a Nash equilibrium with a certain binary payoff entails the existence of an equilibrium with the same payoff in pure, finite-state strategies.Comment: 22 pages, revised versio

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy

BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease. RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease. CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target

Minimal stress shielding with a Mallory-Head titanium femoral stem with proximal porous coating in total hip arthroplasty

<p>Abstract</p> <p>Background</p> <p>As longevity of cementless femoral components enters the third decade, concerns arise with long-term effects of fixation mode on femoral bone morphology. We examined the long-term consequences on femoral remodeling following total hip arthroplasty with a porous plasma-sprayed tapered titanium stem.</p> <p>Methods</p> <p>Clinical data and radiographs were reviewed from a single center for 97 randomly selected cases implanted with the Mallory-Head Porous femoral component during primary total hip arthroplasty. Measurements were taken from preoperative and long-term follow-up radiographs averaging 14 years postoperative. Average changes in the proximal, middle and diaphyseal zones were determined.</p> <p>Results</p> <p>On anteroposterior radiographs, the proximal cortical thickness was unchanged medially and the lateral zone increased 1.3%. Middle cortical thickness increased 4.3% medially and 1.2% laterally. Distal cortical thickness increased 9.6% medially and 1.9% laterally. Using the anteroposterior radiographs, canal fill at 100 mm did not correlate with bony changes at any level (Spearman's rank correlation coefficient of -0.18, 0.05, and 0.00; p value = 0.09, 0.67, 0.97). On lateral radiographs, the proximal cortical thickness increased 1.5% medially and 0.98% laterally. Middle cortical thickness increased 2.4% medially and 1.3% laterally. Distal cortical thickness increased 3.5% medially and 2.1% laterally. From lateral radiographs, canal fill at 100 mm correlated with bony hypertrophy at the proximal, mid-level, and distal femur (Spearman's rank correlation coefficient of 0.85, 0.33, and 0.28, respectively; p value = 0.001, 0.016, and 0.01, respectively).</p> <p>Conclusion</p> <p>Stress shielding is minimized with the Mallory-Head titanium tapered femoral stem with circumferential proximal plasma-sprayed coating in well-fixed and well-functioning total hip arthroplasty. Additionally, the majority of femora demonstrated increased cortical thickness in all zones around the stem prosthesis. Level of Evidence: Therapeutic Level III.</p

Analysis of Myogenic and Candidate Disease Biomarkers in FSHD Muscle Cells

The UMMS Wellstone Program is a foundation and NIH-funded cooperative research center focusing on identifying biomarkers for facioscapulohumeral muscular dystrophy (FSHD) to gain insight into the molecular pathology of the disease and to develop potential therapies. FSHD is characterized by progressive wasting of skeletal muscles, with weakness often initiating in facial muscles and muscles supporting the scapula and upper arms. While the genetics associated with FSHD are complex, the major form of the disease, FSHD1, is linked to contraction of the D4Z4 repeat region located at chromosome 4q. Recently, a transcript encoded at the distal end of the repeat region, Dux4-fl, normally expressed in embryonic stem cells and germ cells, was also detected in differentiated muscle cells and biopsies from FSHD subjects, giving rise to the hypothesis that DUX4-FL function contributes to muscle weakness. We established a repository of high quality, well-characterized primary and immortalized muscle cell strains from FSHD and control subjects in affected families to provide biomaterials for cell and molecular studies to the FSHD research community. qPCR and immunostaining analyses demonstrate similar growth and differentiation characteristics in cells from FSHD and control subjects within families. We detected Dux4-fl transcript and protein in FSHD cells as recently described; interestingly, we also detected Dux4-fl in muscle cells from a subset of control individuals, suggesting that any Dux4-fl-mediated myopathy would require additional modifying elements. Microarray analysis of FSHD and control muscle cells demonstrated that several genes were upregulated in FSHD cells, including genes that were concurrently identified as downstream targets of Dux4-fl and as candidate FSHD disease genes. Future studies will further characterize the RNA and protein expression of candidate disease genes in cells from FSHD and control subjects, including nonmanifesting subjects with the D4Z4 lesion but no muscle weakness, and utilizing whole transcriptome sequencing (RNAseq) to identify additional candidates

Comparative genomics of isolates of a pseudomonas aeruginosa epidemic strain associated with chronic lung infections of cystic fibrosis patients

Pseudomonas aeruginosa is the main cause of fatal chronic lung infections among individuals suffering from cystic fibrosis (CF). During the past 15 years, particularly aggressive strains transmitted among CF patients have been identified, initially in Europe and more recently in Canada. The aim of this study was to generate high-quality genome sequences for 7 isolates of the Liverpool epidemic strain (LES) from the United Kingdom and Canada representing different virulence characteristics in order to: (1) associate comparative genomics results with virulence factor variability and (2) identify genomic and/or phenotypic divergence between the two geographical locations. We performed phenotypic characterization of pyoverdine, pyocyanin, motility, biofilm formation, and proteolytic activity. We also assessed the degree of virulence using the Dictyostelium discoideum amoeba model. Comparative genomics analysis revealed at least one large deletion (40-50 kb) in 6 out of the 7 isolates compared to the reference genome of LESB58. These deletions correspond to prophages, which are known to increase the competitiveness of LESB58 in chronic lung infection. We also identified 308 non-synonymous polymorphisms, of which 28 were associated with virulence determinants and 52 with regulatory proteins. At the phenotypic level, isolates showed extensive variability in production of pyocyanin, pyoverdine, proteases and biofilm as well as in swimming motility, while being predominantly avirulent in the amoeba model. Isolates from the two continents were phylogenetically and phenotypically undistinguishable. Most regulatory mutations were isolate-specific and 29% of them were predicted to have high functional impact. Therefore, polymorphism in regulatory genes is likely to be an important basis for phenotypic diversity among LES isolates, which in turn might contribute to this strain's adaptability to varying conditions in the CF lung

Compositional reasoning for shared-variable concurrent programs

Scalable and automatic formal verification for concurrent systems is always demanding. In this paper, we propose a verification framework to support automated compositional reasoning for concurrent programs with shared variables. Our framework models concurrent programs as succinct automata and supports the verification of multiple important properties. Safety verification and simulations of succinct automata are parallel compositional, and safety properties of succinct automata are preserved under refinements. We generate succinct automata from infinite state concurrent programs in an automated manner. Furthermore, we propose the first automated approach to checking rely-guarantee based simulations between infinite state concurrent programs. We have prototyped our algorithms and applied our tool to the verification of multiple refinements

Blueberry Progress Reports

The 1982 edition of the Blueberry Progress Reports was prepared for the Maine Blueberry Commission and the University of Maine Blueberry Advisory Committee by researchers with the Maine Life Sciences and Agriculture Experiment Station and Maine Cooperative Extension Service at the University of Maine, Orono. Projects in this report include: 1. Introduction 2. Blueberry IPM Program 3. Guthion Drift Study 4. Control, Biology, and Ecology of Insects 5. Blueberry Diseases: Incidence and Control 6. Physiology and Culture of the Lowbush Blueberry 7. Weed Control in Lowbush Blueberries 8. Pruning Blueberrie

Critical temperature of an anisotropic superconductor containing both nonmagnetic and magnetic impurities

The combined effect of both nonmagnetic and magnetic impurities on the superconducting transition temperature is studied theoretically within the BCS model. An expression for the critical temperature as a function of potential and spin-flip scattering rates is derived for a two-dimensional superconductor with arbitrary in-plane anisotropy of the superconducting order parameter, ranging from isotropic s-wave to d-wave (or any pairing state with nonzero angular momentum) and including anisotropic s-wave and mixed (d+s)-wave as particular cases. This expression generalizes the well-known Abrikosov-Gor'kov formula for the critical temperature of impure superconductors. The effect of defects and impurities in high temperature superconductors is discussed.Comment: 4 eps figure